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WO2023178378A1 - Mip inhibitors - Google Patents

Mip inhibitors Download PDF

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Publication number
WO2023178378A1
WO2023178378A1 PCT/AU2023/050201 AU2023050201W WO2023178378A1 WO 2023178378 A1 WO2023178378 A1 WO 2023178378A1 AU 2023050201 W AU2023050201 W AU 2023050201W WO 2023178378 A1 WO2023178378 A1 WO 2023178378A1
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Prior art keywords
alkyl
optionally substituted
compound
membered
group
Prior art date
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PCT/AU2023/050201
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French (fr)
Inventor
Ulrike Holzgrabe
Mitali Sarkar-Tyson
Nicolas SCHEUPLEIN
Theresa LOHR
Anja HASENKOPF
Aleksandra DEBOWSKI
Nicole BZDYL
Jonathan Baell
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DMTC Limited
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Priority claimed from AU2022900706A external-priority patent/AU2022900706A0/en
Application filed by DMTC Limited filed Critical DMTC Limited
Publication of WO2023178378A1 publication Critical patent/WO2023178378A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present disclosure generally relates to pipecolic acid derived compounds.
  • the present disclosure relates to pipecolic acid derived compounds of Formula (I).
  • the present disclosure also relates to formulations and compositions comprising the pipecolic acid derive compounds of Formula (I).
  • the present disclosure also relates to use of these compounds, compositions and/or formulations in treating and/or preventing a disease or condition mediated by a pathogen which is responsive to inhibition of macrophage infectivity potentiator (Mip) proteins.
  • Mip macrophage infectivity potentiator
  • the present disclosure also relates to use of these compounds, compositions and/or formulations in treating and/or preventing a disease or condition mediated in which Mip protein is a virulence factor.
  • Immunophilins are a superfamily of ubiquitous, highly conserved proteins which have been identified across all kingdoms of life (Gothel & Marahiel 1999). The majority of immunophilins exhibit peptidyl-prolyl cis-trans isomerase activity (PPIase) activity which catalyses the rate limiting cis-trans isomerisation of peptidyl-prolyl bonds (Kiefhaber et al. 1990). PPIases are required for efficient folding of numerous proteins and are therefore critical in a range of physiological processes including signal transduction, cell cycle regulation, and protein chaperoning (Lu, Hanes & Hunter 1996; Somarelli & Herrera 2007; Norville et al. 2011).
  • PPIase peptidyl-prolyl cis-trans isomerase activity
  • immunophilins are divided into three distinct families. Although all three bind to immunosuppressive drugs which inhibit their enzymatic activity, they are unrelated in structure and amino acid sequence (Siekierka et al. 1989; Rahfeld et al. 1994). Cyclophilins bind to cyclosporin A; parvulins bind to juglone; and FK506 binding proteins (FKBPs) bind to FK506 and rapamycin.
  • FKBPs FK506 binding proteins
  • Macrophage infectivity potentiator (Mip) proteins and Mip like proteins (ML1) belong to the family of FK506-binding proteins (FKBPs), which form part of the immunophilin superfamily.
  • FKBPs FK506-binding proteins
  • Mip proteins are classically approximately 28 kDa in size, consisting of two distinct domains; an N-terminal dimerization domain, linked to a C-terminal domain containing the FKBP fold exhibiting PPIase activity.
  • Mip proteins and Mip like proteins will be both be referred to as Mip from herein.
  • the first Mip protein was isolated from Legionella pneumophila, an environmental pathogen known to be the causative agent of Legionnaires disease (Engleberg et al., 1984).
  • the lack of a 24 kDa FKBP protein in an L. pneumophila strain was found to reduce the ability of the bacteria to infect macrophages 10- to 100-fold and was therefore named the macrophage infectivity potentiator protein (Mip) (Cianciotto et al., 1989, Fischer et al., 1992).
  • LpMip is a homodimeric protein comprised of two 22.8 kDa monomers (Riboldi- Tunnicliffe et al. 2001).
  • LpMip a site specific mutation was introduced within the mip gene (LPG0791), producing a null mip mutant (Cianciotto et al. 1990).
  • the mutant strain was 80-fold less infective than the wild-type strain within both human macrophages and macrophage-like (U937) cells. Intra-tracheal inoculation with the mutant strain resulted in slower disease progression and less lethal outcomes within a guinea pig model in comparison to wild-type and a complemented mutant. It was proposed that mip was required for full virulence and represented the first genetically defined L. pneumophila virulence factor. Further study demonstrated that LpMip was also implicated in infection of explanted lung epithelial cells (Cianciotto, Stamos & Kamp 1995).
  • Ceymann et al. (2008) solved the crystal structure of free LpMip and the LpMip- rapamycin complex by means of nuclear magnetic resonance spectroscopy. It was determined that binding was mediated by the rapamycin pipecoline moiety in conjunction with the LpMip hydrophobic pocket. Due to the structural similarity between Mip-rapamycin and FKBP12-rapamycin complexes, it was proposed that rapamycin derived non- immunosuppressive FKBP12 inhibitors may represent a starting point for Mip inhibitor development (Ceymann et al. 2008). This subsequently formed the basis of rationally designed small-molecular pipecolic acid derived Mip inhibitors (Juli et al. 2011). Inhibitors were tested against L.
  • the BpMip is a monomeric protein which lacks an N-terminal dimerisation domain. However, it exhibits 40% sequence identity with LpMip, and its C-terminal PPIase domain is highly homologous to those found within other Mips (Cianciotto et al. 1989; Ceymann et al. 2008; Norville et al. 2011). Norville et al. (2011) produced an in-frame deletion B. pseudomallei mip (BPSS1823) mutant which was significantly attenuated within a BALB/c murine model of infection. Infection studies were performed within both epithelial (A549) and macrophage-like (J774A.1) cells.
  • a high- throughput structural biology platform was utilised to perform rapid, strategic investigation of the BpMip crystal structure and enable subsequent inhibitor design (Begley et al. 2014).
  • Inhibitor efficacy was tested against B. pseudomallei within a macrophage-like (J774A.1) model of infection. Results showed a reduction of the cytotoxicity caused by B. pseudomallei in the presence of inhibitors, demonstrating that Mip is a novel anti- virulence target in this pathogen (Begley et al. 2014).
  • the Mip protein of Neisseria gonorrhoeae is important for invasion and persistence within macrophages, and is expressed during infection (Leuzzi et al., 2005, Starnino et al., 2010).
  • An analysis of 21 clinical N. gonorrhoeae isolates showed presence of the Mip protein in all tested clinical isolates, with high levels of conservation between isolates, indicating that the NgMip could be an important virulence factor (Stamino et al., 2010).
  • Sampson and Gotschlich (1992) first described a PPIase protein, inhibit-able by FK506 in Neisseria meningitidis.
  • N. meningitidis encodes two FKBPs: the Mip discussed above (now referred to as NmMipl), and a putative 110-amino acid Mip which has not yet been discussed in the literature, which will be referred to as NmMip2.
  • the NgMip is a surface-exposed 29 kDa lipoprotein, capable of PPIase activity which is inhibited by rapamycin.
  • the C-terminal PPIase domain has high homology to other bacterial Mips, including the LpMip (43.8% amino acid similarity) and the Trypanosoma cruzi Mip (42.3%).
  • the gene NMB 1567 which encodes for the meningococcal NmMipl is shown to be highly up-regulated during meningococci growth in blood.
  • N. meningitidis MC58 mutants lacking NMB1567 were sensitive to killing during the blood infection time course (Echenique-Rivera et al., 2011).
  • NmMipl contributes to the intracellular survival of meningococci within the human host.
  • NmMipl is known to be found on the outer membrane of the bacterium, and is capable of inducing antibodies that activated complement-mediated killing of the meningococci (Hung et al., 2011).
  • NmMipl contains a putative dimerization leader sequence found also in NgMip, which is similar to the dimerization domain of LpMip with the exception of the two methionine residues (Leuzzi et al., 2005).
  • the Mip protein of Coxiella burnetii was identified and shown to exhibit PPIase activity by Mo et al. (1995).
  • the amino acid sequence of CbMip shows similarity to LpMip (46%) and BpMip (43%) with a molecular mass of 25.5 kDa.
  • Secondary structure analysis has indicated that the protein predominantly adopts a beta-strand structure (Tse et al. 2014), however the crystal structure is yet to be solved. Very little is also known regarding the role of CbMip as a virulence factor although it has been shown to be immunogenic in both experimental and natural infections (Seshu et al. 1997).
  • Mips represent potential broad spectrum anti-virulence targets.
  • the pipecolic acid domain of rapamycin is responsible for the PPIase inhibition of Mip.
  • the other part of rapamycin binds to mTOR, a serine/threonine protein kinase, resulting in immunosuppression.
  • the pipecolic moiety has previously been used to generate non- immunosuppressive small molecule inhibitors.
  • X is selected from O, S, and NR 4 ;
  • a 1 , A 2 , A 3 and A 4 are each independently selected from the group consisting of CR' 2 , NR' , S and O, wherein each R' is independently selected from the group consisting of H, halogen, C 1-10 alkyl, OC 1-10 alkyl, C 1-10 haloalkyl, OC 1-10 haloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, OC 2-10 alkenyl, OC 2-10 alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C 1- 10 alkyl-3-10-membered-carbocyclyl, C 1-10 alkyl-3-10-membered-heterocyclyl, each of which is optionally substituted;
  • R 1 and R 3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl;
  • R 2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted;
  • R 4 is selected from the group consisting of H, C 1-10 alkyl, carbocyclyl, C 1-10 alkyl-carbocyclyl, heteroalkyl, heterocyclyl, and C 1-10 alkyl-heterocyclyl, each of which is optionally substituted.
  • the present inventors have surprisingly identified that by introducing substituents, and in particular bulkier substituents, at R 2 increased the stabilization of the pipecolic ester and in some cases the amide moiety (if present) against metabolic processes and/or provided the compounds with better occupation of a pocket at the Mip binding side responsible for the PPIase activity, resulting in improved activity and/or stability. Additionally, by varying the substitution at R 1 and/or R 3 , one or more further advantages were provided, including increased compound stability and/or activity as demonstrated by one or more embodiments or examples described herein. Other advantages of the presently claimed compounds are also described herein.
  • composition comprising a compound of Formula (I) as defined above, and a pharmaceutically acceptable excipient.
  • a method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein comprising administering to the subject an effective amount of a compound of Formula (I) as defined above, or a pharmaceutical composition as defined above.
  • a compound of Formula (I) as defined above or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
  • Mip macrophage infectivity potentiator
  • a compound of Formula (I) as defined above or a pharmaceutical composition as defined above for use in treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
  • Mip macrophage infectivity potentiator
  • a method of treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor comprising administering to the subject a compound of Formula (I) as defined above or a pharmaceutical composition as defined above.
  • Mip macrophage infectivity potentiator
  • a compound of Formula (I) as defined above or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
  • Mip macrophage infectivity potentiator
  • compound of Formula (I) as defined above or a pharmaceutical composition as defined above for use in treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
  • Mip macrophage infectivity potentiator
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • Figure 1 shows inhibition of C. burnetii growth in the presence of Mip compounds measured by Fold differences in Genome Equivalents (GE)
  • GE Genome Equivalents
  • Figure 2 shows that post-exposure treatment with Mip compound AN296 inhibit C. burnetii growth measured by Fold differences in GE (A) Treatment administered 2 days post- infection B) Treatment administered 3 days post-infection.
  • Figure 3 shows that treatment with Mip compounds inhibit C. burnetii growth in axenic media
  • A Treatment with compounds AN131, AN132, AN133 and ANCH37;
  • B Treatment with compounds AN296, AN263, AN259 and AN258;
  • C Treatment with compounds NJS227 and NJS224.
  • Figure 4 shows the Mip compounds inhibit C. burnetii during log-phase growth.
  • A Addition of AN296 during log phase growth at days 2 and 3 days inhibits C. burnetii growth;
  • B Treatment with compounds AN296, AN263, AN259 and AN258;
  • C Treatment with compounds NJS227 and NJS224.
  • Figure 5 shows the in vivo toxicity studies of lead candidate drugs AN296 and AN258 using Galleria mellonella model. Results demonstrates minimal toxicity of both candidates.
  • Figure 6 shows the in vivo efficacy studies of lead candidate drugs AN296 and AN258 using Galleria mellonella model. Results demonstrates increased survival of C. burnetii infected G. mellonella in the presence of candidate drugs.
  • Figure 7 shows that inhibitors of CbMip affect intracellular replication of C. burnetii.
  • a and B Intracellular replication of C. burnetii-NMII in the presence of CbMip inhibitor SF235 (purple square), AN296 (blue triangle) or vehicle control (black circle).
  • Figure 8 shows the targeted inhibition of CbMip reduces C. burnetii replication in axenic media in a dose-dependent manner.
  • A Bioluminescent was measured as an indicator of C. burnetii-lux replication.
  • C. burnetii-lux was inoculated at a concentration of 1 x 10 6 GE/mL into ACCM-2 media with 100 ⁇ M of CbMip inhibitors SF235 (grey square), AN296 (closed triangle) or vehicle control (open circle), and growth was monitored over 5 days. Data is presented as RLU (relative light units) with error bars representing the standard deviation (SD) from three independent experiments.
  • B C.
  • Figure 9 shows that delayed dosing with AN296 impairs C. burnetii replication in axenic media. Bioluminescence was measured as an indicator of C. burnetii-lux replication. The strain was inoculated at a concentration of 1 x 10 6 GE/mL into ACCM-2 media and growth was monitored over 5 days. Cultures were dosed with (A+B) 100 ⁇ M or (C+D) 50 ⁇ M of AN296 (closed circle) or vehicle control (open circle) on (A+C) day 2 or (B+D) day 3 of the growth curve. Data is presented as RLU (relative light units) with error bars represent standard error of the mean from four independent experiments.
  • RLU relative light units
  • FIG. 10 shows AN296 is highly potent against virulent C. burnetii-NMI in axenic media. Growth curve of C. burnetii-NMI in the presence of CbMip inhibitors.
  • C. burnetii-NMI was inoculated at a concentration of 1 x 10 4 CFU/mL into 5 mL of ACCM-2 media supplemented with 0.50 mM tryptophan and containing 100 ⁇ M of CbMip inhibitors, SF235 (grey square), AN296 (closed triangle), or vehicle control (open circle), and growth was monitored over 7 days by enumerating the number of colony forming units per mL in the culture on days 0, 1, 2, 3, 4 and 7.
  • first Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher-numbered item (e.g., a “third” item).
  • the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed.
  • the item may be a particular object, thing, or category.
  • “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required.
  • “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C.
  • “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.
  • range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, 4.75, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.
  • the term “subject” refers to any organism susceptible to a disease or condition that requires therapy.
  • the subject can be a mammal, primate, livestock (e.g., sheep, cow, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster).
  • livestock e.g., sheep, cow, horse, pig
  • companion animal e.g., dog, cat
  • laboratory animal e.g., mouse, rabbit, rat, guinea pig, hamster
  • the subject is a mammal.
  • the subject is human.
  • the disease or condition is mediated by a pathogen, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
  • the disease or condition is Q fever.
  • the term “treating” or “treatment” includes alleviation of the symptoms associated with a specific disease or condition and reducing and/or eliminating said symptoms.
  • the term “treating Q fever” refers to alleviating the symptoms associated with Q fever and/or eliminating the symptoms associated with Q fever.
  • the term “preventing” or “prevention” includes prophylaxis of the specific disorder or condition.
  • the term “preventing Q fever” refers to preventing the onset or duration of the symptoms associated with Q fever.
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof can be administered in a therapeutically effective amount.
  • therapeutically effective amount refers to a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated.
  • the result can be the reduction and/or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system.
  • one result may be the reduction of one or more symptoms associated with Q fever.
  • ⁇ ективное amount refers to an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects.
  • therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.
  • therapeutically effective amount includes, for example, a prophylactically effective amount.
  • a prophylactically effective amount is an amount sufficient to prevent Q fever.
  • an effective amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the compound and any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to an adjusted (e.g., reduced) amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.
  • onset of activity refers to the length of time to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated following the administration of the compound of Formula (I).
  • duration refers to the length of time that the therapeutic continues to be therapeutically effective, i.e., alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated.
  • onset, peak, and duration of therapy may vary depending on factors such as the patient, the condition of the patient, and the route of administration.
  • the compounds of the present disclosure may contain chiral (asymmetric) centers or the molecule as a whole may be chiral.
  • the individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.
  • halo or “halogen” whether employed alone or in compound words such as haloalkyl, represents fluorine, chlorine, bromine or iodine.
  • the alkyl when used in compound words such as haloalkyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different.
  • haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups.
  • Further examples of haloalkyl groups include -CF 3 , -CCl 3 , and -CH 2 CF 3 , -CF 2 CF 3 and -CH 2 CHFCI.
  • alkyl represents straight chain (i.e. linear) or branched chain hydrocarbon groups.
  • alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl groups.
  • the alkyl group is of 1 to 10 carbon atoms (i.e. C 1-10 alkyl).
  • the alkyl group is of 1 to 6 carbon atoms (i.e. C 1-6 alkyl).
  • heteroalkyl represents straight chain (i.e. linear) or branched chain hydrocarbon groups which are analogous to an alkyl group, but in which one or more carbon atoms is/are replaced by one or more heteroatoms selected from nitrogen, sulfur, and oxygen.
  • alkenyl represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon double bond.
  • alkenyl groups include ethylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.
  • the alkenyl group is of 2 to 10 carbon atoms (i.e. C 2-10 alkenyl).
  • the alkenyl group is of 2 to 6 carbon atoms (i.e. C 2-6 alkenyl)
  • alkynyl represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond.
  • alkenyl groups include , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups.
  • the alkynyl group is of 2 to 10 carbon atoms (i.e. C 2-10 alkynyl).
  • the alkynyl group is of 2 to 6 carbon atoms (i.e. C 2- 6 alkynyl).
  • haloalkyl represents to an alkyl group having at least one halogen substituent, where “alkyl” and “halogen” are as described above.
  • the haloalkyl group may have at least one, two or three halogen substituents.
  • haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups.
  • haloalkyl groups include -CF 3 , -CCl 3 , and -CH 2 CF 3 , -CF 2 CF 3 and -CH 2 CHFCI.
  • the haloalkyl group is of 1 to 10 carbon atoms (i.e. C 1- whaloalkyl).
  • the haloalkyl group is of 1 to 6 carbon atoms (i.e. C 1- 6 haloalkyl).
  • the terms “carbocyclyl” and “carbocycle” whether used alone, or in compound words such as alkylcarbocyclyl, represents a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings.
  • the carbocyclyl group is of 3 to 20 carbon atoms (i.e. C 3-20 -membered carbocyclyl).
  • the carbocyclyl group is of 3 to 10 carbon atoms (i.e.
  • C 3-10 -membered carbocyclyl examples include aryl groups such as phenyl, naphthyl, anthracenyl or fluorenyl, saturated groups such as cycloalkyl and cycloalkenyl groups e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl groups, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl.
  • the polycyclic ring system includes bicyclic and tricyclic ring systems.
  • cycloalkyl refers to a monocyclic or polycyclic carbocyclic ring system of varying sizes, e.g., from about 3 to about 20 carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl.
  • the polycyclic ring system includes bicyclic and tricyclic ring systems.
  • heterocyclyl refers to a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and wherein the ring system may be aromatic such as a “heteroaryl” group, non-aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings.
  • Heterocyclyl groups containing a suitable nitrogen atom include the corresponding N-oxides.
  • the heterocyclyl group is of 3 to 20 atoms (i.e. 3-20-membered heterocyclyl).
  • the heterocyclyl group is of 3 to 10 atoms (i.e. 3-10-membered heterocyclyl).
  • the heteroatom may preferably be N, O or S.
  • monocyclic non-aromatic heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, py-razolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thi-omorpholinyl and azepanyl.
  • bicyclic heterocyclyl groups in which one of the rings is non-aromatic include dihydrobenzofuranyl, indanyl, indolinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl.
  • monocyclic aromatic heterocyclyl groups also referred to as monocyclic heteroaryl groups
  • bicyclic aromatic heterocyclyl groups include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole.
  • the polycyclic ring system includes bicyclic and tricyclic ring systems.
  • an “aromatic” group means a cyclic group having 4m+2 % electrons, where m is an integer equal to or greater than 1.
  • aromatic is used interchangeably with “aryl” to refer to an aromatic group, regardless of the valency of aromatic group.
  • aryl represents an monocyclic or polycyclic aromatic carbocyclic ring system.
  • the aryl group is of 3 to 20 carbon atoms (i.e., an aromatic 3-20 membered carbocyclyl).
  • the aryl group is of 3 to 10 carbon atoms (i.e., an aromatic 3-10 membered carbocyclyl).
  • Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl or fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
  • heteroaryl represents a monocyclic or polycyclic aromatic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and may be substituted and/or contain fused rings.
  • Heteroaryl groups containing a suitable nitrogen atom include the corresponding N- oxides.
  • the heteroaryl group is of 3 to 20 atoms (i.e. 3-20-membered heteroaryl).
  • the heteroaryl group is of 3 to 10 atoms (i.e. 3-10-membered heteroaryl).
  • Examples of monocyclic heteroaryl groups include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl.
  • bicyclic heteroaryl groups include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. All regioisomers are contemplated, e.g. 2-pyridyl, 3-pyridyl and 4- pyridyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
  • “- C 1-10 alkyl-” represents an C 1-10 alkyl linker group in which the C 1-10 alkyl group is as defined supra.
  • “-O C 1-10 alkyl-” represents an alkoxy linker group in which the C 1-10 alkyl group is as defined supra.
  • “-C 2-10 alkenyl” represents an C 2-10 alkenyl linker group in which the C 2-10 alkenyl group is as defined supra.
  • “-OC 2-10 alkenyl-” represents an alkenyloxy linker group in which the C 2-10 alkenyl group is as defined supra.
  • saturated refers to a group where all available valence bonds of the backbone atoms are attached to other atoms
  • saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine, and the like.
  • the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms.
  • the term “optionally substituted” means that a functional group is either substituted or unsubstituted, at any available position.
  • substituted refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent).
  • substituent i.e., substituent
  • unsubstituted refers to a group that does not have any further groups attached thereto or substituted therefore.
  • the present disclosure relates to compounds of Formula (I) and pharmaceutically acceptable salts thereof. Salts may be formed in the case of embodiments of the compound of Formula (I), which contain a suitable acidic or basic group. Suitable salts of the compound of Formula (I) include those formed with organic or inorganic acids or bases.
  • the phrase “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts.
  • Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis- (2-hydroxy-3-naph
  • Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine.
  • organic bases for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di-
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. It will also be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport.
  • the compound of Formula (I) is a hydrochloride salt.
  • solvates a complex with solvents in which they are reacted or from which they are precipitated or crystallized.
  • solvates For example, a complex with water is known as a "hydrate”.
  • pharmaceutically acceptable solvate or “solvate” refer to an association of one or more solvent molecules and a compound of the present disclosure.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be understood that the present disclosure encompasses solvated forms, including hydrates, of the compounds of Formula (I) and salts thereof.
  • stereoisomer refers to compounds having the same molecular formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three-dimensional orientations of their atoms in space.
  • enantiomers refers to two compounds that are stereoisomers in that they are non- superimposable mirror images of one another. Relevant stereocenters may be denoted with (R)- or (S)- configuration.
  • the present disclosure provides compounds of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: as described in any of the embodiments below.
  • the present disclosure also provides a macrophage infectivity potentiator (Mip) protein inhibitor compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, as described in any of the embodiments below.
  • Mip macrophage infectivity potentiator
  • X is selected from O, S and NR 4 .
  • X is O or NR 4 .
  • X is O.
  • X is NR 4 .
  • the compound of Formula (I) is selected from the group consisting of: wherein A 1 , A 2 , A 3 , A 4 , L 1 , L 2 , R 1 , R 2 , R 3 and R 4 are as described herein.
  • R 4 may be selected from the group consisting of H, C 1-10 alkyl, carbocyclyl, C 1-10 alkyl-carbocyclyl, heteroalkyl, heterocyclyl, and C 1-10 alkyl-heterocyclyl, each of which is optionally substituted. In one embodiment, R 4 is selected from the group consisting of H and C 1-10 alkyl. In one embodiment, R 4 is H.
  • a 1 , A 2 , A 3 and A 4 are each connected to form a 6-membered heterocycl, wherein depending on the nature of each of A 1 , A 2 , A 3 and A 4 , can be optionally interrupted and/or optionally substituted.
  • a 1 , A 2 , A 3 and A 4 are each independently selected from the group consisting of CR' 2 , NR' , S and O, wherein R is described herein.
  • a 1 , A 2 A 3 and A 4 are each independently selected from the group consisting CR' 2 , NR' , S and O, wherein R is as described herein.
  • a 1 , A 2 and A 3 are each CH 2 , and A 3 is selected from the group consisting CR' 2 , NR' , S and O, wherein R is as described herein.
  • each R is independently selected from the group consisting of H, halogen, C 1-10 alkyl, O C 1-10 alkyl, C 1-10 haloalkyl, O C 1-10 haloalkyl, C 2-10 alkenyl, C 2- 10 alk ynyl, OC 2-10 alkenyl, OC 2-10 alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C 1- 10 alkyl-3 - 10-membered-carbocyclyl, C 1- 10 alkyl-3 - 10-membered- heterocyclyl, each of which is optionally substituted.
  • each R is independently selected from the group consisting of H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1- 6 haloalkyl, and OC 1-6 haloalkyl, each of which is optionally substituted. In one embodiment, each R is independently selected from the group consisting of H or halogen.
  • a 1 , A 2 , A 3 and A 4 are each independently selected from the group consisting of CR' 2 , NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C 1-10 alkyl, O C 1-10 alkyl, C 1-10 haloalkyl, O C 1-10 haloalkyl, C 2- 10 alk enyl, C 2-10 alkynyl, OC 2-10 alkenyl, OC 2-10 alkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C 1-10 alkyl-3-10-membered-carbocyclyl, C 1-10 alkyl- 3-10-membered- heterocyclyl, each of which is optionally substituted.
  • a 1 , A 2 A 3 and A 4 are each independently selected from the group consisting CR' 2 , NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, and OC 1-6 haloalkyl, each of which is optionally substituted.
  • a 1 , A 2 and A 3 are each CH 2 , and A 3 is selected from the group consisting CR' 2 , NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, and OC 1-6 haloalkyl, each of which is optionally substituted.
  • a 1 , A 2 A 3 and A 4 are each CH 2 .
  • the orientation of the linkers L 1 and L 2 within the compound of Formula (I) are undefined. That is, L 1 and L 2 may be attached at either side within the compound of Formula (I).
  • the compound of Formula (I) when L 2 is wherein represents the attachment point of L 2 to the rest of the compound, the compound of Formula (I) may be selected from:
  • L 1 and L 2 are independently selected from one another.
  • L 1 is present and selected from the group consisting of -C 1- 10 alkyl-, -N( C 1-10 alkyl)-, -O C 1-10 alkyl-, -C 2-10 alkenyl-, and -OC 2-10 alkenyl- wherein each C 1- 10 alkyl or C 2-10 alkenyl is uninterrupted or interrupted and optionally substituted.
  • L 1 is - C 1-10 alkyl- wherein C 1-10 alkyl is uninterrupted or interrupted and optionally substituted.
  • each C 1-10 alkyl or C 2-10 alkenyl may be uninterrupted or interrupted and optionally substituted.
  • each C 1-10 alkyl or C 2-10 alkenyl of L 1 and L 2 are uninterrupted and unsubstituted. In the instance where C 1-10 alkyl or C 2-10 alkenyl are not substituted with one or more R 7 , it will be understood that a hydrogen atom will remain as the substitution.
  • L 1 is absent or selected from - C 1-10 alkyl or -N( C 1-10 alkyl)-. In one embodiment, L 1 is absent or selected from -C 1-6 alkyl- or -N(C 1-6 alkyl)-. In one embodiment, L 1 is absent or selected from-CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, or -N/CH 2 )-. In one embodiment, L 1 is absent. In one embodiment, L 1 is - C 1-10 alkyl-. In one embodiment, L 1 is -C 1-6 alkyl-. In one embodiment, L 1 is -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -. In one embodiment, L 1 is -CH 2 -.
  • L 2 is
  • L 2 is -CH 2 CH 2 CH 2 -, and the compound of Formula (I) is
  • the compound of Formula (I) is selected from:
  • L 2 is and the compound of Formula (I) is selected from:
  • Formula (I) is selected from:
  • L 1 is absent and L 2 is and the compound of
  • Formula (I) is selected from:
  • L 1 is -CH 2 -
  • X is O or NR 4
  • the compound of Formula (I) is selected from the group consisting of:
  • L 1 is -CH 2 -
  • L 2 is X is O or NR 4
  • the compound of Formula (I) is selected from the group consisting of:
  • L 1 is absent
  • X is O or NR 4
  • the compound of Formula (I) is selected from the group consisting of:
  • L 1 is -CH 2 -
  • L 2 is X is O or NR 4
  • the compound of Formula (I) is selected from the group consisting of:
  • R 1 and R 3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. That is, R 1 may be an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. Similarly, R 3 may be an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. In one embodiment, R 1 is an optionally substituted carbocyclyl. In one embodiment, R 1 is an optionally substituted heterocyclyl. In one embodiment, R 3 is an optionally substituted carbocyclyl. In one embodiment, R 3 is an optionally substituted heterocyclyl. R 1 and R 3 may be the same (e.g.
  • R 1 is an optionally substituted carbocyclyl and R 3 is an optionally substituted carbocyclyl) or R 1 and R 3 may be different (e.g. R 1 is an optionally substituted carbocyclyl and R 3 is an optionally substituted heterocyclyl) (e.g. the R 1 and R 3 substituents are independently selected from one another). In one embodiment, R 1 is an optionally substituted carbocyclyl and R 3 is an optionally substituted heterocyclyl.
  • R 1 and R 3 are each independently selected from an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. That is, R 1 may be an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. Similarly, R 3 may be an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. In one embodiment, R 1 is an optionally substituted 3-10-membered carbocyclyl. In one embodiment, R 1 is an optionally substituted 3-10-membered heterocyclyl.
  • R 3 is an optionally substituted 3-10-membered carbocyclyl. In one embodiment, R 3 is an optionally substituted 3-10-membered heterocyclyl. In one embodiment, R 1 is an optionally substituted 3-10-membered carbocyclyl and R 3 is an optionally substituted 3-10- membered heteroaryl.
  • R 1 and R 3 are each independently selected from an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. That is, R 1 may be an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. Similarly, R 3 may be an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. In one embodiment, R 1 is an optionally substituted monocyclic carbocyclyl. In one embodiment R 3 is an optionally substituted monocyclic heterocyclyl. In one embodiment, R 1 is an optionally substituted monocyclic carbocyclyl and R 3 is an optionally substituted monocyclic heterocyclyl.
  • R 1 and R 3 are each independently selected from an optionally substituted aryl or an optionally substituted heteroaryl. That is, R 1 may be an optionally substituted aryl or an optionally substituted heteroaryl. Similarly, R 3 may be an optionally substituted aryl or an optionally substituted heteroaryl. In one embodiment, R 1 is an optionally substituted aryl. In one embodiment, R 1 is an optionally substituted heteroaryl. In one embodiment, R 3 is an optionally substituted aryl. In one embodiment, R 3 is an optionally substituted heteroaryl. In one embodiment, R 1 is an optionally substituted aryl and R 3 is an optionally substituted heteroaryl.
  • R 1 and R 3 are each independently selected from an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. That is, R 1 may be an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. Similarly, R 3 may be an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. In one embodiment, R 1 is an optionally substituted monocyclic aryl. In one embodiment, R 1 is an optionally substituted monocyclic heteroaryl. In one embodiment, R 3 is an optionally substituted monocyclic aryl. In one embodiment, R 3 is an optionally substituted monocyclic heteroaryl. In one embodiment, R 1 is an optionally substituted monocyclic aryl and R 3 is an optionally substituted monocyclic heteroaryl.
  • R 1 and R 3 are each independently selected from an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl. That is, R 1 may be an optionally substituted 3-10-membered aryl or an optionally substituted 3-10- membered heteroaryl. Similarly, R 3 may be an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl. In one embodiment, R 1 is an optionally substituted 3-10-membered aryl. In one embodiment, R 1 is an optionally substituted 3-10- membered heteroaryl. In one embodiment, R 3 is an optionally substituted 3-10-membered aryl. In one embodiment, R 3 is an optionally substituted 3-10-membered heteroaryl. In one embodiment R 1 is an optionally substituted 3-10-membered aryl and R 3 is an optionally substituted 3-10-membered heteroaryl.
  • R 1 and R 3 are each independently selected from an optionally substituted 5-6-membered aryl or an optionally substituted 5-6-membered heteroaryl. That is, R 1 may be an optionally substituted 5-6-membered aryl or an optionally substituted 5-6- membered heteroaryl. Similarly, R 3 may be an optionally substituted 5-6-membered aryl or an optionally substituted 5-6-membered heteroaryl. In one embodiment, R 1 is an optionally substituted 5-6-membered aryl. In one embodiment, R 1 is an optionally substituted 5-6- membered heteroaryl. In one embodiment, R 3 is an optionally substituted 5-6-membered aryl. In one embodiment, R 3 is an optionally substituted 5-6-membered heteroaryl. In one embodiment R 1 is an optionally substituted 5-6-membered aryl and R 3 is an optionally substituted 3-10-membered heteroaryl.
  • R 1 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
  • R 1 is an optionally substituted phenyl.
  • R 3 is an optionally substituted phenyl or an optionally substituted 5-6-membered heteroaryl.
  • R 1 is an optionally substituted phenyl and R 3 is an optionally substituted 5-6- membered heteroaryl.
  • R 3 is an optionally substituted N-heterocyclyl. In one embodiment, R 3 is an optionally substituted N-heteroaryl. In one embodiment, R 3 is an optionally substituted 3-10-membered-N-heteroaryl.
  • N- heterocyclyl and N-heteroaryl represents a nitrogen containing heterocyclyl and a nitrogen containing heteroaryl, respectively.
  • R 3 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Each of which is optionally substituted.
  • R 3 is a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
  • R 3 is an optionally substituted pyridyl.
  • R 1 is an optionally substituted phenyl and R 3 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
  • R 1 is an optionally substituted phenyl and R 3 is a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
  • R 3 is a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl
  • R 1 is an optionally substituted phenyl and R 3 is an optionally substituted pyridyl. Further examples of R 1 and/or R 3 include, but are not limited to: each of which is optionally substituted, wherein represents the attachment point to the rest of the compound.
  • R 3 is independently selected from the group consisting of: each of which is optionally substituted, wherein represents the attachment point of R 3 to the rest of the compound.
  • R 1 is an optionally substituted phenyl and R 3 is independently selected from the group consisting of: each of which is optionally substituted, wherein represents the attachment point of R 1 to the rest of the compound.
  • the compound of Formula (I) is wherein: A 5 , A 6 , A 7 , A 8 and A 9 are each independently selected from CR 5 , N or N-oxide; and
  • X, L 1 , L 2 , R 1 , R 2 and R 5 are as described herein. It will be appreciated that A 5 , A 6 , A 7 , A 8 , and A 9 form an aromatic ring. In one embodiment, A 6 is N or N-oxide and each of A 5 , A 7 , A 8 , and A 9 is independently CR 5 . R 2
  • R 2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted.
  • R 2 is an optionally substituted alkyl.
  • R 2 is an optionally substituted alkenyl.
  • R 2 is an optionally substituted alkynyl.
  • R 2 is an optionally substituted carbocyclyl.
  • R 2 is an optionally substituted alkylcarbocyclyl.
  • R 2 is an optionally substituted heteroalkyl.
  • R 2 is an optionally substituted heterocyclyl.
  • R 2 is an optionally substituted alky lheterocy cly 1.
  • R 2 is selected from the group consisting of C 1-10 alkyl, cycloalkyl, C 1-10 alkylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 1-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is an optionally substituted C 1-10 alkyl.
  • R 2 is an optionally substituted cycloalkyl. In one embodiment, R 2 is an optionally substituted C 1- 10 alk ylcycloalkyl. In one embodiment, R 2 is an optionally substituted C 1-10 alkylheterocyclyl. In one embodiment, R 2 is an optionally substituted aryl. In one embodiment, R 2 is an optionally substituted C 1-10 alkylaryl. In one embodiment, R 2 is an optionally substituted heteroaryl. In one embodiment, R 2 is an optionally substituted C 1-10 alkylheteroaryl.
  • the present inventors have surprisingly identified that by introducing a bulkier R 2 substituent (e.g. an optionally substituted alkyl or an optionally substituted alkylaryl) in the middle chain of the compound connecting the lateral pyridine ring with the pipecolic moiety, highly active PPIase inhibitors were identified which have potent inhibitory properties against the macrophage infectivity potentiator (Mip) protein of several bacterial pathogens.
  • a bulkier R 2 substituent e.g. an optionally substituted alkyl or an optionally substituted alkylaryl
  • R 2 is selected from the group consisting of C 1-6 alkyl, aryl, C 1- 6 alkylaryl, heteroaryl, and C 1-6 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is an optionally substituted C 1-6 alkyl.
  • R 2 is an optionally substituted aryl.
  • R 2 is an optionally substituted C 1-6 alkylaryl.
  • R 2 is an optionally substituted heteroaryl.
  • R 2 is an optionally substituted C 1-6 alkylheteroaryl.
  • R 2 is selected from the group consisting of alkyl, aryl, alkylaryl, heteroaryl or alkyheteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 1-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl or C 1- 10 alk ylheteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 1-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, or C 1-6 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 1-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting ofC 1-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 1-6 alkyl, phenyl, C 1-6 alkylphenyl, 5-6-membered heteroaryl or C 1- 6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 2 comprises a 5-6 membered heteroaryl
  • the 5-6- membered heteroaryl may be selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
  • R 2 comprises a 5-6 membered heteroaryl
  • the 5-6-membered heteroaryl may be an optionally substituted pyridyl.
  • R 2 is selected from the group consisting of C 1-6 alkyl, phenyl, C 1- 6 alkylphenyl, pyridyl, or C 1-6 alkylpyridyl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 3-20 alkyl, aryl, alkylaryl, heteroaryl or alkyheteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl or C 1-10 alkylheteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, or C 1-6 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1- 6 alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6- membered aryl, C 1-6 alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6- membered heteroaryl, each of which is optionally substituted. In one embodiment, R 2 is selected from the group consisting of C 3-6 alkyl, phenyl, C 1-6 alkylphenyl, 5-6-membered heteroaryl or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 3-6 alkyl, phenyl, C 1- 6 alkylphenyl, pyridyl, or C 1-6 alkylpyridyl, each of which is optionally substituted. In one example, R 2 is selected from the group consisting of: each of which is optionally substituted, wherein represents the attachment point of R 2 to the rest of the compound.
  • the compound of the present disclosure is a compound of Formula (I), with the proviso that where R 2 is methyl, the compound is not selected from the group consisting of:
  • each of R’, R 1 , R 2 , R 3 and R 4 may be optionally substituted. In one embodiment, each of R’, R 1 , R 2 , R 3 and R 4 may be optionally substituted with one or more R 5 . In the instance where R’, R 1 , R 2 , R 3 and R 4 is not substituted with one or more R 5 , then it will be understood that a hydrogen atom will remain as the substitution.
  • R’ is substituted with one, two, three, four, five, or more, R 5 substituents. In one embodiment, R’ is substituted with one R 5 substituent. In one embodiment, R’ is substituted with two R 5 substituents. In one embodiment, R is substituted with three R 5 substituents. In one embodiment, R is substituted with four R 5 substituents. In one embodiment, R is substituted with five R 5 substituents. In one embodiment, R is substituted with more than five R 5 substituents.
  • R 1 is substituted with one, two, three, four, five, or more, R 5 substituents. In one embodiment, R 1 is substituted with one R 5 substituent. In one embodiment, R 1 is substituted with two R 5 substituents. In one embodiment, R 1 is substituted with three R 5 substituents. In one embodiment, R 1 is substituted with four R 5 substituents. In one embodiment, R 1 is substituted with five R 5 substituents. In one embodiment, R 1 is substituted with more than five R 5 substituents.
  • R 2 is substituted with one, two, three, four, five, or more, R 5 substituents. In one embodiment, R 2 is substituted with one R 5 substituent. In one embodiment, R 2 is substituted with two R 5 substituents. In one embodiment, R 2 is substituted with three R 5 substituents. In one embodiment, R 2 is substituted with four R 5 substituents. In one embodiment, R 2 is substituted with five R 5 substituents. In one embodiment, R 2 is substituted with more than five R 5 substituents.
  • R 3 is substituted with one, two, three, four, five, or more, R 5 substituents. In one embodiment, R 3 is substituted with one R 5 substituent. In one embodiment, R 3 is substituted with two R 5 substituents. In one embodiment, R 3 is substituted with three R 5 substituents. In one embodiment, R 3 is substituted with four R 5 substituents. In one embodiment, R 3 is substituted with five R 5 substituents. In one embodiment, R 3 is substituted with more than five R 5 substituents.
  • R 4 is substituted with one, two, three, four, five, or more, R 5 substituents. In one embodiment, R 4 is substituted with one R 5 substituent. In one embodiment, R 4 is substituted with two R 5 substituents. In one embodiment, R 4 is substituted with three R 5 substituents. In one embodiment, R 4 is substituted with four R 5 substituents. In one embodiment, R 4 is substituted with five R 5 substituents. In one embodiment, R 4 is substituted with more than five R 5 substituents.
  • R , R 1 , R 2 , R 3 or R 4 when any of R , R 1 , R 2 , R 3 or R 4 is substituted with one or more R 5 substituents, the one or more substituents may be the same substituent or a different substituent (e.g., the R 5 substituents are independently selected from one another).
  • each R 5 is independently selected from the group consisting of H, halogen, C 1-10 alkyl, O C 1-10 alkyl, C 1- whaloalkyl, O C 1-10 haloalkyl, C 2-10 alkenyl, OC 2-10 alkenyl, C 2-10 alkynyl, and OC 2-10 alkynyl.
  • R 5 when R 5 is C 1-10 alkyl, O C 1-10 alkyl, C 1-10 haloalkyl, OC 1- 10 haloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, OC 2-10 alkenyl, OC 2-10 alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Cmoalkyl-3-10-membered-carbocyclyl, C 1-10 alkyl- 3-10-membered-heterocyclyl, each C 1-10 alkyl, C 1-10 haloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, 3-10 membered-carbocyclyl, and 3 -10-membered -heterocyclyl may be optionally substituted with one or more R 7 substituents.
  • R 5 when R 5 is C 1-10 alkyl, O C 1-10 alkyl, C 1- 10 haloalkyl, O C 1-10 haloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, OC 2-10 alkenyl, OC 2-10 alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C 1-10 alkyl-3-10-membered-carbocyclyl, C 1-10 alkyl-3-10-membered-heterocyclyl, each C 1-10 alkyl, C 1-10 haloalkyl, C 2-10 alkenyl, C 2- 10 alk ynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl may be optionally substituted with one, two, three, four, five or more than five R 7 substituents. It will be understood that, when R 5 is substituted with one or more R 7 substituents,
  • each R 5 may be independently selected from the group consisting of H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, OC 1-6 haloalkyl, C 2-6 alkenyl, OC 2-6 alkenyl, C 2-6 alkynyl, OC 2-6 alkynyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • each R 6 is independently selected from the group consisting of H, C 1-6 alkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C 1-6 alkyl-3-10- membered-carbocyclyl, and C 1-6 alkyl-3-10-membered-heterocyclyl.
  • each C 1-6 alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl may be optionally substituted with one or more R 7 substituents.
  • each C 1-6 alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl may be optionally substituted with one, two, three, four, five or more than five R 7 substituents. It will be understood that, when R 6 is substituted with one or more R 7 substituents, the one or more substituents may be the same substituent or a different substituent (e.g., the R 7 substituents are independently selected from one another).
  • each R 8 is independently selected from the group consisting of H and C 1-6 alkyl. In one embodiment, R 8 is H. In one embodiment, R 8 is C 1-6 alkyl.
  • each R 1 and R 3 are independently optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, - NH 2 , -N(H)C 1-6 alkyl, orC 1-6 alkylNH 2 .
  • R 1 is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -NH 2 , - N(H)C 1-6 alkyl, orC 1-6 alkylNH 2 .
  • R 1 is optionally substituted with H.
  • R 1 is optionally substituted with halogen (e.g. fluorine).
  • R 1 is optionally substituted with C 1-6 haloalkyl (e.g. -CF 3 ).
  • R 1 is optionally substituted with -NO 2 .
  • R 1 is optionally substituted with - Nth.
  • R 1 is optionally substituted with -N(H)C 1-6 alkyl.
  • R 1 is an aryl optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, - NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 1 is an aryl optionally substituted by one or more groups selected from H, halogen, or C 1-6 haloalkyl.
  • R 1 is a 3-10-membered aryl optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, - SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 1 is a 3-10- membered aryl optionally substituted by one or more groups selected from H, halogen, or C 1- 6 haloalkyl.
  • R 1 is phenyl optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, - NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 1 is phenyl optionally substituted by one or more groups selected from H, halogen, or C 1-6 haloalkyl.
  • R 3 is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -OH, -NO 2 , - NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is optionally substituted with H.
  • R 3 is optionally substituted with halogen (e.g. fluorine).
  • R 3 is optionally substituted with C 1-6 haloalkyl (e.g. -CF 3 ).
  • R 3 is optionally substituted with -NO 2 .
  • R 3 is optionally substituted with - NH 2 . In one embodiment, R 3 is optionally substituted with OC 1-6 alkyl. In one embodiment, R 3 is optionally substituted with -OH. In one embodiment, R 3 is optionally substituted with C 1-6 alkylNH 2 .
  • R 3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, - SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is an aryl or heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1-6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 3-10-membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1- 6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 3-10-membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1- 6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1-6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1-6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , - CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 3-10- membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1-6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, - OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C 1-6 haloalkyl, OC 1-6 alkyl, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 2 is optionally substituted with one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1- 6 haloalkyl, OC 1-6 haloalkyl, C 2-6 alkenyl, OC 2-6 alkenyl, C 2-6 alkynyl, OC 2-6 alkynyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 2 is substituted with H, halogen, OC 1-6 alkyl, C 1-6 haloalkyl, OC 2-6 alkynyl, -NO 2 , NH 2 , -OH, -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • R 2 is substituted with H.
  • R 2 is substituted with halogen.
  • R 2 is substituted with OC 1-6 alkyl.
  • R 2 is substituted with C 1-6 haloalkyl.
  • R 2 is substituted with OC 2-6 alkynyl.
  • R 2 is substituted with -NO 2 .
  • R 2 is substituted with -NH 2 .
  • R 2 is substituted with -N(H)C 1-6 alkyl.
  • R 2 is substituted with C 1-6 alkylNH 2 .
  • R 2 is selected from the group consisting of C 1-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted with one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, OC 1-6 haloalkyl, C 2- 6 alkenyl, OC 2-6 alkenyl, C 2-6 alkynyl, OC 2-6 alkynyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1- 6 alkyl, or C 1-6 alkylNH 2 .
  • R 2 is selected from the group consisting of C 1- 10 alk yl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted with H, halogen, OC 1-6 alkyl, C 1-6 haloalkyl, OC 2-6 alkynyl, -NO 2 , NH 2 , -OH, - N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • the compound of Formula (I) is wherein n is 0 to 5;
  • the compound of Formula (I) is selected from the group consisting of: wherein n is 0 to 5; Ring B is a N-heteroaryl; and
  • the compound of Formula (I) is selected from the group consisting of: wherein n is 0 to 5; and
  • the compound of Formula (I) is: wherein X, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , L 1 , L 2 , R 1 , R 2 and R 5 are as described herein.
  • the compound of Formula (I) is selected from the group consisting of:
  • n is 0 to 5;
  • the compound of Formula (I) is: wherein X, A 1 , A 2 , A 3 , A 4 , L 1 , R 1 , R 2 and R 3 are as described herein.
  • the compound of Formula (I) is selected from:
  • a 1 , A 2 , A 3 , A 4 , L 1 , R 1 , R 2 and R 3 are as described herein.
  • the compound of Formula (I) is selected from:
  • the compound of Formula (I) is selected from the group consisting of:
  • n 0 to 5;
  • the compound of Formula (I) is wherein X, L 1 , L 2 , R 1 , R 2 and R 3 are as described herein.
  • the compound of Formula (I) is wherein n is 0 to 5;
  • the compound of Formula (I) is selected from the group consisting of: wherein n is 0 to 5; and X, L 1 , L 2 , R 1 , R 2 and R 5 are as described herein.
  • the compound of Formula (I) is: wherein: n is 0 to 5; and A 1 , A 2 , A 3 , A 4 , A 5 , X, L 1 , L 2 , R 2 and R 5 are as described herein.
  • the compound of Formula (I) is selected from the group consisting of:
  • n is independently 0 to 5;
  • the compound of Formula (I) is: wherein X, L 1 , R 1 , R 2 and R 3 are as described herein.
  • the compound of Formula (I) is selected from: wherein L 1 , R 1 , R 2 and R 3 are as described herein. In one embodiment, the compound of Formula (I) is selected from: wherein R 1 , R 2 and R 3 are as described herein.
  • the compound of Formula (I) is selected from the group consisting of: wherein n is 0 to 5; and
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 3-10-membered heteroaryl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 5-6-membered heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted pyridyl or N-oxide thereof, pyrazolyl or imadzolyl; and X, A 1 , A 2 , A 3 , A 4 , L 1 and R 1 , are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 5-6-membered heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5-
  • 6-membered aryl 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl; and X, L 1 and R 1 , are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 3-10-membered heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 5-6-membered heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is selected from the group consisting of C 3-6 alkyl, 5-6-membered aryl, C 1-6 alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C 1-6 alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted pyridyl or N-oxide thereof, pyrazolyl or imadzolyl;
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is wherein
  • R 2 is selected from the group consisting of C 3-10 alkyl, cycloalkyl, C 1- 10 alk ylcycloalkyl, heteroalkyl, heterocyclyl, C 1-10 alkylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 3 is an optionally substituted 5-6-membered heteroaryl
  • X, L 1 and R 1 are as described herein.
  • the compound of Formula (I) is selected from the group consisting of:
  • the compound of Formula (I) is selected from the group consisting of:
  • the compound of Formula (I) is selected from the group consisting of:
  • the compounds of Formula (I) as described herein also include, where applicable, a pharmaceutically acceptable salt, solvate, stereoisomer or N- oxide thereof.
  • compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer thereof demonstrate inhibitory activity against macrophage infectivity potentiator (Mip) protein. Such inhibition of Mip protein can provide a therapeutic effect.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof finds use in the therapy of a disease or condition, for example Q fever.
  • a method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
  • Mip macrophage infectivity potentiator
  • Mip macrophage infectivity potentiator
  • a method of treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
  • Mip macrophage infectivity potentiator
  • the pathogen may be a bacterial pathogen.
  • the bacterial pathogen is a Gram-negative bacterium.
  • the Gram-negative bacterium is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
  • the pathogen is Coxiella burnetii
  • the disease or condition is Q fever.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof for use in therapy of Q fever.
  • a method of treating and/or preventing Q fever in a subject comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof in the manufacture of a medicament for the treatment and/or prevention of Q fever in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
  • Mip macrophage infectivity potentiator
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use as a PPIase inhibitor for use as a PPIase inhibitor.
  • a method of treating and/or preventing a disease or condition mediated by a PPIase inhibitor comprising administering to the subject of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
  • Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a PPIase inhibitor.
  • a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in treating and/or preventing a disease or condition mediated by a PPIase inhibitor for use in treating and/or preventing a disease or condition mediated by a PPIase inhibitor.
  • compositions and formulations are provided.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof thereof may in some embodiments be administered alone, it is more typically administered as part of a pharmaceutical composition or formulation.
  • the present disclosure also provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable diluents, carriers or excipients (collectively referred to herein as “excipient” materials).
  • the present disclosure also provides pharmaceutical formulations or compositions, both for veterinary and for human medical use, which comprise compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilisers, or the like.
  • the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
  • Examples of pharmaceutical formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (including fine particle dusts or mists that may be generated by means of various types of metered dose pressurised aerosols), nebulisers or insufflators, rectal, intraperitoneal and topical (including dermal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.
  • parenteral including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular
  • inhalation including fine particle dusts or mists that may be generated by means of various types of metered dose pressurised aerosols
  • nebulisers or insufflators rectal, intraperitoneal and topical (including dermal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.
  • the pharmaceutical formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with the excipient that constitutes one or more necessary ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
  • composition is formulated for oral delivery.
  • pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, pills or tablets each containing a predetermined amount of the active ingredient; as a powder or granules, as a solution or a suspension in an aqueous liquid or non-aqueous liquid, for example as elixirs, tinctures, suspensions or syrups; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may also be presented as a bolus, electuary or paste.
  • a tablet may be made for example by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active, or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may be optionally coated or scored, and may be formulated so as to provide slow or controlled release of the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof.
  • the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof can, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may also be administered liposomally.
  • compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavouring agents such as those well known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents, and lubricants such as those known in the art.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Disintegrators include without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof can also be delivered through the oral cavity by sublingual and/or buccal administration. Moulded tablets, compressed tablets, or freeze-dried tablets are exemplary forms that may be used.
  • compositions include those formulating a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as cellulose (avicel) or polyethylene glycols (PEGs). Such formulations can also include an excipient to aid mucosal adhesion such as hydroxyl propyl cellulose (HPC), hydroxyl propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer, and agents to control release such as polyacrylic copolymer.
  • fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins.
  • high molecular weight excipients such as cellulose (avicel) or polyethylene glycols
  • Lubricants, glidants, flavours, colouring agents, and stabilisers may also be added for ease of fabrication and use.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • the composition is formulated for parenteral delivery.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injections solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1.3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1.3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • the formulation may be a sterile, lyophilized composition that is suitable for reconstitution in an aqueous vehicle prior to injection.
  • a formulation suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, which may for example be formulated to be isotonic with the blood of the recipient.
  • the compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may for example be formulated in compositions including those suitable for inhalation to the lung, by aerosol, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with a carrier that constitutes one or more accessory ingredients.
  • compositions are prepared by bringing the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with a liquid carrier to form a solution or a suspension, or alternatively, bring the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with formulation components suitable for forming a solid, optionally a particulate product, and then, if warranted, shaping the product into a desired delivery form.
  • Solid formulations of the present disclosure when particulate, will typically comprise particles with sizes ranging from about 1 nanometer to about 500 microns. In general, for solid formulations intended for intravenous administration, particles will typically range from about 1 nm to about 10 microns in diameter.
  • the composition may contain compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof that are nanoparticulate having a particulate diameter of below 1000 nm, for example, between 5 and 1000 nm, especially 5 and 500 nm, more especially 5 to 400 nm, such as 5 to 50 nm and especially between 5 and 20 nm.
  • the composition contains compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof with a mean size of between 5 and 20nm.
  • the compound of Formula (I) is polydispersed in the composition, with PDI of between 1.01 and 1.8, especially between 1.01 and 1.5, and more especially between 1.01 and 1.2.
  • the compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof are monodispersed in the composition.
  • formulations may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavouring agents.
  • compositions of the present disclosure may also include polymeric excipients/additives or carriers, e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2- hydroxypropyl-P-cyclodextrin and sulfobutylether-P-cyclodextrin), polyethylene glycols, and pectin.
  • polymeric excipients/additives or carriers e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,
  • compositions may further include diluents, buffers, citrate, tr 6 halose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations).
  • diluents e.g., buffers, citrate, tr 6 halose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents
  • compositions according to the present disclosure are listed in “Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in “Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.
  • the amount of Mip inhibitor of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof (i.e. active ingredient) that is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, including the type, species, age, weight, sex, and medical condition of the subject being treated, and the renal and hepatic function of the subject, and the particular condition, disorder or disease being treated, as well as its severity.
  • An ordinary skilled physician or clinician can readily determine and prescribe the effective amount of the drug required to prevent or treat the condition, disorder or disease.
  • Dosages of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, when used for the indicated effects, may range between, for example, about 0.01 mg per kg of body weight per day (mg/kg/day) to about 1000 mg/kg/day.
  • the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is between about 0.01 and 1000, 0.1 and 500, 0.1 and 100, 1 and 50 mg/kg/day.
  • the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is between about 0.01 and 1000 mg/kg/day.
  • the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is between about 0.1 and 100 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is greater than about 0.01, 0.1, 1, 10, 20, 50, 75, 100, 500, 1000 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is greater than about 0.01 mg/kg/day.
  • the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is less than about 5000, 1000, 75, 50, 20, 10, 1, 0.1 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is less than about 1000 mg/kg/day.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may for example be administered as a single daily dose, or otherwise the total daily dosage may be administered in divided doses of two, three, or four times daily.
  • the Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may be dosed less frequently than once per day, for example once per two days, three days, four days, five days, six days, or once per week.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may be used as the sole active agent in a medicament
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may be used in combination with one or more further therapeutic agents.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with one or more further therapeutic agents.
  • the present disclosure therefore also provides a combination of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and a further therapeutic agent.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a combination of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, a further therapeutic agent, and a pharmaceutically acceptable excipient.
  • Such one or more further therapeutic agents may, for example, be antibacterial (e.g. an antibiotic), antimicrobial, antifungal and/or anti- viral agents.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with an antibacterial agent.
  • An antibacterial agent is an antibiotic.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with an antibiotic.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with a broad-spectrum antibiotic. Examples of an antibiotic include doxycycline.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with an antifungal agent.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with a an antifungal antibiotic.
  • an antifungal antibiotic include rapamycin
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with a vaccine.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with an immunomodulator.
  • immunomodulators include, but are not limited to, immunosuppressants, cytokine inhibitors, antibodies, and immunostimulants. The immunomodulator may suppress inflammation and/or immune activation (e.g., cell proliferation and homing to tissues) of airways.
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with an anti-inflammatory agent.
  • An example of an anti-inflammatory drug is a nonsteroidal anti-inflammatory drug (NSAID).
  • NSAID nonsteroidal anti-inflammatory drug
  • a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof is used in combination with a nonsteroidal anti-inflammatory drug NSAID.
  • the Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and the one or more further therapeutic/pharmaceutically active agents may be administered simultaneously, subsequently or separately.
  • they may be administered as part of the same composition, or administered as separate compositions.
  • the further therapeutic agent may be formulated for administration by any suitable route, for example orally, intravenously, subcutaneously, intramuscularly, intranasally, and/or by inhalation.
  • the further therapeutic agents when employed in combination with a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may be used for example in those amounts indicated in the Physicians’ Desk Reference or as otherwise determined by one of ordinary skill in the art.
  • X is selected from O, S, and NR 4 ;
  • R 1 and R 3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl;
  • R 2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted;
  • R 4 is selected from the group consisting of H, C 1-10 alkyl, carbocyclyl, C 1-10 alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C 1-10 alkyl-heterocyclyl, each of which is optionally substituted.
  • X is selected from O, S, and NR 4 ;
  • R 1 and R 3 are each independently selected from a carbocyclyl or a heterocyclyl
  • R 2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl;
  • R 4 is selected from the group consisting of H, C 1-10 alkyl, carbocyclyl, C 1-10 alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C 1-10 alkyl-heterocyclyl; wherein each of R 1 , R 2 , R 3 and R 4 is optionally substituted with one or more R 5 ; each R 5 is independently selected from the group consisting of H, halogen, C 1-10 alkyl, O C 1-10 alkyl, C 1-10 haloalkyl, O C 1-10 haloalkyl, C 2-10 alkenyl, C 2-10 alkynyl, OC 2-10 alkenyl, OC 2- 10 alk ynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C 1-10 alkyl-3-10- membered-carbocyclyl, C 1-10 alkyl-3-10-membered-heterocyclyl, -NO 2 ,
  • R 1 and R 3 are each independently selected from an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl.
  • R 3 is independently selected from the group consisting of: each of which is optionally substituted.
  • each R 1 and R 3 are independently optionally substituted by one or more groups selected from H, halogen, C 1- 6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, -NO 2 , -CN, -SO 2 H, -OH, -NH 2 , -N(H)C 1-6 alkyl, orC 1- 6 alkylNH 2 .
  • R 2 is selected from the group consisting of C 1-10 alkyl, cycloalkyl, C 1-10 alkylcycloalkyl, heteroalkyl, heterocyclyl, C 1- 10 alk ylheterocyclyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 1-10 alkyl, aryl, C 1-10 alkylaryl, heteroaryl, and C 1-10 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is selected from the group consisting of C 1-6 alkyl, aryl, C 1-6 alkylaryl, heteroaryl, and C 1-6 alkylheteroaryl, each of which is optionally substituted.
  • R 2 is optionally substituted with one or more groups selected from H, halogen, C 1-6 alkyl, OC 1-6 alkyl, C 1-6 haloalkyl, OC 1- 6 haloalkyl, C 2-6 alkenyl, OC 2-6 alkenyl, C 2-6 alkynyl, OC 2-6 alkynyl, -NO 2 , -CN, -SO 2 H, -OH, - NH 2 , -N(H)C 1-6 alkyl, or C 1-6 alkylNH 2 .
  • a pharmaceutical composition comprising a compound of any one of paragraphs 1 to 22, and a pharmaceutically acceptable excipient.
  • a method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein comprising administering to the subject an effective amount of compound of any one of paragraphs 1 to 22, or a pharmaceutical composition of paragraph 23.
  • Mop macrophage infectivity potentiator
  • Mip macrophage infectivity potentiator
  • a method of treating and/or preventing a disease or condition mediated by a Gram- negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor comprising administering to the subject a compound of any one of paragraphs 1 to 22, or a pharmaceutical composition of paragraph 23.
  • Mop macrophage infectivity potentiator
  • the Gram-negative bacteria is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
  • Mip macrophage infectivity potentiator
  • Inhibitors of the Mip protein were largely derived for the pipecolic acid moiety of the immunosuppressive rapamycin. Omitting the other parts of the rapamycin molecule result in Mip inhibitors of a wide concentration range, depending on the bacterial origin of the Mip protein (Juli et al., 2014 and Seufert et al., 2016.). Instead of more or less randomly varying the substituents, we looked closer to the different binding mode of the inhibitors of the BpMip using X-ray analysis and decided to combine both binding modes in order to arrest the Mip protein, which is undergoing great conformational changes upon the catalysis of the peptidyl-prolyl isomerisation.
  • Scheme 2 Synthesis scheme for the R/S mixed pipecolic ester derivatives S/R,S'-10a, S/R,S-10b, S/R,S-10c, and S/R,S-10d.
  • Reagents and conditions (a) NaNO 2 , H 2 SO 4 (1 M), H 2 O, 0 °C rt; (b) K 2 CO 3 , allyl bromide, DMF, rt; (c) i) NaHCO 3 , DCM, rt ii) BnSO 2 Cl, NMM, DCM, 0 °C, iii) LiOH (1 M), DCM, rt; (d) EDC HCl, DMAP, DCM, 0 °C rt; (e) Pd(PPh 3 ) 4 , morpholine, THF, 0°C ⁇ rt; (f) 3 -picolylamine, EDC HCl, HOBt, DCM, 0 °C rt.
  • the diastereomeric mixtures S/R,S-10a-d were synthesized (Scheme 2).
  • the substances were also intended to be used as a reference for stereochemically pure compounds to test whether the preference for the S-configuration at the C2 position of the pipecolic acid observed by Seufert et al (2016) also applies in the presence of an additional side chain.
  • they should be used to see if the diastereomers can be analytically distinguished from each other.
  • the allyl protecting group of 15a-d was cleaved under palladium catalysis with Pd(PPh 3 ) 4 and morpholine, resulting in 16a-d.
  • 3 -picolylamine was attached to the carboxylic acid by amide coupling using EDC ⁇ HCl and HOBt to give R/S,S-10a-d.
  • racemic (R/S)- 1-(benzylsulfonyl)piperidine-2-carboxylate (14) a basic hydrolysis was chosen, which would lead to racemization in the case of enantiomerically pure reactants. This synthetic step was omitted in subsequent synthetic routes to obtain the distinct stereo isomers.
  • Reagents and conditions (a) NaNO 2 , H 2 SO 4 (2M), 0 C; (b) i) KOH, MeOH, 40°C, ii) 10% Pd/C, H 2 (15 bar), MW 400W, isopropanol, iii) EDC ⁇ HCl, HOBt, 3 -picolylamine, DCM, 0 °C ⁇ rt; (c) NaBH 4 , MeOH, 0°C; (d) 3-, EDC HCl, HOBt, DCM, 0 °C ⁇ rt; (e) EDC ⁇ HCl, DMAP, DCM, 0°C rt; (f) i) trifluoroacetic acid (TFA), DCM, 0°C ⁇ rt, 2h, ii) BnSO 2 Cl, NMM, DCM, rt.
  • TFA trifluoroacetic acid
  • Scheme 4 Synthesis scheme for the (S)-pipecolic acid amide derivatives as stereochemically pure compounds.
  • Reagents and conditions (a) allyl alcohol, SOCh, 0 °C ⁇ 60 °C; (b) N-Boc-(S)- pipecolic acid, EDC HCl + HOBt or HBTU, DIPEA, DCM, 0 °C ⁇ rt; (c) i) TFA, DCM, 0 °C ⁇ rt, ii) BnSCECl or 4-F-BnSO 2 Cl, NMM, DCM, 0°C rt; (d) i) Pd(PPh 3 ) 4 , morpholine, THF, 0°C rt, ii) 3 -picolylamine, EDC HCl + HOBt or HBTU, DIPEA, DCM, 0 °C ⁇ rt; (e) m-CPBA, EtOAc, rt.
  • the N-oxidcs S/R,S-22a, S,S-22g, and S,S-22d were prepared by oxidation of the respective inhibitor with meto-chloroperoxybenzoic acid (m-CPBA) in EtOAc.
  • m-CPBA meto-chloroperoxybenzoic acid
  • the amino acids were O-protected by using thionyl chloride and allyl alcohol to give the allyl esters 23.
  • the Boc-protection group of the amides 24 was cleaved with TFA.
  • the N- sulfonamides 25 were formed using the respective sulfonyl chloride and NMM, and the allyl function was again cleaved under palladium catalysis.
  • the stereochemically pure amides S,S-21a, S,R-21a, S,S-21e, S,R-21e, S,S-21d, and S,S-21g were obtained by amidation with 3 -picolylamine.
  • Scheme 5 Synthesis scheme of an alternative pathway for stereochemically pure S- pipecolic amide derivatives 5, 5-2111, S,S-21i, and S,S-28i that allows a late variation of the sulfonamide moiety.
  • Reagents and conditions (a) 3 -picolylamine, HBTU, DIPEA, DCM, 0°C rt; (b) i) TFA, rt; ii) A-Boc-(S)-pipecolic acid, HBTU, DIPEA, DCM, 0°C ⁇ rt; (c) i) TFA, rt; ii) BnSO 2 Cl / 4-F-BnSO 2 Cl, NMM or DIPEA, DCM, 0°C rt.
  • the compounds were again amide coupled to the Boc-protected S-pipccolic acid with HBTU and DIPEA to give S,S-27h and S,S-27i.
  • the A- sulfonamides were formed with the respective sulfonyl chloride derivative to give S,S-21h, S,S-21i, and S,S-28i.
  • R/S,S-15d was synthesized according to general procedure A using allyl (S)-2- hydroxy-4-methylpentanoate (940 mg, 5.46 mmol), ( R/S)-1-(benzylsulfonyl)piperidine-2- carboxylic acid (1.50 g, 5.46 mmol), EDC ⁇ HCl (1.26 g, 6.55 mmol), and DMAP (340 mg, 2.73 mmol) in dry DCM (200 mL).
  • R/S,S-15a was synthesized following general procedure A using 220 mg (1.07 mmol) of S-13a, (R/S)-1-(benzylsulfonyl)piperidine-2-carboxylic acid (302 mg, 1.07 mmol), EDC ⁇ HCl (245 mg, 1.28 mmol), and DMAP (66 mg, 0.54 mmol) in dry DCM (40 mL).
  • R/S,S-15b 300 mg, 0.71 mmol was deprotected according to general procedure D using 60 mg of Pd(PPh 3 ) 4 (0.05 mmol) and 70 ⁇ L of morpholine (0.75 mmol) in dry THF (10 mL).
  • the intermediate product was further reacted with 3 -picolylamine (80 ⁇ L, 0.85 mmol), EDC ⁇ HCl (164 mg, 0.85 mmol), and HOBt (48 mg, 0.37 mmol) in dry DCM (30 mL) according to general procedure A.
  • R/S,S-15c (720 mg, 1.65 mmol) was deprotected using 150 mg of Pd(Ph 3 ) 4 (150 mg, 0.08 mmol) and morpholine (150 ⁇ L, 17.3 mmol) in dry THF (20 mL) following general procedure D.
  • the intermediate product (656 mg, 1.65 mmol) was further reacted with 3 -picolylamine (208 ⁇ L, 1.98 mmol), EDC ⁇ HCl (382 mg, 1.98 mmol), and HOBt (116 mg, 0.42 mmol) in dry DCM (70 mL) according to general procedure A.
  • R/S,S-15d (900 mg, 2.05 mmol) was deprotected using 160 mg of Pd(PPh 3 ) 4 (0.14 mmol) and morpholine (200 ⁇ L, 2.15 mmol) in dry THF (20 mL) following general procedure D.
  • the intermediate product (819 mg, 2.05 mmol) was then reacted with 3- picolylamine (273 ⁇ L, 2.48 mmol), EDC ⁇ HCl (473 mg, 2.48 mmol), and HOBt (138 mg, 1.03 mmol) in dry DCM (80 mL) according to general procedure A.
  • Compound R-17d was prepared according to general procedure A using 1.89 g (R)-2- hydroxy-4-methoxypentanoic acid (14.3 mmol), HOBt (1.93 g, 14.3 mmol), EDC ⁇ HCl (2.74 g, 15.2 mmol), and 3 -picolylamine (2.20 mL, 21.5 mmol) in dry DCM (200 mL).
  • S,S-20d was prepared following general procedure A using 1.36 g of S-17d (6.10 mmol), (S)-1-Boc-piperidine-2-carboxylic acid (1.54 g, 6.71 mmol), EDC HCl (1.75 g, 9.15 mmol), and DMAP (0.15 g, 1.22 mmol) in dry DCM (200 mL). After stirring at rt for 4 h and workup according to general procedure A, the crude oily product was purified by column chromatography (SiO 2 , DCM/MeOH 15:1). Compound S,S-20d was obtained as a colorless oil (1.29 g, 2.97 mmol, 49%).
  • R-23a Allyl-d-phenylalaninate hydrochloride ( R-23a)
  • R-23a was synthesized according to general procedure G using 1.00 g of D- phenylalanine (6.05 mmol) and thionyl chloride (1.00 mL, 13.3 mmol) in allyl alcohol (12 mL, 181.6 mmol).
  • S,R-25a was allyl-deprotected according to general procedure D using Pd(PPh 3 ) 4 (50 mg, 0.04 mmol) and morpholine (40 ⁇ L, 0.44 mmol) in dry THF (10 mL).
  • the carboxylic acid intermediate 150 mg, 0.40 mmol
  • isomerization of the pipecolic acid stereocenter occurred.
  • Diastereomeric ratio 58:42.
  • Example 4 Synthesis of MIPS-0052721, MIPS-0052756, MIPS-0052488, MIPS-0052581, MIPS-0052658, MIPS-0052695, MIPS-005275
  • Anhydrous DCM was purchased in an anhydrous form and stored under nitrogen.
  • PS refers to commercial petroleum spirits with a boiling point range of 60-80 °C. All column (flash) chromatography was performed on silica gel SiO 2 (40-63 ⁇ m, normal phase liquid chromatogrphay) or C18 spherical (20-35 pm, 100 ⁇ , reverse phase liquid chromatography) using automated chromatography systems (Biotage Isolera/Selekt) unless otherwise indicated.
  • Analytical TLC was performed using aluminium backed 0.2 mm thick silica gel 60 GF254 plates. The TLC plates were visualised using a 254 nm UV lamp, otherwise stained with 10% phosphomolybdic Acid (PMA) in ethanol.
  • PMA phosphomolybdic Acid
  • Liquid chromatography-Mass spectrometry was performed using an UHPLC/MS 1260/6120, detection at 254 nm and 214 nm. Liquid chromatography-Mass spectrometry (LC-MS) was performed using ESI and APCI LC-MS. Each method used 254 nm detector and a reverse phase C8(2) 5 ⁇ 50 ⁇ 4.6 mm 100A column. The column temperature was 30 °C and the injection volume, 5 ⁇ L. The eluent system used was solvent A (H 2 O 0.1% formic acid) and solvent B (MeCN 0.1% formic acid). The gradient for ESI was 5 to 100% B over A over 4 min then eluted 100% B for 6 min. The gradient for APCI was 5 to 100% B over A over 2 min then eluted 100% B for 2 min.
  • EDCI.HCl (0.62 g, 3.24 mmol), HOBt (0.44 g, 3.24 mmol) and DIPEA (1.2 mL, 6.75 mmol) were added to a solution of Boc-Leu-OH (0.63 g, 2.70 mmol) in anhydrous DMF (14 mL) at 0 °C and let stir for 10 min.
  • (1 -Methyl-1H-pyrazol-4-yl)methanamine (0.30 g, 2.70 mmol) was then added and let stir for 16 h at rt. After this time, the reaction was poured into water (10 mL) and EtOAc (10 mL) was added.

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Abstract

The present disclosure generally relates to pipecolic acid derived compounds, in particular pipecolic acid derived compounds of Formula (I), and to formulations and compositions comprising the same. The present disclosure also relates to methods and uses of these compounds, compositions and/or formulations in treating and/or preventing a disease or condition mediated by a pathogen which is responsive to inhibition of macrophage infectivity potentiator (Mip) proteins, and/or a disease or condition mediated in which Mip protein is a virulence factor.

Description

MIP INHIBITORS
FIELD
The present disclosure generally relates to pipecolic acid derived compounds. In particular, the present disclosure relates to pipecolic acid derived compounds of Formula (I). The present disclosure also relates to formulations and compositions comprising the pipecolic acid derive compounds of Formula (I). The present disclosure also relates to use of these compounds, compositions and/or formulations in treating and/or preventing a disease or condition mediated by a pathogen which is responsive to inhibition of macrophage infectivity potentiator (Mip) proteins. The present disclosure also relates to use of these compounds, compositions and/or formulations in treating and/or preventing a disease or condition mediated in which Mip protein is a virulence factor.
BACKGROUND
Immunophilins are a superfamily of ubiquitous, highly conserved proteins which have been identified across all kingdoms of life (Gothel & Marahiel 1999). The majority of immunophilins exhibit peptidyl-prolyl cis-trans isomerase activity (PPIase) activity which catalyses the rate limiting cis-trans isomerisation of peptidyl-prolyl bonds (Kiefhaber et al. 1990). PPIases are required for efficient folding of numerous proteins and are therefore critical in a range of physiological processes including signal transduction, cell cycle regulation, and protein chaperoning (Lu, Hanes & Hunter 1996; Somarelli & Herrera 2007; Norville et al. 2011). On the basis of binding partners, immunophilins are divided into three distinct families. Although all three bind to immunosuppressive drugs which inhibit their enzymatic activity, they are unrelated in structure and amino acid sequence (Siekierka et al. 1989; Rahfeld et al. 1994). Cyclophilins bind to cyclosporin A; parvulins bind to juglone; and FK506 binding proteins (FKBPs) bind to FK506 and rapamycin.
Macrophage infectivity potentiator (Mip) proteins and Mip like proteins (ML1) belong to the family of FK506-binding proteins (FKBPs), which form part of the immunophilin superfamily. A number of studies have shown that deletion of Mips result in pleiotrophic effects in bacteria which ultimately results in reduced virulence of pathogens. This suggests that Mips are an attractive anti-virulence target to combat a broad range of bacterial pathogens. Mip proteins are classically approximately 28 kDa in size, consisting of two distinct domains; an N-terminal dimerization domain, linked to a C-terminal domain containing the FKBP fold exhibiting PPIase activity. These proteins are believed to form a homodimer of approximately 60 kDa (Helbig et al., 2001; Leuzzi et al., 2005). ML1 proteins possess the FKBP fold domain but lack the dimerization domain, resulting in an approximately 12-14 kDa protein with PPIase activity (Norville et al. 2011). For simplicity, Mip proteins and Mip like proteins (ML1) will be both be referred to as Mip from herein.
Legionella pneumophila Mip
The first Mip protein was isolated from Legionella pneumophila, an environmental pathogen known to be the causative agent of Legionnaires disease (Engleberg et al., 1984). The lack of a 24 kDa FKBP protein in an L. pneumophila strain was found to reduce the ability of the bacteria to infect macrophages 10- to 100-fold and was therefore named the macrophage infectivity potentiator protein (Mip) (Cianciotto et al., 1989, Fischer et al., 1992). LpMip is a homodimeric protein comprised of two 22.8 kDa monomers (Riboldi- Tunnicliffe et al. 2001). To investigate the function of LpMip, a site specific mutation was introduced within the mip gene (LPG0791), producing a null mip mutant (Cianciotto et al. 1990). The mutant strain was 80-fold less infective than the wild-type strain within both human macrophages and macrophage-like (U937) cells. Intra-tracheal inoculation with the mutant strain resulted in slower disease progression and less lethal outcomes within a guinea pig model in comparison to wild-type and a complemented mutant. It was proposed that mip was required for full virulence and represented the first genetically defined L. pneumophila virulence factor. Further study demonstrated that LpMip was also implicated in infection of explanted lung epithelial cells (Cianciotto, Stamos & Kamp 1995).
Ceymann et al. (2008) solved the crystal structure of free LpMip and the LpMip- rapamycin complex by means of nuclear magnetic resonance spectroscopy. It was determined that binding was mediated by the rapamycin pipecoline moiety in conjunction with the LpMip hydrophobic pocket. Due to the structural similarity between Mip-rapamycin and FKBP12-rapamycin complexes, it was proposed that rapamycin derived non- immunosuppressive FKBP12 inhibitors may represent a starting point for Mip inhibitor development (Ceymann et al. 2008). This subsequently formed the basis of rationally designed small-molecular pipecolic acid derived Mip inhibitors (Juli et al. 2011). Inhibitors were tested against L. pneumophila within a human macrophage-like (U937) model of infection. Results suggested that the PPIase domain confers bacterial adhesion to extracellular lung tissue targets rather than prolonging survival or enabling replication within macrophages (Juli et al. 2011). However, it has been suggested that this may be attributable to low level residual PPIase activity (Wintermeyer et al. 1995).
Burkholderia pseudomallei Mip
The BpMip is a monomeric protein which lacks an N-terminal dimerisation domain. However, it exhibits 40% sequence identity with LpMip, and its C-terminal PPIase domain is highly homologous to those found within other Mips (Cianciotto et al. 1989; Ceymann et al. 2008; Norville et al. 2011). Norville et al. (2011) produced an in-frame deletion B. pseudomallei mip (BPSS1823) mutant which was significantly attenuated within a BALB/c murine model of infection. Infection studies were performed within both epithelial (A549) and macrophage-like (J774A.1) cells. Results indicated that BpMip was not required for epithelial cell adhesion, although it was required for intracellular replication and survival within both cell types. It was demonstrated that BpMip was implicated in a wider range of virulence-associated phenotypes than reported for other pathogens, including bacterial motility, protease production, and pH tolerance (Norville et al. 2011). In 2014, a high- throughput structural biology platform was utilised to perform rapid, strategic investigation of the BpMip crystal structure and enable subsequent inhibitor design (Begley et al. 2014). Inhibitor efficacy was tested against B. pseudomallei within a macrophage-like (J774A.1) model of infection. Results showed a reduction of the cytotoxicity caused by B. pseudomallei in the presence of inhibitors, demonstrating that Mip is a novel anti- virulence target in this pathogen (Begley et al. 2014).
Neisseria species Mip
The Mip protein of Neisseria gonorrhoeae (NgMip) is important for invasion and persistence within macrophages, and is expressed during infection (Leuzzi et al., 2005, Starnino et al., 2010). An analysis of 21 clinical N. gonorrhoeae isolates showed presence of the Mip protein in all tested clinical isolates, with high levels of conservation between isolates, indicating that the NgMip could be an important virulence factor (Stamino et al., 2010). Sampson and Gotschlich (1992) first described a PPIase protein, inhibit-able by FK506 in Neisseria meningitidis. This protein was found to have an identity of 97% to the C- terminal PPIase domain of the NgMip. However, it has recently been discovered that N. meningitidis encodes two FKBPs: the Mip discussed above (now referred to as NmMipl), and a putative 110-amino acid Mip which has not yet been discussed in the literature, which will be referred to as NmMip2.
The NgMip is a surface-exposed 29 kDa lipoprotein, capable of PPIase activity which is inhibited by rapamycin. The C-terminal PPIase domain has high homology to other bacterial Mips, including the LpMip (43.8% amino acid similarity) and the Trypanosoma cruzi Mip (42.3%). The gene NMB 1567 which encodes for the meningococcal NmMipl is shown to be highly up-regulated during meningococci growth in blood. N. meningitidis MC58 mutants lacking NMB1567 were sensitive to killing during the blood infection time course (Echenique-Rivera et al., 2011). This result along with the constant up-regulation during infection indicates that NmMipl contributes to the intracellular survival of meningococci within the human host. NmMipl is known to be found on the outer membrane of the bacterium, and is capable of inducing antibodies that activated complement-mediated killing of the meningococci (Hung et al., 2011). NmMipl contains a putative dimerization leader sequence found also in NgMip, which is similar to the dimerization domain of LpMip with the exception of the two methionine residues (Leuzzi et al., 2005).
Coxiella burnetii Mip
The Mip protein of Coxiella burnetii, the causative agent of Q fever, was identified and shown to exhibit PPIase activity by Mo et al. (1995). The amino acid sequence of CbMip shows similarity to LpMip (46%) and BpMip (43%) with a molecular mass of 25.5 kDa. Secondary structure analysis has indicated that the protein predominantly adopts a beta-strand structure (Tse et al. 2014), however the crystal structure is yet to be solved. Very little is also known regarding the role of CbMip as a virulence factor although it has been shown to be immunogenic in both experimental and natural infections (Seshu et al. 1997).
Inhibitors to Mip Proteins
Although the exact target(s) of Mips are yet to be elucidated, their structure and function have been well studied. The biological significance of Mips makes them an attractive target for novel antimicrobials. The dual-domain concept has formed the basis for development of a number of PPIase inhibitors with higher levels of host-pathogen selectivity (Wang & Etzkorn 2006; Norville et al. 2011; Begley et al. 2014). Several strategies have been employed in the identification of novel inhibitors, including screening of FK506 and rapamycin homologues, and crystallography based rational drug design (Ceymann et al. 2008; Juli et al. 2011; Begley et al. 2014). Due to their highly conserved enzymatic domains, Mips represent potential broad spectrum anti-virulence targets.
The pipecolic acid domain of rapamycin is responsible for the PPIase inhibition of Mip. The other part of rapamycin binds to mTOR, a serine/threonine protein kinase, resulting in immunosuppression. The pipecolic moiety has previously been used to generate non- immunosuppressive small molecule inhibitors. By using a rational drug design approach a library of inhibitors was generated that showed potent activity against LpMip and BpMip in the nanomolar range (Juli et al. 2011, Seufert et al. 2016).
These inhibitors were analysed in vitro using a macrophage cell infection model for B. pseudomallei (Begley et al. 2014). The level of lactate dehydrogenase (LDH) released as a result of cell death was measured. The compounds CJ37, CJ40, CJ168 and CJ183 were tested. They reduced cytotoxicity between 20-40%. A BpMip mutant was used as a positive control and resulted in similar levels of cytotoxicity confirming specificity to Mip. No cytotoxicity was evident when the compounds were incubated with macrophage cells absent of bacteria. This provided evidence that this series of molecules does not adversely affect the biological function of mammalian macrophages. Therefore, the small pipecolic acid library which has previously been established contains compounds that are potent inhibitors of BpMip.
Despite demonstrating potent activity, the compounds described above are of limited application owing to their unstable nature . It is therefore desirable to develop compounds with improved stability and/or inhibitory activity against macrophage infectivity potentiator (Mip) proteins, or at least provide the public with a useful alternative.
SUMMARY
The present inventors have surprisingly found that compounds of Formula (I) demonstrate inhibitory activity against macrophage infectivity potentiator (Mip) protein.
In one aspect, there is provided compound of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,
Figure imgf000008_0001
wherein:
X is selected from O, S, and NR4;
A1, A2, A3 and A4 are each independently selected from the group consisting of CR' 2, NR' , S and O, wherein each R' is independently selected from the group consisting of H, halogen, C1-10alkyl, OC1-10alkyl, C1-10haloalkyl, OC1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1- 10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, each of which is optionally substituted;
L1 and L2 are each independently absent or selected from the group consisting of -C1- 10alk yl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O- ,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH-, -C(=O)NH( C1-10alkyl)-, - S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -N( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH(CI- 10alkyl)-, and -OS(=O)2-, wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted;
R1 and R3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl;
R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted;
R4 is selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl-carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl, each of which is optionally substituted.
In another aspect, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,
Figure imgf000009_0001
wherein X, L1, L2, R1, R2 and R3 are described above and herein.
According to some embodiments or examples described herein, the present inventors have surprisingly identified that by introducing substituents, and in particular bulkier substituents, at R2 increased the stabilization of the pipecolic ester and in some cases the amide moiety (if present) against metabolic processes and/or provided the compounds with better occupation of a pocket at the Mip binding side responsible for the PPIase activity, resulting in improved activity and/or stability. Additionally, by varying the substitution at R1 and/or R3, one or more further advantages were provided, including increased compound stability and/or activity as demonstrated by one or more embodiments or examples described herein. Other advantages of the presently claimed compounds are also described herein.
In another aspect, there is provided a pharmaceutical composition comprising a compound of Formula (I) as defined above, and a pharmaceutically acceptable excipient.
In another aspect, there is provided a method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein, the method comprising administering to the subject an effective amount of a compound of Formula (I) as defined above, or a pharmaceutical composition as defined above.
In another aspect, there is provided use of a compound of Formula (I) as defined above or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In another aspect, there is provided a compound of Formula (I) as defined above or a pharmaceutical composition as defined above, for use in treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In another aspect, there is provided a method of treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor, comprising administering to the subject a compound of Formula (I) as defined above or a pharmaceutical composition as defined above.
In another aspect, there is provided use of a compound of Formula (I) as defined above or a pharmaceutical composition as defined above, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
In another aspect, there is provided of compound of Formula (I) as defined above or a pharmaceutical composition as defined above, for use in treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
BRIEF DESCRIPTION OF FIGURES
Whilst it will be appreciated that a variety of embodiments of the disclosure may be utilised, in the following, we describe a number of examples of the disclosure with reference to the following drawings. Figure 1 shows inhibition of C. burnetii growth in the presence of Mip compounds measured by Fold differences in Genome Equivalents (GE) (A) compounds AN296, AN263, AN258 and AN259; (B) compounds AN258 and NJS227.
Figure 2 shows that post-exposure treatment with Mip compound AN296 inhibit C. burnetii growth measured by Fold differences in GE (A) Treatment administered 2 days post- infection B) Treatment administered 3 days post-infection.
Figure 3 shows that treatment with Mip compounds inhibit C. burnetii growth in axenic media (A) Treatment with compounds AN131, AN132, AN133 and ANCH37; (B) Treatment with compounds AN296, AN263, AN259 and AN258; (C) Treatment with compounds NJS227 and NJS224.
Figure 4 shows the Mip compounds inhibit C. burnetii during log-phase growth. (A) Addition of AN296 during log phase growth at days 2 and 3 days inhibits C. burnetii growth; (B) Treatment with compounds AN296, AN263, AN259 and AN258; (C) Treatment with compounds NJS227 and NJS224.
Figure 5 shows the in vivo toxicity studies of lead candidate drugs AN296 and AN258 using Galleria mellonella model. Results demonstrates minimal toxicity of both candidates.
Figure 6 shows the in vivo efficacy studies of lead candidate drugs AN296 and AN258 using Galleria mellonella model. Results demonstrates increased survival of C. burnetii infected G. mellonella in the presence of candidate drugs.
Figure 7 shows that inhibitors of CbMip affect intracellular replication of C. burnetii. (A and B) Intracellular replication of C. burnetii-NMII in the presence of CbMip inhibitor SF235 (purple square), AN296 (blue triangle) or vehicle control (black circle). (A) THP-1 cells with 50 μM of inhibitor (n = 6) and (B) HeLa cells with 100 μM of inhibitor (n = 3). Error bars represent standard error of the mean. *, p < 0.05: **, p < 0.01; ****, p < 0.0001. p values were determined using two-way ANOVA, followed by Dunnett’s multiple comparison post-test.
Figure 8 shows the targeted inhibition of CbMip reduces C. burnetii replication in axenic media in a dose-dependent manner. (A) Bioluminescent was measured as an indicator of C. burnetii-lux replication. C. burnetii-lux was inoculated at a concentration of 1 x 106 GE/mL into ACCM-2 media with 100 μM of CbMip inhibitors SF235 (grey square), AN296 (closed triangle) or vehicle control (open circle), and growth was monitored over 5 days. Data is presented as RLU (relative light units) with error bars representing the standard deviation (SD) from three independent experiments. (B) C. burnetii-lux replication over 5 days in the presence of 25 μM, 50 μM or 100 μM of SF235 or AN296. Data is presented as RLU relative to the vehicle control with error bars representing SD from at least three independent experiments. (C) Colony forming units per mL was determined for C. burnetii-NMII after 4 days of growth in the presence of 25 μM, 50 μM or 100 μM of SF235 or AN296. Data is presented as the fold increase in CFU/mL relative to day 0 with error bars representing the SD from at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. p values were determined using two-way ANOVA followed by Dunnett’s multiple comparison post-test.
Figure 9 shows that delayed dosing with AN296 impairs C. burnetii replication in axenic media. Bioluminescence was measured as an indicator of C. burnetii-lux replication. The strain was inoculated at a concentration of 1 x 106 GE/mL into ACCM-2 media and growth was monitored over 5 days. Cultures were dosed with (A+B) 100 μM or (C+D) 50 μM of AN296 (closed circle) or vehicle control (open circle) on (A+C) day 2 or (B+D) day 3 of the growth curve. Data is presented as RLU (relative light units) with error bars represent standard error of the mean from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. p values were determined using two-way ANOVA, followed by Sidak’s multiple comparison post-test. (E) C. burnetii-lux replication after dosing with 50 μM or 100 μM of AN296 on day 2 or day 3 of the 5 day growth curve. Data is presented as RLU relative to the control with error bars representing SD from at least four independent experiments. ****, p < 0.0001. p values were determined using two-way ANOVA, followed by Tukey’s multiple comparison post-test.
Figure 10 shows AN296 is highly potent against virulent C. burnetii-NMI in axenic media. Growth curve of C. burnetii-NMI in the presence of CbMip inhibitors. (A) C. burnetii-NMI was inoculated at a concentration of 1 x 104 CFU/mL into 5 mL of ACCM-2 media supplemented with 0.50 mM tryptophan and containing 100 μM of CbMip inhibitors, SF235 (grey square), AN296 (closed triangle), or vehicle control (open circle), and growth was monitored over 7 days by enumerating the number of colony forming units per mL in the culture on days 0, 1, 2, 3, 4 and 7. Data is presented as logio CFU/mL with error bars representing the SD from at least three independent experiments. ***, p < 0.001; ****, p < 0.0001. p values were determined using two-way ANOVA followed by Dunnett’s multiple comparison post-test. (B) C. burnetii-NMI was inoculated at a concentration of 1 x 104 GE/mL into 5 mL of ACCM-2 media supplemented with 0.50 mM tryptophan. After 3 days, cultures were inoculated with 100 μM of CbMip inhibitors, SF235 (grey square), AN296 (closed triangle), or vehicle control (open circle). Growth was monitored over 7 days by enumerating the number of colony forming units per mL in the culture on days 0, 1, 2, 3, 4 and 7. Data is presented as logio CFU/mL with error bars representing the SD from at least three independent experiments; ****, p < 0.0001. p values were determined using two-way ANOVA followed by Dunnett’s multiple comparison post-test.
DETAILED DESCRIPTION
General definitions
In the following description, reference is made to the accompanying drawings which form a part hereof, and which is shown, by way of illustration, several embodiments. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present disclosure.
With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.
Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.
Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, hydrogels, processes, and compositions, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher-numbered item (e.g., a “third” item).
As used herein, the phrase “at least one of”, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of” means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.
As used herein, the term “about”, unless stated to the contrary, typically refers to a range of up to +/- 10% of the designated value, and includes smaller ranges therein, for example +/- 5% or +/- 1% of the designated value. It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.
Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, 4.75, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the term “subject” refers to any organism susceptible to a disease or condition that requires therapy. For example, the subject can be a mammal, primate, livestock (e.g., sheep, cow, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one example, the subject is a mammal. In one embodiment, the subject is human. In one embodiment, the disease or condition is mediated by a pathogen, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein. In one embodiment, the disease or condition is Q fever.
As used herein, the term “treating” or “treatment” includes alleviation of the symptoms associated with a specific disease or condition and reducing and/or eliminating said symptoms. For example, as used herein, the term “treating Q fever” refers to alleviating the symptoms associated with Q fever and/or eliminating the symptoms associated with Q fever. As used herein, the term “preventing” or “prevention” includes prophylaxis of the specific disorder or condition. For example, as used herein, the term “preventing Q fever” refers to preventing the onset or duration of the symptoms associated with Q fever.
As would be understood by the person skilled in the art, a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, can be administered in a therapeutically effective amount. The term “therapeutically effective amount”, as used herein, refers to a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The result can be the reduction and/or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. For example, one result may be the reduction of one or more symptoms associated with Q fever. The term, “effective amount”, as used herein, refers to an amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. In one embodiment, a prophylactically effective amount is an amount sufficient to prevent Q fever. It is understood that “an effective amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the compound and any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to an adjusted (e.g., reduced) amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.
The term “onset” of activity, as used herein, refers to the length of time to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated following the administration of the compound of Formula (I). The term “duration” refers to the length of time that the therapeutic continues to be therapeutically effective, i.e., alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The person skilled in the art would be aware that onset, peak, and duration of therapy may vary depending on factors such as the patient, the condition of the patient, and the route of administration.
The compounds of the present disclosure may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.
The term “halo” or “halogen” whether employed alone or in compound words such as haloalkyl, represents fluorine, chlorine, bromine or iodine. Further, when used in compound words such as haloalkyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCl3, and -CH2CF3, -CF2CF3 and -CH2CHFCI.
As used herein, the term “alkyl” whether used alone, or in compound words such as haloalkyl, cycloalkyl, alkylcycloalkyl, heteroalkyl, alkylheterocyclyl, alkylheteroaryl, and alkylaryl, represents straight chain (i.e. linear) or branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl groups. In one example, the alkyl group is of 1 to 10 carbon atoms (i.e. C1-10alkyl). In another example, the alkyl group is of 1 to 6 carbon atoms (i.e. C1-6alkyl).
As used herein, the term “heteroalkyl” represents straight chain (i.e. linear) or branched chain hydrocarbon groups which are analogous to an alkyl group, but in which one or more carbon atoms is/are replaced by one or more heteroatoms selected from nitrogen, sulfur, and oxygen.
As used herein, the term “alkenyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include ethylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups. In one example, the alkenyl group is of 2 to 10 carbon atoms (i.e. C2-10alkenyl). In another example, the alkenyl group is of 2 to 6 carbon atoms (i.e. C2-6alkenyl)
As used herein, the term “alkynyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond. Examples of alkenyl groups include , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups. In one example, the alkynyl group is of 2 to 10 carbon atoms (i.e. C2-10alkynyl). In another examples, the alkynyl group is of 2 to 6 carbon atoms (i.e. C2- 6alkynyl).
As used herein, the term “haloalkyl” represents to an alkyl group having at least one halogen substituent, where “alkyl” and “halogen” are as described above. For example, the haloalkyl group may have at least one, two or three halogen substituents. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCl3, and -CH2CF3, -CF2CF3 and -CH2CHFCI. In one example, the haloalkyl group is of 1 to 10 carbon atoms (i.e. C1- whaloalkyl). In another example, the haloalkyl group is of 1 to 6 carbon atoms (i.e. C1- 6haloalkyl).
As used herein, the terms “carbocyclyl” and “carbocycle” whether used alone, or in compound words such as alkylcarbocyclyl, represents a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings. In one example, the carbocyclyl group is of 3 to 20 carbon atoms (i.e. C3-20-membered carbocyclyl). In another example, the carbocyclyl group is of 3 to 10 carbon atoms (i.e. C3-10-membered carbocyclyl). Examples of such groups include aryl groups such as phenyl, naphthyl, anthracenyl or fluorenyl, saturated groups such as cycloalkyl and cycloalkenyl groups e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl groups, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
As used herein, the term “cycloalkyl” whether used alone, or in compound words such as alkylcycloalkyl, refers to a monocyclic or polycyclic carbocyclic ring system of varying sizes, e.g., from about 3 to about 20 carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
As used herein, the term “heterocyclyl” whether used alone or in compound words such as alkylheterocyclyl, refers to a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and wherein the ring system may be aromatic such as a “heteroaryl” group, non-aromatic, saturated, or unsaturated, and may be substituted and/or contain fused rings. Heterocyclyl groups containing a suitable nitrogen atom include the corresponding N-oxides. In one example, the heterocyclyl group is of 3 to 20 atoms (i.e. 3-20-membered heterocyclyl). In another example, the heterocyclyl group is of 3 to 10 atoms (i.e. 3-10-membered heterocyclyl). The heteroatom may preferably be N, O or S. Examples of monocyclic non-aromatic heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, py-razolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thi-omorpholinyl and azepanyl. Examples of bicyclic heterocyclyl groups in which one of the rings is non-aromatic include dihydrobenzofuranyl, indanyl, indolinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl. Examples of monocyclic aromatic heterocyclyl groups (also referred to as monocyclic heteroaryl groups) include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl (e.g. the radical derived from pyridine), triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic aromatic heterocyclyl groups (also referred to as bicyclic heteroaryl groups) include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
As will be understood, an “aromatic” group means a cyclic group having 4m+2 % electrons, where m is an integer equal to or greater than 1. As used herein, “aromatic” is used interchangeably with “aryl” to refer to an aromatic group, regardless of the valency of aromatic group.
As used herein, the term “aryl” whether used alone, or in compound words such as alkylaryl, represents an monocyclic or polycyclic aromatic carbocyclic ring system. In one example, the aryl group is of 3 to 20 carbon atoms (i.e., an aromatic 3-20 membered carbocyclyl). In another example, the aryl group is of 3 to 10 carbon atoms (i.e., an aromatic 3-10 membered carbocyclyl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl or fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
As used herein, the term “heteroaryl” whether used alone, or in compound words such as alkylheteroaryl, represents a monocyclic or polycyclic aromatic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and may be substituted and/or contain fused rings. Heteroaryl groups containing a suitable nitrogen atom include the corresponding N- oxides. In one example, the heteroaryl group is of 3 to 20 atoms (i.e. 3-20-membered heteroaryl). In another example, the heteroaryl group is of 3 to 10 atoms (i.e. 3-10-membered heteroaryl). Examples of monocyclic heteroaryl groups include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic heteroaryl groups include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. All regioisomers are contemplated, e.g. 2-pyridyl, 3-pyridyl and 4- pyridyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.
“- C1-10alkyl-” represents an C1-10alkyl linker group in which the C1-10alkyl group is as defined supra.
“-O C1-10alkyl-” represents an alkoxy linker group in which the C1-10alkyl group is as defined supra.
“-C2-10alkenyl” represents an C2-10alkenyl linker group in which the C2-10alkenyl group is as defined supra.
“-OC2-10alkenyl-” represents an alkenyloxy linker group in which the C2-10alkenyl group is as defined supra.
“-C(=O)-” represents a carbonyl linker group.
“-C(=O) C1-10alkyl-” represents an alkanoyl linker group in which the C1-10alkyl group is as defined supra. “-C(=O)O-” represents an ester linking group.
“-C(=O)O( C1-10alkyl)-” represents an alkyl ester linking group in which the C1-10alkyl group is as defined supra.
“-OC(=O)( C1-10alkyl)-” represents alkanoate linker group in which the C1-10alkyl group is as defined supra.
“-C(=O)NH-” represents an amide linker group.
“-C(=O)NH( C1-10alkyl)-” represents an alkyl amide linker group in which the C1- 10alk yl group is defined supra.
“-S(=O)2-” represents a sulfone linker group.
“-S(=O)NH-” represents a sulfmamide linker group.
“-S(=O)NH( C1-10alkyl)-” represents an alkyl sulfmamide linker group in which the C1-10alkyl group is as defined supra.
“-S(=O)2NH-” represents a sulfonamide linker group.
“-S(=O)2NH( C1-10alkyl)-” represents an alkyl sulfonamide linker group in which the C1-10alkyl group is as defined supra.
“-OS(=O)2-” represents a sulfonate ester linker group.
“-O-” represents an ether linker group.
“-NH-” represents an amine linker group
“-S-” represents a sulfide linker.
As used herein, the term “saturated” refers to a group where all available valence bonds of the backbone atoms are attached to other atoms Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine, and the like.
As used herein, the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms. Representative examples include, but are not limited to, alkenes (e.g., -CH2-CH2CH=CH), phenyl, pyrrole, and the like.
As used herein, the term “optionally substituted” means that a functional group is either substituted or unsubstituted, at any available position.
As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent). As used herein, the term “unsubstituted” refers to a group that does not have any further groups attached thereto or substituted therefore.
The present disclosure relates to compounds of Formula (I) and pharmaceutically acceptable salts thereof. Salts may be formed in the case of embodiments of the compound of Formula (I), which contain a suitable acidic or basic group. Suitable salts of the compound of Formula (I) include those formed with organic or inorganic acids or bases.
As used herein, the phrase “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts. Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis- (2-hydroxy-3-naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. It will also be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport. In one example, the compound of Formula (I) is a hydrochloride salt. Those skilled in the art of organic and/or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". As used herein, the phrase “pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a compound of the present disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be understood that the present disclosure encompasses solvated forms, including hydrates, of the compounds of Formula (I) and salts thereof.
Those skilled in the art of organic and/or medicinal chemistry will appreciate that the compounds of Formula (I) and salts thereof may be present in amorphous form, or in a crystalline form. It will be understood that the present disclosure encompasses all forms and polymorphs of the compounds of Formula (I) and salts thereof.
As used herein, the term “stereoisomer” refers to compounds having the same molecular formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three-dimensional orientations of their atoms in space. As used herein, the term “enantiomers” refers to two compounds that are stereoisomers in that they are non- superimposable mirror images of one another. Relevant stereocenters may be denoted with (R)- or (S)- configuration.
Compounds of Formula (I)
The present disclosure provides compounds of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof:
Figure imgf000023_0001
as described in any of the embodiments below.
The present disclosure also provides a macrophage infectivity potentiator (Mip) protein inhibitor compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, as described in any of the embodiments below.
X
In the above Formula (I), X is selected from O, S and NR4. In one embodiment, X is O or NR4. In one embodiment, X is O. In another embodiment, X is NR4.
In some embodiments, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000024_0001
wherein A1, A2, A3, A4, L1, L2, R1, R2, R3 and R4 are as described herein.
In the above Formula (I), where X is NR4, R4 may be selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl-carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl, each of which is optionally substituted. In one embodiment, R4 is selected from the group consisting of H and C1-10alkyl. In one embodiment, R4 is H.
A1, A2, A3 and A4
In the above Formula (I), A1, A2, A3 and A4 are each connected to form a 6-membered heterocycl, wherein depending on the nature of each of A1, A2, A3 and A4, can be optionally interrupted and/or optionally substituted. In one embodiment, A1, A2, A3 and A4 are each independently selected from the group consisting of CR'2, NR' , S and O, wherein R is described herein.
In one embodiment, A1, A2 A3 and A4 are each independently selected from the group consisting CR'2, NR' , S and O, wherein R is as described herein.
In one embodiment, A1, A2 and A3 are each CH2, and A3 is selected from the group consisting CR'2, NR' , S and O, wherein R is as described herein. In the above Formula (I), each R is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2- 10alk ynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1- 10alkyl-3 - 10-membered-carbocyclyl, C1- 10alkyl-3 - 10-membered- heterocyclyl, each of which is optionally substituted. In one embodiment, each R is independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1- 6haloalkyl, and OC1-6haloalkyl, each of which is optionally substituted. In one embodiment, each R is independently selected from the group consisting of H or halogen.
In one embodiment, A1, A2, A3 and A4 are each independently selected from the group consisting of CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2- 10alk enyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl- 3-10-membered- heterocyclyl, each of which is optionally substituted.
In one embodiment, A1, A2 A3 and A4 are each independently selected from the group consisting CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, and OC1-6haloalkyl, each of which is optionally substituted.
In one embodiment, A1, A2 and A3 are each CH2, and A3 is selected from the group consisting CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, and OC1-6haloalkyl, each of which is optionally substituted. In one embodiment, A1, A2 A3 and A4 are each CH2.
L1 and L2
In the above Formula (I), L1 and L2 represent linker moieties and are each independently absent or selected from the group consisting of - C1-10alkyl-, -O C1-10alkyl-, -C2- 10alk enyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O-,-C(=O)O( C1-10alkyl)-, - OC(=O)( C1-10alkyl)-, -C(=O)NH- , -C(=O)NH( C1-10alkyl)-, -N( C1-10alkyl)- -S(=O)NH-, - S(=O)NH( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and -OS(=O)2- wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted. That is, L1 may be absent, - C1-10alkyl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2- 10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O-,-C(=O)O( C1-10alkyl)-, -OC(=O)(C1- 10alkyl)-, -C(=O)NH- , -C(=O)NH( C1-10alkyl)-, -N( C1-10alkyl)-, -S(=O)NH-, -S(=O)NH(C1- 10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and -OS(=O)2- wherein each C1- 10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted. Similarly, L2 may be absent, - C1-10alkyl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, - C(=O) C1-10alkyl-, -C(=O)O-,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH- , - C(=O)NH( C1-10alkyl)-, -N( C1-10alkyl)-, -S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -S(=O)2-, - S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and -OS(=O)2- wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted.
Unless otherwise stated or structurally depicted, it will be appreciated that the orientation of the linkers L1 and L2 within the compound of Formula (I) are undefined. That is, L1 and L2may be attached at either side within the compound of Formula (I). For example, in the above Formula (I), when L2 is -C(=O)NH-, the compound of Formula (I) may be selected from:
Figure imgf000026_0001
In another example, in the above Formula (I), when L2 is -C(=O)NH( C1-10alkyl)-, for example -C(=O)NH-CH2-, the compound of Formula (I) may be selected from:
Figure imgf000026_0002
In another example, in the above Formula (I), when L2 is wherein
Figure imgf000027_0001
represents the attachment point of L2 to the rest of the compound, the compound of
Figure imgf000027_0003
Formula (I) may be selected from:
Figure imgf000027_0002
When L1 is absent, it will be understood that that there is a direct bond between the central sulfur atom of the sulfonyl group and R1. Similarly, when L2 is absent, it will be understood that there is a direct bond between R3 and the carbon atom attached to R2.
In one embodiment, L1 and L2 are different (e.g. L1 is - C1-10alkyl- and L2 is - C(=O)NH( C1-10alkyl)-, wherein each - C1-10alkyl is optionally substituted). In other words, L1 and L2 are independently selected from one another.
In one embodiment, L1 is present and selected from the group consisting of -C1- 10alkyl-, -N( C1-10alkyl)-, -O C1-10alkyl-, -C2-10alkenyl-, and -OC2-10alkenyl- wherein each C1- 10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted. In one embodiment, L1 is - C1-10alkyl- wherein C1-10alkyl is uninterrupted or interrupted and optionally substituted.
In one embodiment, L2 is present and selected from the group consisting of -C1- 10alkyl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O- ,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH- , -C(=O)NH( C1-10alkyl)-, - S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and - OS(=O)2- wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted. In one embodiment, L2 is selected from the group consisting of -C1- 10alkyl-, -C(=O)NH- or -C(=O)NH( C1-10alkyl)- wherein C1-10alkyl is uninterrupted or interrupted and optionally substituted. In one embodiment, L2 is selected from the group consisting of -C(=O)NH- or -C(=O)NH( C1-10alkyl)- wherein C1-10alkyl is uninterrupted or interrupted and optionally substituted. In one embodiment, L2 is -C(=O)NH( C1-10alkyl)- wherein C1-10alkyl is uninterrupted or interrupted and optionally substituted.
When present in either L1 and/or L2, each C1-10alkyl or C2-10alkenyl may be uninterrupted or interrupted and optionally substituted. In one embodiment, each C1-10alkyl or C2-10alkenyl may be uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -NH-, -C(=O)NH-, -S-, and -S(=O)2-, and optionally substituted with one or more R7. In one embodiment, each C1-10alkyl or C2-10alkenyl of L1 and L2 are uninterrupted and unsubstituted. In the instance where C1-10alkyl or C2-10alkenyl are not substituted with one or more R7, it will be understood that a hydrogen atom will remain as the substitution.
In one embodiment, L1 is absent or selected from - C1-10alkyl or -N( C1-10alkyl)-. In one embodiment, L1 is absent or selected from -C1-6alkyl- or -N(C1-6alkyl)-. In one embodiment, L1 is absent or selected from-CH2-, -CH2CH2-, -CH2CH2CH2-, or -N/CH2)-. In one embodiment, L1 is absent. In one embodiment, L1 is - C1-10alkyl-. In one embodiment, L1 is -C1-6alkyl-. In one embodiment, L1 is -CH2-, -CH2CH2- or -CH2CH2CH2-. In one embodiment, L1 is -CH2-.
In one embodiment, L2 is -C(C=O)NH-, -C(=O)NH( C1-10alkyl)- or
Figure imgf000028_0001
In one embodiment, L2 is -C(C=O)NH_. In one embodiment, L2 is -C(=O)NH(C1- 10alkyl)-. In one embodiment, L2 is -C(=O)NH(C1-6alkyl)-. In one embodiment, L2 is -
C(=O)NH-CH2-. In one embodiment, L2 is
Figure imgf000028_0002
In one embodiment, L2 is -CH2CH2CH2-, and the compound of Formula (I) is
Figure imgf000028_0003
In one embodiment, L2 is -C(C=O)NH- and the compound of Formula (I) is
Figure imgf000029_0001
In one embodiment, L2 is -C(=O)NH-CH2-, and the compound of Formula (I) is selected from:
Figure imgf000029_0002
In one embodiment, L2 is and the compound of Formula (I) is
Figure imgf000029_0003
selected from:
Figure imgf000029_0004
In one embodiment, L1 is -CH2- and L2 is -C(=O)NH-CH2-, and the compound of Formula (I) is selected from:
Figure imgf000030_0001
In one embodiment, L1 is -CH2- and L2 is and the compound of
Figure imgf000030_0004
Formula (I) is selected from:
Figure imgf000030_0002
In one embodiment, L1 is absent and L2 is -C(=O)NH-CH2-, and the compound of
Formula (I) is selected from:
Figure imgf000030_0003
In one embodiment, L1 is absent and L2 is and the compound of
Formula (I) is selected from:
Figure imgf000030_0005
Figure imgf000031_0001
In another example, L1 is -CH2-, L2 is -C(=O)NH-CH2-, X is O or NR4, and the compound of Formula (I) is selected from the group consisting of:
Figure imgf000031_0002
In another example, L1 is -CH2-, L2 is
Figure imgf000031_0003
X is O or NR4, and the compound of Formula (I) is selected from the group consisting of:
Figure imgf000031_0004
Figure imgf000032_0004
In another example, L 1 is absent, L2 is -C(=O)NH-CH2-, X is O or NR4, and the compound of Formula (I) is selected from the group consisting of:
Figure imgf000032_0003
In another example, L1 is -CH2-, L2 is
Figure imgf000032_0002
X is O or NR4, and the compound of Formula (I) is selected from the group consisting of:
Figure imgf000032_0001
Figure imgf000033_0001
R1 and R3
In the above Formula (I), R1 and R3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. That is, R1 may be an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. Similarly, R3 may be an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. In one embodiment, R1 is an optionally substituted carbocyclyl. In one embodiment, R1 is an optionally substituted heterocyclyl. In one embodiment, R3 is an optionally substituted carbocyclyl. In one embodiment, R3 is an optionally substituted heterocyclyl. R1 and R3 may be the same (e.g. R1 is an optionally substituted carbocyclyl and R3 is an optionally substituted carbocyclyl) or R1 and R3 may be different (e.g. R1 is an optionally substituted carbocyclyl and R3 is an optionally substituted heterocyclyl) (e.g. the R1 and R3 substituents are independently selected from one another). In one embodiment, R1 is an optionally substituted carbocyclyl and R3 is an optionally substituted heterocyclyl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. That is, R1 may be an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. Similarly, R3 may be an optionally substituted 3-10-membered carbocyclyl or an optionally substituted 3-10-membered heterocyclyl. In one embodiment, R1 is an optionally substituted 3-10-membered carbocyclyl. In one embodiment, R1 is an optionally substituted 3-10-membered heterocyclyl. In one embodiment, R3 is an optionally substituted 3-10-membered carbocyclyl. In one embodiment, R3 is an optionally substituted 3-10-membered heterocyclyl. In one embodiment, R1 is an optionally substituted 3-10-membered carbocyclyl and R3 is an optionally substituted 3-10- membered heteroaryl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. That is, R1 may be an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. Similarly, R3 may be an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl. In one embodiment, R1 is an optionally substituted monocyclic carbocyclyl. In one embodiment R3 is an optionally substituted monocyclic heterocyclyl. In one embodiment, R1 is an optionally substituted monocyclic carbocyclyl and R3 is an optionally substituted monocyclic heterocyclyl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted aryl or an optionally substituted heteroaryl. That is, R1 may be an optionally substituted aryl or an optionally substituted heteroaryl. Similarly, R3 may be an optionally substituted aryl or an optionally substituted heteroaryl. In one embodiment, R1 is an optionally substituted aryl. In one embodiment, R1 is an optionally substituted heteroaryl. In one embodiment, R3 is an optionally substituted aryl. In one embodiment, R3 is an optionally substituted heteroaryl. In one embodiment, R1 is an optionally substituted aryl and R3 is an optionally substituted heteroaryl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. That is, R1 may be an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. Similarly, R3 may be an optionally substituted monocyclic aryl or an optionally substituted monocyclic heteroaryl. In one embodiment, R1 is an optionally substituted monocyclic aryl. In one embodiment, R1 is an optionally substituted monocyclic heteroaryl. In one embodiment, R3 is an optionally substituted monocyclic aryl. In one embodiment, R3 is an optionally substituted monocyclic heteroaryl. In one embodiment, R1 is an optionally substituted monocyclic aryl and R3 is an optionally substituted monocyclic heteroaryl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl. That is, R1 may be an optionally substituted 3-10-membered aryl or an optionally substituted 3-10- membered heteroaryl. Similarly, R3 may be an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl. In one embodiment, R1 is an optionally substituted 3-10-membered aryl. In one embodiment, R1 is an optionally substituted 3-10- membered heteroaryl. In one embodiment, R3 is an optionally substituted 3-10-membered aryl. In one embodiment, R3 is an optionally substituted 3-10-membered heteroaryl. In one embodiment R1 is an optionally substituted 3-10-membered aryl and R3 is an optionally substituted 3-10-membered heteroaryl.
In one embodiment, R1 and R3 are each independently selected from an optionally substituted 5-6-membered aryl or an optionally substituted 5-6-membered heteroaryl. That is, R1 may be an optionally substituted 5-6-membered aryl or an optionally substituted 5-6- membered heteroaryl. Similarly, R3 may be an optionally substituted 5-6-membered aryl or an optionally substituted 5-6-membered heteroaryl. In one embodiment, R1 is an optionally substituted 5-6-membered aryl. In one embodiment, R1 is an optionally substituted 5-6- membered heteroaryl. In one embodiment, R3 is an optionally substituted 5-6-membered aryl. In one embodiment, R3 is an optionally substituted 5-6-membered heteroaryl. In one embodiment R1 is an optionally substituted 5-6-membered aryl and R3 is an optionally substituted 3-10-membered heteroaryl.
In one embodiment, R1 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidinyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted.
In one embodiment, R1 is an optionally substituted phenyl. In one embodiment, R3 is an optionally substituted phenyl or an optionally substituted 5-6-membered heteroaryl. In one embodiment, R1 is an optionally substituted phenyl and R3 is an optionally substituted 5-6- membered heteroaryl.
In one embodiment, R3 is an optionally substituted N-heterocyclyl. In one embodiment, R3 is an optionally substituted N-heteroaryl. In one embodiment, R3 is an optionally substituted 3-10-membered-N-heteroaryl. As used herein, the term “N- heterocyclyl” and “N-heteroaryl” represents a nitrogen containing heterocyclyl and a nitrogen containing heteroaryl, respectively.
In one embodiment, R3 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Each of which is optionally substituted. In one embodiment, R3 is a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted. In one embodiment R3 is an optionally substituted pyridyl.
In one embodiment, R1 is an optionally substituted phenyl and R3 is an optionally substituted phenyl or a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted. In one embodiment, R1 is an optionally substituted phenyl and R3 is a 5-6-membered heteroaryl selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted. In one embodiment, R1 is an optionally substituted phenyl and R3 is an optionally substituted pyridyl. Further examples of R1 and/or R3 include, but are not limited to:
Figure imgf000036_0001
each of which is optionally substituted, wherein represents the attachment point to the rest of the compound.
In one embodiment, R3 is independently selected from the group consisting of:
Figure imgf000036_0002
Figure imgf000037_0001
each of which is optionally substituted, wherein represents the attachment point of R3 to the rest of the compound.
In one embodiment, R1 is an optionally substituted phenyl and R3 is independently selected from the group consisting of:
Figure imgf000037_0002
each of which is optionally substituted, wherein represents the attachment point of R1 to
Figure imgf000037_0003
the rest of the compound.
In one embodiment, the compound of Formula (I) is
Figure imgf000037_0004
wherein: A5, A6, A7, A8 and A9 are each independently selected from CR5, N or N-oxide; and
X, L1, L2, R1, R2 and R5 are as described herein. It will be appreciated that A5, A6, A7, A8, and A9 form an aromatic ring. In one embodiment, A6 is N or N-oxide and each of A5, A7, A8, and A9 is independently CR5. R2
In the above Formula (I), R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted. In one embodiment, R2 is an optionally substituted alkyl. In one embodiment, R2 is an optionally substituted alkenyl. In one embodiment, R2 is an optionally substituted alkynyl. In one embodiment, R2 is an optionally substituted carbocyclyl. In one embodiment, R2 is an optionally substituted alkylcarbocyclyl. In one embodiment, R2 is an optionally substituted heteroalkyl. In one embodiment, R2 is an optionally substituted heterocyclyl. In one embodiment, R2 is an optionally substituted alky lheterocy cly 1.
In one embodiment, R2 is selected from the group consisting of C1-10alkyl, cycloalkyl, C1-10alkylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C1-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is an optionally substituted C1-10alkyl. In one embodiment, R2 is an optionally substituted cycloalkyl. In one embodiment, R2 is an optionally substituted C1- 10alk ylcycloalkyl. In one embodiment, R2 is an optionally substituted C1-10alkylheterocyclyl. In one embodiment, R2 is an optionally substituted aryl. In one embodiment, R2 is an optionally substituted C1-10alkylaryl. In one embodiment, R2 is an optionally substituted heteroaryl. In one embodiment, R2 is an optionally substituted C1-10alkylheteroaryl.
According to at least some embodiments or examples described herein, the present inventors have surprisingly identified that by introducing a bulkier R2 substituent (e.g. an optionally substituted alkyl or an optionally substituted alkylaryl) in the middle chain of the compound connecting the lateral pyridine ring with the pipecolic moiety, highly active PPIase inhibitors were identified which have potent inhibitory properties against the macrophage infectivity potentiator (Mip) protein of several bacterial pathogens.
In one embodiment, R2 is selected from the group consisting of C1-6alkyl, aryl, C1- 6alkylaryl, heteroaryl, and C1-6alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is an optionally substituted C1-6alkyl. In one embodiment, R2 is an optionally substituted aryl. In one embodiment, R2 is an optionally substituted C1-6alkylaryl. In one embodiment, R2 is an optionally substituted heteroaryl. In one embodiment, R2 is an optionally substituted C1-6alkylheteroaryl.
In one embodiment, R2 is selected from the group consisting of alkyl, aryl, alkylaryl, heteroaryl or alkyheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C1-10alkyl, aryl, C1-10alkylaryl, heteroaryl or C1- 10alk ylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C1-6alkyl, aryl, C1-6alkylaryl, heteroaryl, or C1-6alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C1-6alkyl, 5-6-membered aryl, C1-6alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
In one embodiment, R2 is selected from the group consisting ofC1-6alkyl, 5-6-membered aryl, C1-6alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C1-6alkyl, phenyl, C1-6alkylphenyl, 5-6-membered heteroaryl or C1- 6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
In one embodiment, where R2 comprises a 5-6 membered heteroaryl, the 5-6- membered heteroaryl may be selected from the group consisting of pyridyl, pyrimidyl furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyridazenyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl, each of which is optionally substituted. For example, where R2 comprises a 5-6 membered heteroaryl, the 5-6-membered heteroaryl may be an optionally substituted pyridyl. In one embodiment, R2 is selected from the group consisting of C1-6alkyl, phenyl, C1- 6alkylphenyl, pyridyl, or C1-6alkylpyridyl, each of which is optionally substituted.
In one embodiment, R2 is selected from the group consisting of C3-20alkyl, aryl, alkylaryl, heteroaryl or alkyheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl or C1-10alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, or C1-6alkylheteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1- 6alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
In one embodiment, R2 is selected from the group consisting of C3-6alkyl, 5-6- membered aryl, C1-6alkyl-5-6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6- membered heteroaryl, each of which is optionally substituted. In one embodiment, R2 is selected from the group consisting of C3-6alkyl, phenyl, C1-6alkylphenyl, 5-6-membered heteroaryl or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
In one embodiment, R2 is selected from the group consisting of C3-6alkyl, phenyl, C1- 6alkylphenyl, pyridyl, or C1-6alkylpyridyl, each of which is optionally substituted. In one example, R2 is selected from the group consisting of:
Figure imgf000040_0001
each of which is optionally substituted, wherein represents the attachment point of R2 to the rest of the compound.
In some embodiments, the compound of the present disclosure is a compound of Formula (I), with the proviso that where R2 is methyl, the compound is not selected from the group consisting of:
Figure imgf000040_0002
R5 to R8
In the above Formula (I), each of R’, R1, R2, R3 and R4 may be optionally substituted. In one embodiment, each of R’, R1, R2, R3 and R4 may be optionally substituted with one or more R5. In the instance where R’, R1, R2, R3 and R4 is not substituted with one or more R5, then it will be understood that a hydrogen atom will remain as the substitution.
In some embodiments, R’ is substituted with one, two, three, four, five, or more, R5 substituents. In one embodiment, R’ is substituted with one R5 substituent. In one embodiment, R’ is substituted with two R5 substituents. In one embodiment, R is substituted with three R5 substituents. In one embodiment, R is substituted with four R5 substituents. In one embodiment, R is substituted with five R5 substituents. In one embodiment, R is substituted with more than five R5 substituents.
In some embodiments, R1 is substituted with one, two, three, four, five, or more, R5 substituents. In one embodiment, R1 is substituted with one R5 substituent. In one embodiment, R1 is substituted with two R5 substituents. In one embodiment, R1 is substituted with three R5 substituents. In one embodiment, R1 is substituted with four R5 substituents. In one embodiment, R1 is substituted with five R5 substituents. In one embodiment, R1 is substituted with more than five R5 substituents.
In some embodiments, R2 is substituted with one, two, three, four, five, or more, R5 substituents. In one embodiment, R2 is substituted with one R5 substituent. In one embodiment, R2 is substituted with two R5 substituents. In one embodiment, R2 is substituted with three R5 substituents. In one embodiment, R2 is substituted with four R5 substituents. In one embodiment, R2 is substituted with five R5 substituents. In one embodiment, R2 is substituted with more than five R5 substituents.
In some embodiments, R3 is substituted with one, two, three, four, five, or more, R5 substituents. In one embodiment, R3 is substituted with one R5 substituent. In one embodiment, R3 is substituted with two R5 substituents. In one embodiment, R3 is substituted with three R5 substituents. In one embodiment, R3 is substituted with four R5 substituents. In one embodiment, R3 is substituted with five R5 substituents. In one embodiment, R3 is substituted with more than five R5 substituents.
In some embodiments, R4 is substituted with one, two, three, four, five, or more, R5 substituents. In one embodiment, R4 is substituted with one R5 substituent. In one embodiment, R4 is substituted with two R5 substituents. In one embodiment, R4 is substituted with three R5 substituents. In one embodiment, R4 is substituted with four R5 substituents. In one embodiment, R4 is substituted with five R5 substituents. In one embodiment, R4 is substituted with more than five R5 substituents.
It will be understood that, when any of R , R1, R2, R3 or R4 is substituted with one or more R5 substituents, the one or more substituents may be the same substituent or a different substituent (e.g., the R5 substituents are independently selected from one another).
In the above Formula (I), each R5 is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2- 10alk ynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C i- 10alkyl-3 - 10-membered-carbocyclyl, C i- 10alkyl-3 - 10-membered- heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, - SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, - N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6. In some embodiments, each R5 is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1- whaloalkyl, O C1-10haloalkyl, C2-10alkenyl, OC2-10alkenyl, C2-10alkynyl, OC2-10alkynyl, -NO2, - CN -N(R6)2, -OR6, -S(=O)2R6, or -N(R6)C(=O)R6 In some embodiments, each R5 is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1- whaloalkyl, O C1-10haloalkyl, C2-10alkenyl, OC2-10alkenyl, C2-10alkynyl, and OC2-10alkynyl.
In the above Formula (I), when R5 is C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, Cmoalkyl-3-10-membered-carbocyclyl, C1-10alkyl- 3-10-membered-heterocyclyl, each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3 -10-membered -heterocyclyl may be optionally substituted with one or more R7 substituents. In some embodiments, when R5 is C1-10alkyl, O C1-10alkyl, C1- 10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2- 10alk ynyl, 3-10 membered-carbocyclyl, and 3-10-membered-heterocyclyl may be optionally substituted with one, two, three, four, five or more than five R7 substituents. It will be understood that, when R5 is substituted with one or more R7 substituents, the one or more substituents may be the same substituent or a different substituent (e.g., the R7 substituents are independently selected from one another).
In some embodiments, each R5 may be independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, OC1-6haloalkyl, C2-6alkenyl, OC2-6alkenyl, C2-6alkynyl, OC2-6alkynyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In the above Formula (I), each R6 is independently selected from the group consisting of H, C1-6alkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-6alkyl-3-10- membered-carbocyclyl, and C1-6alkyl-3-10-membered-heterocyclyl. In the above Formula (I), when R6 is C1-6alkyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C1-6alkyl-3-10-membered-carbocyclyl, and C1-6alkyl-3-10- membered-heterocyclyl, each C1-6alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl may be optionally substituted with one or more R7 substituents. In some embodiments, when R6 is C1-6alkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-6alkyl-3-10-membered-carbocyclyl, and C1-6alkyl-3-10-membered- heterocyclyl, each C1-6alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl may be optionally substituted with one, two, three, four, five or more than five R7 substituents. It will be understood that, when R6 is substituted with one or more R7 substituents, the one or more substituents may be the same substituent or a different substituent (e.g., the R7 substituents are independently selected from one another).
In the above Formula (I), each R7 may be independently selected from the group consisting of halogen, -NO2, -N(R8)2, -CN, =O, -C(=O)OR8, -N(R8)C(=O)R8, -OR8, C1-6alkyl, and -OC1-6alkyl, or R7 together with the carbon it is attached forms a C3-6carbocyclic ring. In the above Formula (I), each R8 is independently selected from the group consisting of H and C1-6alkyl. In one embodiment, R8 is H. In one embodiment, R8 is C1-6alkyl.
In the above Formula (I), each R1 and R3 may be independently optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1- 10alk yl and C1-10haloalkyl is optionally substituted with one or more R7. In some embodiments, each R1 and R3 are independently optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, - NH2, -N(H)C1-6alkyl, orC1-6alkylNH2.
In one embodiment, R1 is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, - OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In some embodiments, R1 is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -NH2, - N(H)C1-6alkyl, orC1-6alkylNH2. In one embodiment, R1 is optionally substituted with H. In one embodiment, R1 is optionally substituted with halogen (e.g. fluorine). In one embodiment, R1 is optionally substituted with C1-6haloalkyl (e.g. -CF3). In one embodiment, R1 is optionally substituted with -NO2. In one embodiment, R1 is optionally substituted with - Nth. In one embodiment, R1 is optionally substituted with -N(H)C1-6alkyl.
In one embodiment, R1 is an aryl optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2- 10alk enyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered- heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, - SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, - N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1- 10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3- 10-membered - heterocyclyl is optionally substituted with one or more R7. In one embodiment, R1 is an aryl optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, OC1- 10alk yl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or - N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R1 is an aryl optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, - NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R1 is an aryl optionally substituted by one or more groups selected from H, halogen, or C1-6haloalkyl.
In one embodiment, R1 is a 3-10-membered aryl optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2- 10alk enyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered- heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, - SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, - N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1- 10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3- 10-membered - heterocyclyl is optionally substituted with one or more R7. In one embodiment, R1 is a 3-10- membered aryl optionally substituted by one or more groups selected from H, halogen, C1- 10alk yl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R1 is a 3-10-membered aryl optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, - SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R1 is a 3-10- membered aryl optionally substituted by one or more groups selected from H, halogen, or C1- 6haloalkyl.
In one embodiment, R1 is phenyl optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2- 10alk enyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10- membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered- heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, - SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, - N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1- whaloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3- 10-membered - heterocyclyl is optionally substituted with one or more R7. In one embodiment, R1 is phenyl optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, OC1- 10alk yl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or - N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R1 is phenyl optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, - NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R1 is phenyl optionally substituted by one or more groups selected from H, halogen, or C1-6haloalkyl.
In one embodiment, R3 is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, - OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In some embodiments, R3 is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -OH, -NO2, - NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is optionally substituted with H. In one embodiment, R3 is optionally substituted with halogen (e.g. fluorine). In one embodiment, R3 is optionally substituted with C1-6haloalkyl (e.g. -CF3). In one embodiment, R3 is optionally substituted with -NO2. In one embodiment, R3 is optionally substituted with - NH2. In one embodiment, R3 is optionally substituted with OC1-6alkyl. In one embodiment, R3 is optionally substituted with -OH. In one embodiment, R3 is optionally substituted with C1-6alkylNH2. In some embodiments, R3 is an aryl or heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1- whaloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, - S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, - S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered- carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R7.
In one embodiment, R3 is an aryl or heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1- 10alk yl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, - SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is an aryl or heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1-6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In some embodiments, R3 is an 3-10-membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1- 10alk yl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2- 10alk enyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1- 10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, - C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6,
N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1- 10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3-10-membered- heterocyclyl is optionally substituted with one or more R7. In one embodiment, R3 is a 3-10- membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1- 10alk yl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is a 3-10-membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1- 6haloalkyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is a 3-10-membered aryl or a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1- 6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In some embodiments, R3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, OC1- 10alk yl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2- 10alk ynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10- membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, - C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, - C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3 -10-membered -heterocyclyl is optionally substituted with one or more R7. In one embodiment, R3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, OC1- 10alk yl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or - N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is phenyl or a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1-6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In some embodiments, R3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl- 3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, -S(=O)N(R6)2, - S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, -S(=O)2R6, - N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1- 10alk yl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered-carbocyclyl, and 3-10- membered-heterocyclyl is optionally substituted with one or more R7. In one embodiment, R3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, - OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is a heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1-6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In some embodiments, R3 is a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1- 10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, -C(=O)N(R6)2, - S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, - S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered- carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R7. In one embodiment, R3 is a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1- 10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is a 3-10-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, - CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is a 3-10- membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1-6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In some embodiments, R3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1- 10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -N02, -CN, =O, -N(R6)2, -C(=O)N(R6)2, - S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, -C(=O)OR6, -S(=O)R6, - S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered- carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R7. In one embodiment, R3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- 10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1- 10alk yl and C1-10haloalkyl is optionally substituted with one or more R7. In one embodiment, R3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, - OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R3 is a 5-6-membered heteroaryl, each of which is optionally substituted by one or more groups selected from H, halogen, C1-6haloalkyl, OC1-6alkyl, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
In the above Formula (I), R2 may be optionally substituted with one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2- 10alk enyl, OC2-10alkenyl, C2-10alkynyl, OC2-10alkynyl, -NO2, -CN -N(R6)2, -OR6, -S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, or C2-10alkynyl is optionally substituted with one or more R7. In some embodiments, R2 is optionally substituted with one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1- 6haloalkyl, OC1-6haloalkyl, C2-6alkenyl, OC2-6alkenyl, C2-6alkynyl, OC2-6alkynyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R2 is substituted with H, halogen, OC1-6alkyl, C1-6haloalkyl, OC2-6alkynyl, -NO2, NH2, -OH, -N(H)C1-6alkyl, or C1-6alkylNH2. In one embodiment, R2 is substituted with H. In one embodiment, R2 is substituted with halogen. In one embodiment, R2 is substituted with OC1-6alkyl. In one embodiment, R2 is substituted with C1-6haloalkyl. In one embodiment, R2 is substituted with OC2-6alkynyl. In one embodiment, R2 is substituted with -NO2. In one embodiment, R2 is substituted with -NH2. In one embodiment, R2 is substituted with -N(H)C1-6alkyl. In one embodiment, R2 is substituted with C1-6alkylNH2.
In the above Formula (I), R2 may be selected from the group consisting of C1-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted with one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1- whaloalkyl, O C1-10haloalkyl, C2-10alkenyl, OC2-10alkenyl, C2-10alkynyl, OC2-10alkynyl, -NO2, - CN -N(R6)2, -OR6, -S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, or C2-10alkynyl is optionally substituted with one or more R7. In some embodiments, R2 is selected from the group consisting of C1-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted with one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, OC1-6haloalkyl, C2- 6alkenyl, OC2-6alkenyl, C2-6alkynyl, OC2-6alkynyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1- 6alkyl, or C1-6alkylNH2. In one embodiment, R2 is selected from the group consisting of C1- 10alk yl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted with H, halogen, OC1-6alkyl, C1-6haloalkyl, OC2-6alkynyl, -NO2, NH2, -OH, - N(H)C1-6alkyl, or C1-6alkylNH2.
In one embodiment, the compound of Formula (I) is
Figure imgf000050_0001
wherein n is 0 to 5; and
X, A1, A2, A3, A4, L1, L2, R2, R3 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000050_0002
wherein n is 0 to 5; Ring B is a N-heteroaryl; and
X, A1, A2, A3, A4, L1, L2, R1, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000051_0002
wherein n is 0 to 5; and
X, A1, A2, A3, A4, L1, L2, R1, R2 and R5 are as described herein. In one embodiment, the compound of Formula (I) is:
Figure imgf000051_0001
wherein X, A1, A2, A3, A4, A5, A6, A7, A8, A9, L1, L2, R1, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000052_0001
wherein: n is 0 to 5; and
X, A1, A2, A3, A4, L1, L2, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is:
Figure imgf000052_0002
wherein X, A1, A2, A3, A4, L1, R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is selected from:
Figure imgf000053_0001
wherein A1, A2, A3, A4, L1, R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is selected from:
Figure imgf000053_0002
Figure imgf000054_0001
wherein A1, A2, A3, A4, R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000055_0001
wherein n is 0 to 5; and
X, L1, L2 R1, R3 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is
Figure imgf000055_0002
wherein X, L1, L2, R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is
Figure imgf000055_0003
wherein n is 0 to 5; and
X, L1, L2, R2, R3 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000056_0001
wherein n is 0 to 5; and X, L1, L2, R1, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is:
Figure imgf000056_0002
wherein: n is 0 to 5; and A1, A2, A3, A4, A5 , X, L1, L2, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000057_0001
wherein: n is independently 0 to 5; and
X, L1, L2, R2 and R5 are as described herein.
In one embodiment, the compound of Formula (I) is:
Figure imgf000057_0002
wherein X, L1, R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is selected from:
Figure imgf000057_0003
wherein L1, R1, R2 and R3 are as described herein. In one embodiment, the compound of Formula (I) is selected from:
Figure imgf000058_0001
wherein R1, R2 and R3 are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000058_0002
wherein n is 0 to 5; and
X, L1, L2 R1, R3 and R5 are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000059_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein. In some embodiments, the compound of Formula (I) is
Figure imgf000059_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000060_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000060_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000061_0002
wherein
L2is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000061_0001
wherein
L2is - -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000062_0001
wherein
L2is - -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000062_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000063_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein. In some embodiments, the compound of Formula (I) is
Figure imgf000063_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 3-10-membered heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000064_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 5-6-membered heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000064_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted pyridyl or N-oxide thereof, pyrazolyl or imadzolyl; and X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000065_0001
wherein
L2is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 5-6-membered heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000065_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000066_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000066_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000067_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5-
6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein. In some embodiments, the compound of Formula (I) is
Figure imgf000067_0002
wherein
L2is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000068_0001
wherein
L2is - -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, A1, A2, A3, A4, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000068_0002
wherein
L2is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000069_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000069_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000070_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted; R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 3-10-membered heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000070_0002
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 5-6-membered heteroaryl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000071_0001
wherein
L2 is -C(=O)NH- or -C(=O)NH( C1-10alkyl)-, wherein each C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-6alkyl, 5-6-membered aryl, C1-6alkyl-5- 6-membered aryl, 5-6-membered heteroaryl, or C1-6alkyl-5-6-membered heteroaryl, each of which is optionally substituted.
R3 is an optionally substituted pyridyl or N-oxide thereof, pyrazolyl or imadzolyl; and
X, L1 and R1, are as described herein.
In some embodiments, the compound of Formula (I) is
Figure imgf000071_0002
wherein
L2is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted;
R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1- 10alk ylcycloalkyl, heteroalkyl, heterocyclyl, C1-10alkylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
R3 is an optionally substituted 5-6-membered heteroaryl; and
X, L1 and R1, are as described herein.
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
In one embodiment, the compound of Formula (I) is selected from the group consisting of:
Figure imgf000087_0002
Figure imgf000088_0001
It will be appreciated that the compounds of Formula (I) as described herein also include, where applicable, a pharmaceutically acceptable salt, solvate, stereoisomer or N- oxide thereof.
Therapeutic Methods and Uses
It has been surprisingly found that compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, demonstrate inhibitory activity against macrophage infectivity potentiator (Mip) protein. Such inhibition of Mip protein can provide a therapeutic effect. In particular, the compounds of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, finds use in the therapy of a disease or condition, for example Q fever.
Accordingly, there is provided a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in therapy of a disease or condition, for example Q fever.
In some embodiments, there is provided a method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein, the method comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
In some embodiments, there is provided use of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In some embodiments, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In some embodiments, there is provided a method of treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor, the method comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
In some embodiments, there is provided use of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
In some embodiments, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence
In some embodiments, the pathogen may be a bacterial pathogen. In one embodiment, the bacterial pathogen is a Gram-negative bacterium.
In one embodiment, the Gram-negative bacterium is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
In one embodiment, the pathogen is Coxiella burnetii, and the disease or condition is Q fever. In one embodiment or example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, finds use in the treatment and/or prevention of Q fever.
In some embodiments, there is provided a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in therapy of Q fever. In some embodiments, there is provided a method of treating and/or preventing Q fever in a subject, the method comprising administering to the subject an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
In some embodiments, there is provided use of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prevention of Q fever in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In some embodiments, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in treating and/or preventing Q fever in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
In some embodiments, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use as a PPIase inhibitor.
In some embodiments, there is provided a method of treating and/or preventing a disease or condition mediated by a PPIase inhibitor, comprising administering to the subject of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof.
In some embodiments, there is provided use of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a PPIase inhibitor.
In some embodiments, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, for use in treating and/or preventing a disease or condition mediated by a PPIase inhibitor.
It will be appreciated that the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, can also be administered or used as a pharmaceutical composition or formulation as described herein.
Compositions and formulations
Whilst a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof thereof may in some embodiments be administered alone, it is more typically administered as part of a pharmaceutical composition or formulation. Thus, the present disclosure also provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and a pharmaceutically acceptable excipient. The pharmaceutical composition comprises one or more pharmaceutically acceptable diluents, carriers or excipients (collectively referred to herein as “excipient” materials).
The present disclosure also provides pharmaceutical formulations or compositions, both for veterinary and for human medical use, which comprise compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilisers, or the like. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
Examples of pharmaceutical formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (including fine particle dusts or mists that may be generated by means of various types of metered dose pressurised aerosols), nebulisers or insufflators, rectal, intraperitoneal and topical (including dermal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.
The pharmaceutical formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with the excipient that constitutes one or more necessary ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
In some embodiments, that composition is formulated for oral delivery. For example, pharmaceutical formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, pills or tablets each containing a predetermined amount of the active ingredient; as a powder or granules, as a solution or a suspension in an aqueous liquid or non-aqueous liquid, for example as elixirs, tinctures, suspensions or syrups; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may also be presented as a bolus, electuary or paste.
A tablet may be made for example by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally coated or scored, and may be formulated so as to provide slow or controlled release of the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof. The compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof can, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may also be administered liposomally.
Exemplary compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavouring agents such as those well known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents, and lubricants such as those known in the art. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Disintegrators include without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. A compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof can also be delivered through the oral cavity by sublingual and/or buccal administration. Moulded tablets, compressed tablets, or freeze-dried tablets are exemplary forms that may be used. Exemplary compositions include those formulating a compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as cellulose (avicel) or polyethylene glycols (PEGs). Such formulations can also include an excipient to aid mucosal adhesion such as hydroxyl propyl cellulose (HPC), hydroxyl propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer, and agents to control release such as polyacrylic copolymer. Lubricants, glidants, flavours, colouring agents, and stabilisers may also be added for ease of fabrication and use. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. For oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
In some embodiments, the composition is formulated for parenteral delivery. Formulations for parenteral administration include aqueous and non-aqueous sterile injections solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1.3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
For example, in one embodiment, the formulation may be a sterile, lyophilized composition that is suitable for reconstitution in an aqueous vehicle prior to injection. In one embodiment, a formulation suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, which may for example be formulated to be isotonic with the blood of the recipient.
The compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof may for example be formulated in compositions including those suitable for inhalation to the lung, by aerosol, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with a carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by bringing the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with a liquid carrier to form a solution or a suspension, or alternatively, bring the compound of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof into association with formulation components suitable for forming a solid, optionally a particulate product, and then, if warranted, shaping the product into a desired delivery form. Solid formulations of the present disclosure, when particulate, will typically comprise particles with sizes ranging from about 1 nanometer to about 500 microns. In general, for solid formulations intended for intravenous administration, particles will typically range from about 1 nm to about 10 microns in diameter. The composition may contain compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof that are nanoparticulate having a particulate diameter of below 1000 nm, for example, between 5 and 1000 nm, especially 5 and 500 nm, more especially 5 to 400 nm, such as 5 to 50 nm and especially between 5 and 20 nm. In one example, the composition contains compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof with a mean size of between 5 and 20nm. In some embodiments, the compound of Formula (I) is polydispersed in the composition, with PDI of between 1.01 and 1.8, especially between 1.01 and 1.5, and more especially between 1.01 and 1.2. In one example, the compounds of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof are monodispersed in the composition.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavouring agents.
The compositions of the present disclosure may also include polymeric excipients/additives or carriers, e.g., polyvinylpyrrolidones, derivatised celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2- hydroxypropyl-P-cyclodextrin and sulfobutylether-P-cyclodextrin), polyethylene glycols, and pectin. The compositions may further include diluents, buffers, citrate, tr6halose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the present disclosure are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in "Handbook of Pharmaceutical Excipients", Third Ed., Ed. A. H. Kibbe, Pharmaceutical Press, 2000.
Dosage
The amount of Mip inhibitor of Formula (I) or a pharmaceutically acceptable salt, solvate, or enantiomer thereof (i.e. active ingredient) that is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, including the type, species, age, weight, sex, and medical condition of the subject being treated, and the renal and hepatic function of the subject, and the particular condition, disorder or disease being treated, as well as its severity. An ordinary skilled physician or clinician can readily determine and prescribe the effective amount of the drug required to prevent or treat the condition, disorder or disease.
Dosages of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, when used for the indicated effects, may range between, for example, about 0.01 mg per kg of body weight per day (mg/kg/day) to about 1000 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is between about 0.01 and 1000, 0.1 and 500, 0.1 and 100, 1 and 50 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is between about 0.01 and 1000 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is between about 0.1 and 100 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is greater than about 0.01, 0.1, 1, 10, 20, 50, 75, 100, 500, 1000 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is greater than about 0.01 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is less than about 5000, 1000, 75, 50, 20, 10, 1, 0.1 mg/kg/day. In one example, the dosage of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is less than about 1000 mg/kg/day.
A Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may for example be administered as a single daily dose, or otherwise the total daily dosage may be administered in divided doses of two, three, or four times daily. In one example, the Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may be dosed less frequently than once per day, for example once per two days, three days, four days, five days, six days, or once per week.
If administered intravenously, an infusion of the compound over a period of time may be used, for example. Furthermore, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
Combinations
Whilst a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may be used as the sole active agent in a medicament, it is also possible for a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, to be used in combination with one or more further therapeutic agents. Accordingly, in one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with one or more further therapeutic agents. The present disclosure therefore also provides a combination of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and a further therapeutic agent. The present disclosure also provides a pharmaceutical composition comprising a combination of a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, a further therapeutic agent, and a pharmaceutically acceptable excipient. Such one or more further therapeutic agents may, for example, be antibacterial (e.g. an antibiotic), antimicrobial, antifungal and/or anti- viral agents.
In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with an antibacterial agent. An example of an antibacterial agent is an antibiotic. Accordingly, in one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with an antibiotic. In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with a broad-spectrum antibiotic. Examples of an antibiotic include doxycycline.
In another example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with an antifungal agent. In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with a an antifungal antibiotic. Examples of an antifungal antibiotic include rapamycin
In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with a vaccine.
In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with an immunomodulator. Examples of immunomodulators include, but are not limited to, immunosuppressants, cytokine inhibitors, antibodies, and immunostimulants. The immunomodulator may suppress inflammation and/or immune activation (e.g., cell proliferation and homing to tissues) of airways. In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with an anti-inflammatory agent. An example of an anti-inflammatory drug is a nonsteroidal anti-inflammatory drug (NSAID). In one example, a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, is used in combination with a nonsteroidal anti-inflammatory drug NSAID.
The Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, and the one or more further therapeutic/pharmaceutically active agents, may be administered simultaneously, subsequently or separately. For example, they may be administered as part of the same composition, or administered as separate compositions. The further therapeutic agent may be formulated for administration by any suitable route, for example orally, intravenously, subcutaneously, intramuscularly, intranasally, and/or by inhalation.
The further therapeutic agents, when employed in combination with a Mip inhibitor of Formula (I), or a pharmaceutically acceptable salt, solvate, or enantiomer thereof, may be used for example in those amounts indicated in the Physicians’ Desk Reference or as otherwise determined by one of ordinary skill in the art.
The present disclosure can also be described by reference to one or more of the following numbered paragraphs:
1. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,
Figure imgf000098_0001
wherein:
X is selected from O, S, and NR4; L1 and L2 are each independently absent or selected from the group consisting of -C1- 10alk yl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O- ,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH- , -C(=O)NH( C1-10alkyl)-, - S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and - OS(=O)2-, wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted;
R1 and R3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl;
R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted; and
R4 is selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl, each of which is optionally substituted.
2. The compound of paragraph 1, wherein:
X is selected from O, S, and NR4;
L1 and L2 are each independently absent or selected from the group consisting of -C1- 10alk yl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O- ,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH- , -C(=O)NH( C1-10alkyl)-, - S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and - OS(=O)2-; wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -NH-, -C(=O)NH-, -S-, and -S(=O)2-, and is optionally substituted with one or more R7;
R1 and R3 are each independently selected from a carbocyclyl or a heterocyclyl;
R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl; and
R4 is selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl; wherein each of R1, R2, R3 and R4 is optionally substituted with one or more R5; each R5 is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2- 10alk ynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10- membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, - C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, - C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered- carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R7; each R6 is independently selected from the group consisting of H, C1-6alkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-6alkyl-3-10-membered-carbocyclyl, and C1-6alkyl-3- 10-membered-heterocyclyl; wherein each C1-6alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7; each R7 is independently selected from the group consisting of halogen, -NO2, - N(R8)2, -CN, =O, -C(=O)OR8, -N(R8)C(=O)R8, -OR8, C1-6alkyl, and -OC1-6alkyl, and each R8 is independently selected from the group consisting of H and C1-6alkyl.
3. The compound of paragraph 1 or paragraph 2, wherein R1 and R3 are each independently selected from an optionally substituted aryl or an optionally substituted heteroaryl.
4. The compound of any one of paragraphs 1 to 3, wherein R1 and R3 are each independently selected from an optionally substituted 3-10-membered aryl or an optionally substituted 3-10-membered heteroaryl.
5. The compound of any one of paragraphs 1 to 4, wherein R1 is an optionally substituted phenyl.
6. The compound of any one of paragraphs 1 to 5, wherein R3 is an optionally substituted phenyl or an optionally substituted 6-membered heteroaryl. 7. The compound of any one of paragraphs 1 to 6, wherein R3 is an optionally substituted pyridyl.
8. The compound of paragraph 7, wherein the pyridyl is pyridine-N-oxide.
9. The compound of paragraph 7 or paragraph 8, wherein R3 is independently selected from the group consisting of:
Figure imgf000101_0001
each of which is optionally substituted.
10. The compound of any one of paragraphs 1 to 9, wherein each R1 and R3 are independently optionally substituted by one or more groups selected from H, halogen, C1- 10alk yl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7.
11. The compound of any one of paragraphs 1 to 10, wherein each R1 and R3 are independently optionally substituted by one or more groups selected from H, halogen, C1- 6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, orC1- 6alkylNH2.
12. The compound of any one of paragraphs 1 to 11, wherein R2 is selected from the group consisting of C1-10alkyl, cycloalkyl, C1-10alkylcycloalkyl, heteroalkyl, heterocyclyl, C1- 10alk ylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted. 13. The compound of any one of paragraphs 1 to 12, wherein R2 is selected from the group consisting of C1-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
14. The compound of any one of paragraphs 1 to 13, wherein R2 is selected from the group consisting of C1-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-6alkylheteroaryl, each of which is optionally substituted.
15. The compound of any one of paragraphs 1 to 14, wherein R2 is optionally substituted with one or more groups selected from H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, OC2-10alkenyl, C2-10alkynyl, OC2-10alkynyl, -NO2, -CN - N(R6)2, -OR6, -S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl, C1-10haloalkyl, C2- 10alk enyl, or C2-10alkynyl is optionally substituted with one or more R7.
16. The compound of any one of paragraphs 1 to 15, wherein R2 is optionally substituted with one or more groups selected from H, halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, OC1- 6haloalkyl, C2-6alkenyl, OC2-6alkenyl, C2-6alkynyl, OC2-6alkynyl, -NO2, -CN, -SO2H, -OH, - NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
17. The compound of any one of paragraphs 1 to 16, wherein L1 is - C1-10alkyl-.
18. The compound of any one of paragraphs 1 to 17, wherein L2 is - C1-10alkyl-, - C(=O)NH- or -C(=O)NH( C1-10alkyl)-.
19. The compound of any one of paragraphs 1 to 18, wherein L2 is -C(=O)NH(C1- 10alk yl)-.
20. The compound of any one of paragraphs 1 to 19, wherein X is O or NH.
21. The compound of any one of paragraphs 1 to 20, wherein the compound of Formula (I) is selected from the group consisting of:
Figure imgf000103_0002
or a pharmaceutically acceptable salt, solvate, stereoisomer or N-oxide thereof.
22. The compound of any one of paragraphs 1 to 21, wherein the compound of Formula
(I) is selected from the group consisting of:
Figure imgf000103_0001
Figure imgf000104_0001
or a pharmaceutically acceptable salt, solvate, or N-oxide thereof.
23. A pharmaceutical composition comprising a compound of any one of paragraphs 1 to 22, and a pharmaceutically acceptable excipient.
24. A method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein, the method comprising administering to the subject an effective amount of compound of any one of paragraphs 1 to 22, or a pharmaceutical composition of paragraph 23.
25. The method of paragraph 24, wherein the pathogen is a bacterial pathogen.
26. The method of paragraph 25, wherein the bacterial pathogen is a Gram-negative bacterium.
27. The method of any one of paragraphs 24 to 26, wherein the Gram- negative bacterium is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii. 28. The method of any one of paragraphs 24 to 27, wherein the pathogen is Coxiella burnetii, and the disease or condition is Q fever.
29. Use of a compound of any one of paragraphs 1 to 22 or a pharmaceutical composition of paragraph 23 in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
30. A compound of any one of paragraphs 1 to 22 or a pharmaceutical composition of paragraph 23, for use in treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
31. A method of treating and/or preventing a disease or condition mediated by a Gram- negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor, comprising administering to the subject a compound of any one of paragraphs 1 to 22, or a pharmaceutical composition of paragraph 23.
32. The method of paragraph 31, wherein the Gram-negative bacteria is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
33. The method of paragraph 31 or paragraph 32, wherein the Gram-negative bacteria is Coxiella burnetii, and the disease or condition is Q fever.
34. Use of a compound of any one of paragraphs 1 to 22 or a pharmaceutical composition of paragraph 23, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
35. A compound of any one of paragraphs 1 to 22 or a pharmaceutical composition of paragraph 23, for use in treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
The present application claims priority from AU2022900706 filed on 21 March 2022, the entire contents of which are incorporated herein by reference.
The present disclosure will now be described with reference to the following examples which illustrate some particular aspects of the present disclosure. However, it is to be understood that the particularity of the following description of the present disclosure is not to supersede the generality of the preceding description of the present disclosure.
EXAMPLES
General materials and methods
Unless otherwise stated, all reagents were obtained from commercial sources.
Overview
Inhibitors of the Mip protein were largely derived for the pipecolic acid moiety of the immunosuppressive rapamycin. Omitting the other parts of the rapamycin molecule result in Mip inhibitors of a wide concentration range, depending on the bacterial origin of the Mip protein (Juli et al., 2014 and Seufert et al., 2016.). Instead of more or less randomly varying the substituents, we looked closer to the different binding mode of the inhibitors of the BpMip using X-ray analysis and decided to combine both binding modes in order to arrest the Mip protein, which is undergoing great conformational changes upon the catalysis of the peptidyl-prolyl isomerisation. These newly designed compounds were characterized by an additional alkyl and alkylaryl substituent in the middle chain, connecting the lateral pyridine ring with the pipecolic moiety. Taking the different stereoisomers into account, highly active PPIase inhibitors of S,S configuration were identified, which have potent inhibitory properties against the macrophage infectivity potentiator (Mip) protein of several bacterial pathogens. Furthermore, using in vitro cell infection models we demonstrate that these compounds impair the virulence of pathogenic organisms such as Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae and Coxiella burnetii. The compounds reduce the survival of all these pathogens in macrophages. Interestingly the compounds demonstrate bacteriostatic properties against C. burnetii, suggesting that Mip is essential in this organism. These Mip inhibitors described here are therefore novel compounds that reduce the survival of bacterial pathogens in cells and demonstrate broad- spectrum efficacy.
General Procedures
Synthesis of literature compounds: Synthesis of (R/S)- l -(bcnzylsulfonyl)pipcridinc- 2-carboxylic acid (14) was performed according to Juli et al. (2014). Synthesis of structures 9 and 11 has been described by Seufert et al. (2016). 2-Hydroxy-4-phenylbutanoic acid, (S)-2- hydroxy-4-phenylbutanoic acid, (R)-2-hydroxy-4-phenylbutanoic acid, (S)-2-hydroxy-3- methylbutanoic acid, (S)-2-hydroxy-4-methylpentanoic acid, (2S,3S)-2-hydroxy-3- methylpentanoic acid, and (R)-2-hydroxy-4-methylpentanoic acid were prepared according to general procedure F. Allyl (S)-2-hydroxy-3-methylbutanoate, allyl (S)-2-hydroxy-4- methylpentanoate and allyl (2S,3S)-2-hydroxy-3-methylpentanoate, were prepared according to general procedure H.
General procedure A. Esterification and amidation was carried out according to Seufert et al. (2016). First, the corresponding carboxylic acid (1 equiv) was dissolved in dry DCM (30-40 mL per 1 mmol limiting reactant). In the case of esterifications, EDC·HCl (1.2 equiv) and DMAP (0.2-1 equiv) was added under ice cooling. In the case of amidations was added under ice cooling an auxiliary base (DIPEA, NMM or NEt3; 1-4 equiv) and either a combination of EDC·HCl (1.2 equiv) and HOBt (0.2-1 equiv) or HBTU alone (1.2- 2 equiv). After stirring for 15 min, the corresponding amine or alcohol (1-1.2 equiv) was added to the solution under ice cooling. The reaction was allowed to warm up to rt and stirred until completion, monitored by TLC. The mixture was then washed several times with saturated ammonium chloride solution, brine, and water (2 x 50 mL per step). After separation of the phases, the combined organic phases were dried over Na2SO4 and filtered. The organic solvent was removed in vacuo, and the crude product was purified by column chromatography or flash chromatography. General procedure B. According to Seufert et al. (2016) the Boc -protection group was cleaved at rt using an excess of TFA (2-10 mL) in dry DCM (10-20 mL). After completion, monitored by TLC (usually around 24 h), the reaction was neutralized with a sat. NaHCO3 solution and extracted with DCM (3 x 50 mL). After separation of the phases, the combined organic layers were dried over Na2SO4, filtered, and the solvent was removed in vacuo to give the crude product, which was used in the next step without further purification.
General procedure C. According to Seufert et al. (2016), to a solution of the piperidine-derivative (1 equiv) in dry DCM (20 mL per 1 mmol limiting reactant), A- methylmorpholine (NMM) (1.5-3 equiv) or DIPEA (1.5-3 equiv) and the corresponding sulfonyl chloride (phenylmethanesulfonyl chloride or (4-fluorophenyl)methanesulfonyl chloride) (1 equiv) were added and stirred at rt. After the reaction was completed according to TLC monitoring, the organic layer was washed consecutively with 2M HCl (50 mL), water (50 mL), and sat. NaHCO3 solution (50 mL). The phases were separated, and the organic phase was dried over Na2SO4. The desiccant was filtered off, the solvent was removed in vacuo, and the product was purified using column chromatography or flash chromatography.
General procedure D. According to a modified protocol by Franz et al. (2009), the allylester (1 equiv) was dissolved in dry THF (10 mL per 1 mmol limiting reactant) and cooled to 0 °C. Pd(PPh3)4 (0.1 equiv) and morpholine (1.05 equiv) were added, and the mixture was stirred at 0°C for 2 h. The solvent was removed in vacuo, and the residue was dissolved in DCM and washed with 1M HCl (3 x 50 mL). The organic phase was filtered using Celite® to remove excess palladium. The solvent was removed in vacuo, and the crude oily product was purified using column chromatography or flash chromatography.
General procedure E. According to a modified procedure by Patil et al. (2016), the pyridine -bearing compound (1 equiv) was diluted in EtOAc (5 mL per 100 μmol), and m-CPBA (2 equiv) was added slowly under ice cooling. The reaction mixture was immediately allowed to warm to rt and stirred for 2-18 h until completion, indicated by TLC. Subsequently, the reaction solution was directly loaded onto silica gel and purified by flash chromatography . General procedure F. For the synthesis of a-hydroxy acids, according to a modified protocol by Casalme et al. (2017), the amino acid (1 equiv) dissolved in IM H2SO4 (2 equiv) was cooled to 0 °C. A saturated aqueous solution of NaNO2 (6 equiv) was added dropwise over 5 h at 0 °C. The mixture was then allowed to warm to rt and stirred overnight. Subsequently, the reaction solution was diluted with water (50 mL) and extracted with Et2O (4 x 50 mL). The combined organic phases were washed with brine (50 mL), and the solvent was removed in vacuo. The crude product was used for the next step without further purification.
General Procedure G. According to Schwenk et al. (2016) the corresponding amino acid (1 equiv) was suspended in an excess of allyl alcohol (2 mL per 1 mmol amino acid) and cooled to 0 °C. Thionyl chloride (2-3 equiv) was added dropwise, and the mixture was slowly warmed to 60 °C. After 8 h of stirring at 60 °C, the reaction mixture was cooled to rt and stirred for another 12 h. After the addition of cooled Et2O (150 mL), the resulting precipitate was filtered through a glass filter frit, washed further with diethyl ether (2 x 150 mL), and the solvent was removed in vacuo. If necessary, the product was purified by flash chromatography.
General Procedure H. For allyl protection of the a -hydroxy acids, according to Yao et al. (2015), the corresponding a-hydroxy acid was dissolved in dry DMF (15 mL), and Na2CO3 (1.2 equiv) was added. Subsequently, allyl bromide (4 equiv) was carefully added at rt, and the reaction mixture was stirred until completion, monitored by TLC. Subsequently, the reaction mixture was diluted with water (50 mL) and extracted with Et2O (4 x 50 mL). The combined organic phases were washed with 3M HCl (70 mL) and saturated NaHCO3 solution (70 mL) and dried over Na2SO4. After filtration, the solvent was removed in vacuo. If necessary, the product was purified by flash chromatography.
Summary of Library Development and Chemistry
Scheme 1. The general structure of the developed library of Mip inhibitors bearing an additional side chain and prior art structures 9 and 11 comprising no side chain. All compounds synthesized, including their explicit side chains, are shown in Table 1.
Figure imgf000110_0001
By inserting an additional side chain, another stereo center is established. The first objective was to develop a library of pipecolic acid ester derivatives (X = O, Scheme 1) with a variety of additional side chains (R2) and different stereochemical orientations at the two stereocenters (*config. pip. and *config. side chain, Scheme 1) to test the respective influence on the binding affinity to BpMip as measured by FPA. The pyridine moiety (L2R3 = Py, Scheme 1) was kept constant in this series, as this subgroup provided promising properties in terms of solubility, cytotoxicity, and affinity towards BpMip. As a residue at the sulfonamide unit, a benzyl (Z = H) and a para-fluorobenzyl (Z = F, Scheme 1) moiety had turned out to be advantageous for PPIase-inhibition against BpMip. The amide position at the pyridine residue was changed compared to the compound 9 (Scheme 1) for reasons of synthetic feasibility so that the side chain could be introduced by amino acids. This allows for inexpensive reagents and easy preparation of the compounds by amidation and esterification reactions under mild conditions without affecting the stereo-information.
Synthesis of R/S Pipecolic Acid Ester Diastereomeric Mixtures
Scheme 2: Synthesis scheme for the R/S mixed pipecolic ester derivatives S/R,S'-10a, S/R,S-10b, S/R,S-10c, and S/R,S-10d.
Figure imgf000111_0001
Reagents and conditions: (a) NaNO2, H2SO4 (1 M), H2O, 0 °C rt; (b) K2CO3, allyl bromide, DMF, rt; (c) i) NaHCO3, DCM, rt ii) BnSO2Cl, NMM, DCM, 0 °C, iii) LiOH (1 M), DCM, rt; (d) EDC HCl, DMAP, DCM, 0 °C rt; (e) Pd(PPh3)4, morpholine, THF, 0°C→ rt; (f) 3 -picolylamine, EDC HCl, HOBt, DCM, 0 °C rt.
To test different aliphatic and aromatic substituents as side chains, the diastereomeric mixtures S/R,S-10a-d were synthesized (Scheme 2). The substances were also intended to be used as a reference for stereochemically pure compounds to test whether the preference for the S-configuration at the C2 position of the pipecolic acid observed by Seufert et al (2016) also applies in the presence of an additional side chain. In addition, they should be used to see if the diastereomers can be analytically distinguished from each other. L-phenylalanine, L-valine, L-leucine, and L-isoleucine, respectively, were reacted with NaNO2/H2SO4 via diazonium salt in a Sandmeyer-type reaction to convert the amino function to a hydroxyl group. The carboxylic group of the a-hydroxy acids 12a-d was then protected using allyl bromide and K2CO3 to give 13a-d. Subsequently, the ester was formed under Steglich conditions by linking the amides and racemic (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylate (14) with DMAP and NMM. The allyl protecting group of 15a-d was cleaved under palladium catalysis with Pd(PPh3)4 and morpholine, resulting in 16a-d. In a final step, 3 -picolylamine was attached to the carboxylic acid by amide coupling using EDC·HCl and HOBt to give R/S,S-10a-d. For the preparation of racemic (R/S)- 1-(benzylsulfonyl)piperidine-2-carboxylate (14) a basic hydrolysis was chosen, which would lead to racemization in the case of enantiomerically pure reactants. This synthetic step was omitted in subsequent synthetic routes to obtain the distinct stereo isomers. In addition, care had to be taken during synthesis and workup not to use too basic conditions once the pipecolic acid moiety was introduced. To ensure the correct stereo chemistry, pure isomers were used as starting material and synthetic procedures were chosen, which are known for the retention of stereochemistry. When the pure stereo isomers were synthesized, as from the introduction of the second stereo center, differentiation of diastereomers via 1H NMR became possible and was carried out. The absence of diastereomers in the prepared pure stereoisomers in NMR, as evidenced by a single data set, also suggests the absence of enantiomers due to potential racemization.
Synthesis of the Pipecolic Acid Ester Derivatives with a Specific Stereo-Information at the Pipecolic Acid C2 Position
Scheme 3. Synthesis scheme of the pipecolic ester derivatives, which allows the preparation of the explicit stereo isomers.
Figure imgf000112_0001
Reagents and conditions: (a) NaNO2, H2SO4 (2M), 0 C; (b) i) KOH, MeOH, 40°C, ii) 10% Pd/C, H2 (15 bar), MW 400W, isopropanol, iii) EDC·HCl, HOBt, 3 -picolylamine, DCM, 0 °C → rt; (c) NaBH4, MeOH, 0°C; (d) 3-, EDC HCl, HOBt, DCM, 0 °C → rt; (e) EDC·HCl, DMAP, DCM, 0°C rt; (f) i) trifluoroacetic acid (TFA), DCM, 0°C → rt, 2h, ii) BnSO2Cl, NMM, DCM, rt.
The above synthetic pathway was developed to obtain pipecolic ester derivatives with a defined configuration at the C2 position of the piperidine ring and the possibility of a defined configuration at the side chain when the respective amino acid was used (Scheme 3) Initially, the enantiomers of homophenylalanine and leucine were converted into a-hydroxy acids 12d and 12e, as mentioned before. For amide formation, the a-hydroxy acids were then converted with 3 -picolylamine, EDC·HCl, and HOBt to give 17. Deviating from this, for the preparation of S,S/R-10f, 18 was obtained by aldol condensation, subsequent hydrogenation of the double bond to give 19, amide coupling with 3 -picolylamine, and a non-stereoselective reduction of the keto group at the a-position of the amide to the alcohol with NaBH4. Next, the ester was formed under Steglich-conditions linking the amide intermediates with the Boc- protected (S)- or (R) -pipecolic acid to achieve the respective stereo isomers 20. Finally, cleavage of the Boc-group with TFA and an SN2 reaction of the piperidine-nitrogen with phenylmethanesulfonyl chloride lead to the inhibitors S,S-10a, S,R-10a, R,S-10a, R,R-10a, S,S/R-10e, R,S/R-10e, S,S-10e, S,R-10e, S,S/R-10f, S,S-10d, and S,R-10d.
Synthesis of the (S)-Pipecolic Amide Stereochemically Pure Derivatives
Scheme 4: Synthesis scheme for the (S)-pipecolic acid amide derivatives as stereochemically pure compounds.
Figure imgf000113_0001
Reagents and conditions: (a) allyl alcohol, SOCh, 0 °C → 60 °C; (b) N-Boc-(S)- pipecolic acid, EDC HCl + HOBt or HBTU, DIPEA, DCM, 0 °C → rt; (c) i) TFA, DCM, 0 °C → rt, ii) BnSCECl or 4-F-BnSO2Cl, NMM, DCM, 0°C rt; (d) i) Pd(PPh3)4, morpholine, THF, 0°C rt, ii) 3 -picolylamine, EDC HCl + HOBt or HBTU, DIPEA, DCM, 0 °C → rt; (e) m-CPBA, EtOAc, rt.
Due to the high chemical and metabolic stability of amides (X = NH, Scheme 1) the stereochemically pure amides S,S-21a, S,R-21a, S,S-21e, S,R-21e, S,S-21g, and S,S-21d were also prepared via a synthetic route that allows late variation of the pyridine moiety (cf. Scheme 4). The N-oxidcs S/R,S-22a, S,S-22g, and S,S-22d were prepared by oxidation of the respective inhibitor with meto-chloroperoxybenzoic acid (m-CPBA) in EtOAc. The S- configuration at the pipecolic acid demonstrated good properties for compounds with a side chain. Thus, only the configuration at the side chain was subsequently varied. The amino acids were O-protected by using thionyl chloride and allyl alcohol to give the allyl esters 23. After amidation with Boc-protected S-pipccolic acid, the Boc-protection group of the amides 24 was cleaved with TFA. The N- sulfonamides 25 were formed using the respective sulfonyl chloride and NMM, and the allyl function was again cleaved under palladium catalysis. The stereochemically pure amides S,S-21a, S,R-21a, S,S-21e, S,R-21e, S,S-21d, and S,S-21g were obtained by amidation with 3 -picolylamine.
Synthesis of S-Pipecolic Amide Derivatives via an Alternative Pathway for Late
Variation of the Sulfonamide Moiety
Scheme 5: Synthesis scheme of an alternative pathway for stereochemically pure S- pipecolic amide derivatives 5, 5-2111, S,S-21i, and S,S-28i that allows a late variation of the sulfonamide moiety.
Figure imgf000114_0001
Reagents and conditions: (a) 3 -picolylamine, HBTU, DIPEA, DCM, 0°C rt; (b) i) TFA, rt; ii) A-Boc-(S)-pipecolic acid, HBTU, DIPEA, DCM, 0°C → rt; (c) i) TFA, rt; ii) BnSO2Cl / 4-F-BnSO2Cl, NMM or DIPEA, DCM, 0°C rt.
To allow a late variation of the sulfonamide moiety, an alternative synthesis pathway was established for the stereochemically pure pipecolic amide derivatives (Scheme 5). Replacing the allyl protecting group with the Boc group has the additional advantage of being toxicologically safe, which would also be important in a prospective scale-up. The Boc- protected amino acids (S)-A-Boc-4-(prop-2-yn-1-yloxy)phenylalanine and (S)-A-Boc-4- fluorophenylglycine, respectively, were amide coupled to 3 -picolylamine with HBTU and DIPEA to give the amides S-26h and S-26i. After cleavage of the Boc -protection group with TFA, the compounds were again amide coupled to the Boc-protected S-pipccolic acid with HBTU and DIPEA to give S,S-27h and S,S-27i. After cleaving the Boc -protection group again with TFA, the A- sulfonamides were formed with the respective sulfonyl chloride derivative to give S,S-21h, S,S-21i, and S,S-28i.
Example 1: Synthesis of the R/S- Pipecolic Ester Derivatives
Allyl (S)-2-hydroxy-3-phenylpropanoate (S-13a)
The allyl protection of (S)-2-hydroxy-3-phcnylpropanoic acid (800 mg, 4.81 mmol) was carried out following general procedure H using allyl bromide (1.24 mL, 14.44 mmol) and sodium carbonate (560 mg, 5.29 mmol) in dry DMF (15 mL). After workup according to the general procedure, the crude oily product was purified by column chromatography (SiO2, PE/EtOAc = 5:1). Compound S-13a was obtained as a colorless oil (752 mg, 73%). Rf: 0.61 (DCSIL; PE/EtOAc = 5:1; KMnO4). IR (ATR), [cm-1]: 3451, 2935, 1725, 1454, 1176, 1090,
Figure imgf000115_0001
990, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.24-7.14 (m, 5H), 5.88-5.78 (m, 1H), 5.29- 5.20 (ddq, J = 17.2, 10.0, 1.2, 2H), 4.59 (dt, J = 5.9, 1.2, 2H), 4.43-4.39 (m, 1H), 3.07 (dd, J = 13.9, 4.5, 1H), 2.91 (dd, J = 13.9, 6.8, 1H), 2.65 (d, J = 6.2, 1H).13C NMR (CDCl3, δ [ppm]): 173.8, 135.9, 130.9, 129.5 (2C), 128.8 (2C), 127.4, 119.2, 71.2, 66.4, 40.3.
(S)-1-(Allyloxy)-3-methyl-1-oxobutan-2-yl (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylate (R/S,S-15b)
R/S,S-15b was synthesized according to general procedure A using 160 mg (1.01 mmol) of allyl (S)-2-hydroxy-3-methylbutanoate, (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylic acid (14, 286 mg, 1.01 mmol), EDC·HCl (230 mg, 1.21 mmol), and DMAP (70 mg, 0.51 mmol) in dry DCM (40 mL). The crude oily product was purified by column chromatography (SiO2, PE/EtOAc = 10:1). Compound R/S,S-15b was obtained as a yellow oil (1.94 g, 4.58 mmol, 81%). Rf: 0.4 (DCSIL; PE/EtOAc = 10:1; KMnO4), IR (ATR),
Figure imgf000115_0002
[cm-1]: 2952, 2871, 1737, 1455, 1337, 1188, 1125, 1072, 1058, 966, 697. Diastereomeric ratio: 45:55. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.46-7.43 (m, 2H), 7.38-7.33 (m, 3H), 5.96-5.85 (m, 1H), 5.38-5.30 (m, 1H), 5.28-5.23 (m, 1H), 4.97 (d, J = 4.1, 1H), 4.71-4.61 (m, 3H), 4.27 (s, 2H), 3.48-3.45 (m, 1H), 3.30-3.15 (m, 1H), 2.34-2.25 (m, 2H), 1.72-1.30 (m, 5H), 1.06-0.98 (m, 6H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.46-7.43 (m, 2H), 7.38- 7.33 (m, 3H), 5.96-5.85 (m, 1H), 5.38-5.30 (m, 1H), 5.28-5.23 (m, 1H), 4.97 (d, J = 4.1, 1H), 4.71-4.61 (m, 2H), 4.57-4.55 (m, 1H), 4.24-4.22 (m, 2H), 3.48-3.45 (m, 1H), 3.30- 3.15 (m, 1H), 2.34-2.25 (m, 1H), 2.18-2.14 (m, 1H), 1.72-1.30 (m, 5H), 1.06-0.98 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 171.6, 169.0, 131.5, 131.1 (2C), 129.5, 128.7 (2C), 128.6, 119.2, 77.8, 65.9, 59.1, 56.1, 43.6, 30.3, 28.1, 25.2, 20.2, 18.9, 17.2. 13C NMR (CDCl3, δ [ppm]): 171.4, 169.0, 131.5, 131.1 (2C), 129.4, 128.7 (2C), 128.5, 119.2, 77.5, 65.9, 58.8, 55.8, 43.5, 30.3, 27.8, 25.0, 20.2, 18.9, 17.0.
(S)-1-(Allyloxy)-4-methyl-1-oxopentan-2-yl (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylate ( R/S,S-15d)
R/S,S-15d was synthesized according to general procedure A using allyl (S)-2- hydroxy-4-methylpentanoate (940 mg, 5.46 mmol), ( R/S)-1-(benzylsulfonyl)piperidine-2- carboxylic acid (1.50 g, 5.46 mmol), EDC·HCl (1.26 g, 6.55 mmol), and DMAP (340 mg, 2.73 mmol) in dry DCM (200 mL). R/S,S-15d was obtained as a yellow oil (1.94 g, 4.42 mmol, 81%), Rf: 0.35 (DCSIL; PE/EtOAc = 10:1; KMnO4), IR (ATR), [cm-1]: 2953,
Figure imgf000116_0001
2869, 1738, 1455, 1337, 1187, 1125, 1072, 1057, 966, 697. Diastereomeric ratio: 47:53. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.46-7.44 (m, 2H), 7.37-7.34 (m, 3H), 5.95-5.85 (m, 1H), 5.37-5.30 (m, 1H), 5.28-5.23 (m, 1H), 5.14-5.10 (m, 1H), 4.69-4.66 (m, 3H), 4.27 (s, 2H), 3.48-3.45 (m, 1H), 3.28-3.15 (m, 1H), 2.30-2.26 (m, 1H), 1.91-1.30 (m, 8H), 0.98-0.93 (m, 6H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.46-7.44 (m, 2H), 7.37-7.34 (m, 3H), 5.95-5.85 (m, 1H), 5.37-5.30 (m, 1H), 5.28-5.23 (m, 1H), 5.14-5.10 (m, 1H), 4.69-4.66 (m, 3H), 4.24- 4.22 (m, 2H), 3.48-3.45 (m, 1H), 3.28-3.15 (m, 1H), 2.19-2.13 (m, 1H), 1.91-1.30 (m, 8H), 0.98-0.93 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 171.4, 170.0, 131.5, 131.1 (2C), 129.5, 128.7 (2C), 128.6, 119.2, 72.0, 66.1, 58.9, 55.9, 43.6, 39.8, 28.1, 25.2, 24.8, 23.2, 21.8, 20.3. 13C NMR (CDCl3, δ [ppm]): 171.4, 170.0, 131.5, 131.1 (2C), 129.4, 128.6 (2C), 128.5, 119.2, 71.8, 66.1, 58.8, 55.8, 43.4, 39.8, 27.8, 25.2, 24.6, 23.2, 21.4, 20.2. (2S,3S)-1-(Allyloxy)-3-methyl-1-oxopentan-2-yl (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylate (R/S, S- 15c) R/S,S-15c was synthesized following general procedure A using 600 mg (3.52 mmol) of allyl (2S,3S)-2-hydroxy-3-methylpentanoate, (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylic acid (1.00 g, 3.52 mmol), EDC·HCl (810 mg, 4.22 mmol), and DMAP (215 mg, 1.76 mmol) in dry DCM (140 mL). Compound R/S,S-15c was obtained as yellow oil (1.49 g, 3.41 mmol, 97%). Rf: 0.45 (DCSIL; PE/EtOAc = 10:1; KMnO4), IR (ATR), [cm-1]: 2953,
Figure imgf000117_0001
2871, 1737, 1455, 1337, 1188, 1171, 1073, 1057, 966, 697. Diastereomeric ratio: 50:50. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.46-7.43 (m, 2H), 7.37-7.34 (m, 3H), 5.94-5.86 (m, 1H), 5.37-5.31 (m, 1H), 5.28-5.23 (m, 1H), 5.02 (d, J = 4.2, 1H), 4.66-4.62 (m, 3H), 4.27 (s, 2H), 3.48-3.45 (m, 1H), 3.30-3.15 (m, 1H), 2.29-2.26 (m, 1H), 2.08-2.01 (m, 1H), 1.72-1.25 (m, 7H), 1.03-0.99 (dd, J = 10.8, 6.9, 3H), 0.95-0.90 (td, J = 7.4, 3.9, 3H). 1H NMR (CDCl3, 6 [ppm], J [Hz]): 7.46-7.43 (m, 2H), 7.37-7.34 (m, 3H), 5.94-5.86 (m, 1H), 5.37-5.31 (m, 1H), 5.28-5.23 (m, 1H), 4.98 (d, J = 4.2, 1H), 4.66-4.62 (m, 2H), 4.57-4.56 (m, 1H), 4.24- 4.22 (m, 2H), 3.48-3.45 (m, 1H), 3.30-3.15 (m, 1H), 2.19-2.17 (m, 1H), 2.08-2.01 (m, 1H), 1.72-1.25 (m, 7H), 1.03-0.99 (dd, J = 10.8, 6.9, 3H), 0.95-0.90 (td, J = 7.4, 3.9, 3H). 13C NMR (CDCl3, δ [ppm]): 171.5, 169.1, 131.5, 131.1 (2C), 129.5, 128.7 (2C), 128.6, 119.2, 77.3, 66.0, 59.1, 56.1, 43.6, 36.8, 28.1, 25.2, 24.7, 20.2, 15.6, 11.7. 13C NMR (CDCl3, 6 [ppm]): 171.4, 169.0, 131.5, 131.0 (2C), 129.4, 128.6 (2C), 128.6, 119.2, 77.0, 66.0, 58.9, 55.8, 43.5, 36.8, 27.9, 25.1, 24.5, 20.2, 15.6, 11.7.
(S)-1-(Allyloxy)-1-oxo-3-phenylpropan-2-yl (R/S)-1-(benzylsulfonyl)piperidine-2- carboxylate (R/S, S- 15a)
R/S,S-15a was synthesized following general procedure A using 220 mg (1.07 mmol) of S-13a, (R/S)-1-(benzylsulfonyl)piperidine-2-carboxylic acid (302 mg, 1.07 mmol), EDC·HCl (245 mg, 1.28 mmol), and DMAP (66 mg, 0.54 mmol) in dry DCM (40 mL). The crude oily product was purified by column chromatography (SiO2, PE/EtOAc = 10:1). Compound R/S,S-15a was obtained as yellow oil (430 mg, 0.91 mmol, 85%). Rf: 0.5 (DCSIL; PE/EtOAc = 10:1; KMnO4). IR (ATR), [cm-1]: 2963, 2869, 1742, 1455, 1336, 1186, 1175,
Figure imgf000117_0002
1073, 1056, 966, 697. Diastereomeric ratio: 55:45. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.43- 7.20 (m, 10H), 5.93-5.80 (m, 1H), 5.37-5.24 (m, 3H), 4.66-4.62 (m, 3H), 4.66-4.62 (m, 3H), 4.17-4.16 (m, 2H), 3.44-3.41 (m, 1H), 3.32-3.11 (m, 2H), 3.08-3.01 (m, 1H), 2.25-1.97 (m, 1H), 1.74-1.30 (m, 3H), 0.90-0.86 (m, 2H). 13C NMR (CDCl3, 5f [ppm]): 170.9, 168.8, 135.5, 131.4, 131.2 (2C), 129.4 (2C), 129.3), 128.7 (2C), 128.6 (2C), 128.5, 127.4, 119.2, 73.6, 66.4, 58.7, 55.7, 43.4, 37.4, 28.1, 25.2, 20.2. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.43- 7.20 (m, 10H), 5.93-5.80 (m, 1H), 5.37-5.24 (m, 3H), 4.66-4.62 (m, 3H), 4.66-4.62 (m, 3H), 4.17-4.16 (m, 2H), 3.44-3.41 (m, 1H), 3.32-3.11 (m, 2H), 3.08-3.01 (m, 1H), 2.25-1.97 (m, 1H), 1.74-1.30 (m, 3H), 0.90-0.86 (m, 2H). 13C NMR (CDCl3, δ [ppm]): 170.9, 168.8, 135.5, 131.4, 131.2 (2C), 129.4 (2C), 129.3), 128.7 (2C), 128.6 (2C), 128.5, 127.4, 119.2, 73.6, 66.4, 58.7, 55.7, 43.4, 37.4, 28.1, 25.2, 20.2.
(S)-3-Methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)butan-2-yl (R/S)-1- (benzylsulfonyl)piperidine-2-carboxylate (R/S,S-10b)
In a first step, R/S,S-15b (300 mg, 0.71 mmol) was deprotected according to general procedure D using 60 mg of Pd(PPh3)4 (0.05 mmol) and 70 μL of morpholine (0.75 mmol) in dry THF (10 mL). The intermediate product was further reacted with 3 -picolylamine (80 μL, 0.85 mmol), EDC·HCl (164 mg, 0.85 mmol), and HOBt (48 mg, 0.37 mmol) in dry DCM (30 mL) according to general procedure A. Purification using column chromatography (SiO2, PE/EtOAc = 3:1) afforded compound R/S,S-10b as a colorless oil (256 mg, 0.54 mmol, 76%), Rf: 0.5 (DCSIL; EtOAc/PE = 3:2). IR (ATR), [cm-1]: 3360, 2965, 1739, 1672, 1536, 1455,
Figure imgf000118_0001
1334, 1199, 1125, 1058, 951, 697. Diastereomeric ratio: 47:53. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.59-8.57 (m, 1H), 8.52-8.50 (m, 1H), 7.76-7.69 (m, 2H), 7.41-7.35 (m, 5H), 7.30- 7.26 (m, 1H), 5.29 (d, J = 3.0, 1H), 4.47-4.43 (m, 2H), 4.28-4.26 (m, 1H), 4.22 (s, 2H), 3.40-3.35 (m, 1H), 3.13-3.06 (m, 1H), 2.43-2.31 (m, 1H), 2.19-2.17 (m, 1H), 1.64-1.25 (m, 5H), 0.97-0.92 (m, 6H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.59-8.57 (m, 1H), 8.52-8.50 (m, 1H), 7.76-7.69 (m, 2H), 7.41-7.35 (m, 5H), 7.30-7.26 (m, 1H), 5.16 (d, J = 3.0, 1H), 4.47-4.43 (m, 2H), 4.22 (s, 2H), 4.11-4.09 (m, 1H), 3.23-3.20 (m, 1H), 3.02-2.95 (m, 1H), 2.43-2.31 (m, 1H), 2.03 (m, 1H), 1.64-1.25 (m, 5H), 0.97-0.92 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 170.4, 169.4, 148.9, 148.1, 136.8, 134.5, 130.9 (2C), 129.2, 129.0 (2C), 128.9, 123.9, 79.4, 59.6, 57.2, 44.2, 40.6, 31.0, 27.0, 24.6, 20.3, 19.4, 16.9. 13C NMR (CDCl3, 6 [ppm]): 170.2, 169.2, 148.8, 147.8, 136.7, 134.5, 130.9 (2C), 129.2, 129.0 (2C), 128.8, 123.8, 79.1, 59.0, 56.1, 44.1, 40.6, 30.7, 27.0, 24.6, 19.7, 18.7, 16.6. LC/MS (m/z) 474.35 [M+H]+. Purity (HPLC, both diastereomers in total): 99.1%, tR = 7.84 min, 8.12 min. HRMS (m/z) C24H31N3O5S, [M+H]+, calculated 474.20572, found 474.20675, error 2.2 ppm. (2S,3S)-3-Methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl (benzylsulfonyl)piperidine-2-carboxylate (R/S,S-10c)
In a first step, R/S,S-15c (720 mg, 1.65 mmol) was deprotected using 150 mg of Pd(Ph3)4 (150 mg, 0.08 mmol) and morpholine (150 μL, 17.3 mmol) in dry THF (20 mL) following general procedure D. The intermediate product (656 mg, 1.65 mmol) was further reacted with 3 -picolylamine (208 μL, 1.98 mmol), EDC·HCl (382 mg, 1.98 mmol), and HOBt (116 mg, 0.42 mmol) in dry DCM (70 mL) according to general procedure A. Compound R/S,S-10c was obtained as a colorless oil (450 mg, 0.92 mmol, 56%), Rf: 0.5 (DCSIL; EtOAc/PE = 3:2), IR (ATR), [cm-1]: 3359, 2964, 2937, 2877, 1738, 1671, 1536, 1455, 1334, 1175, 1125, 1059, 917, 795, 698. Diastereomeric ratio: 51:49. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.55 (m, 1H), 8.51-8.49 (m, 1H), 7.73-7.65 (m, 2H), 7.40-7.33 (m, 5H), 7.26-7.23 (m, 1H), 5.32 (d, 7 = 3.1, 1H), 4.44 (dd, J = 9.9, 6.0, 2H), 4.26-4.24 (m, 1H), 4.20 (s, 2H), 3.38-3.35 (m, 1H), 3.09-2.93 (m, 1H), 2.17-1.99 (m, 2H), 1.63-1.24 (m, 7H), 0.94-0.87 (m, 6H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.55 (m, 1H), 8.51-8.49 (m, 1H), 7.73-7.65 (m, 2H), 7.40-7.33 (m, 5H), 7.26-7.23 (m, 1H), 5.21 (d, J = 3.1, 1H), 4.44 (dd, J = 9.9, 6.0, 2H), 4.20 (s, 2H), 4.10-4.08 (m, 1H), 3.22-3.19 (m, 1H), 3.09-2.93 (m, 1H), 2.17-1.99 (m, 2H), 1.63-1.24 (m, 7H), 0.94-0.87 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 170.3, 169.2, 149.2, 148.4, 136.4, 134.3, 130.9 (2C), 129.2, 128.8 (2C), 128.7, 123.7, 79.2, 59.6, 57.1, 44.4, 40.7, 37.5, 27.1, 24.6, 24.4, 20.3, 15.8, 12.0. 13C NMR (CDCl3, δ [ppm]): 170.1, 169.0, 149.2, 148.2, 136.3, 134.3, 130.9 (2C), 129.0, 128.8 (2C), 128.7, 123.6, 78.8, 59.0, 56.2, 44.1, 40.6, 37.5, 27.0, 24.6, 24.4, 19.7, 15.1, 11.7. LC/MS (m/z) 488.40 [M+H]+. Purity (HPLC, both diastereomers in total): 97.3%, tR = 8.17 min, 8.47 min. HRMS (m/z) C25H33N3O5S, [M+H]+, calculated 488.22137, found 488.22225, error 1.8 ppm.
(S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl (R/S)-1- (benzylsulfonyl)piperidine-2-carboxylate (R/S, S -10d )
In a first step, R/S,S-15d (900 mg, 2.05 mmol) was deprotected using 160 mg of Pd(PPh3)4 (0.14 mmol) and morpholine (200 μL, 2.15 mmol) in dry THF (20 mL) following general procedure D. The intermediate product (819 mg, 2.05 mmol) was then reacted with 3- picolylamine (273 μL, 2.48 mmol), EDC·HCl (473 mg, 2.48 mmol), and HOBt (138 mg, 1.03 mmol) in dry DCM (80 mL) according to general procedure A. Compound R/S,S-1Od was obtained as a colorless oil (721 mg, 1.48 mmol, 72%), Rf: 0.5 (DCSIL; EtOAc/PE = 3:2), IR (ATR), [cm-1]: 3357, 2952, 2870, 1738, 1672, 1536, 1455, 1334, 1174, 1125, 1057, 921, 697. Diastereomeric ratio: 60:40. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.55 (m, 1H), 8.51-8.49 (m, 1H), 7.69-7.65 (m, 2H), 7.40-7.33 (m, 5H), 7.28-7.24 (m, 1H), 5.38-5.34 (m, 1H), 4.45-4.41 (m, 2H), 4.21-4.20 (m, 3H), 3.37-3.34 (m, 1H), 3.06-2.99 (m, 1H), 2.16 (m, 1H), 1.80-1.74 (m, 2H), 1.67-1.24 (m, 5H), 1.06-1.02 (m, 1H), 0.92-0.90 (m, 6H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.55 (m, 1H), 8.51-8.49 (m, 1H), 7.69-7.65 (m, 2H), 7.40-
7.33 (m, 5H), 7.28-7.24 (m, 1H), 5.27-5.24 (m, 1H), 4.45-4.41 (m, 2H), 4.21-4.20 (m, 2H), 4.11-4.09 (m, 1H), 3.21-3.19 (m, 1H), 3.06-2.99 (m, 1H), 1.99-1.97 (m, 1H), 1.80-1.74 (m, 2H), 1.67-1.24 (m, 5H), 1.06-1.02 (m, 1H), 0.92-0.90 (m, 6H). 13C NMR (CDCl3, δ [ppm]):
170.5, 170.3, 149.0, 148.2, 136.5, 134.3, 130.9 (2C), 129.2, 129.0 (2C), 128.9, 123.8, 74.0,
59.5, 56.8, 44.1, 41.1, 40.7, 27.1, 24.9, 24.7, 23.1, 21.7, 20.2. 13C NMR (CDCl3, δ [ppm]): 170.3, 170.2, 149.0, 148.0, 136.4, 134.3, 130.9 (2C), 129.0, 128.9 (2C), 128.8, 123.7, 73.9, 59.0, 56.1, 44.1, 40.9, 40.5, 27.1, 24.7, 24.6, 23.1, 21.5, 19.7. LC/MS (m/z) 488.45 [M+H]+. Purity (HPLC, both diastereomers in total): 98.4%, tR = 8.26 min, 8.47 min. HRMS (m/z) C25H33N3O5S, [M+H]+, calculated 488.22137, found 488.22150, error 0.3 ppm.
(S)-1-Oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl-(R/S)-1- (benzylsulfonyl)piperidine-2-carboxylate (R/S, S- 10a )
The cleavage of the allyl ester of R/S,S-15a (400 mg, 0.85 mmol) was carried out following general procedure D using 50 mg of Pd(PPh3)4 (0.04 mmol) and morpholine (100 μL, 0.89 mmol) in dry THF (10 mL). For the subsequent amidation of the intermediate product (337 mg, 0.85 mmol) general procedure A was applied using 100 μL of 3- picolylamine (1.02 mmol), EDC·HCl (230 mg, 1.02 mmol), and HOBt (58 mg, 0.43 mmol) in dry DCM (40 mL). Purification using column chromatography (SiO2, PE/EtOAc = 3:1) afforded compound R/S,S-1Oa as a colorless oil (542 mg, 1.11 mmol, 56%), Rf: 0.5 (DCSIL; EtOAc/PE = 3:2). IR (ATR), [cm-1]: 3361, 2941, 1737, 1697, 1539, 1455, 1315, 1188,
Figure imgf000120_0001
1122, 1056, 932, 695, 610. Diastereomeric ratio: 58:42. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.52 (m, 2H), 7.77-7.58 (m, 2H), 7.38-7.17 (m, 11H), 5.67 (dd, J = 9.2, 3.9, 1H), 4.50-
4.33 (m, 2H), 4.18-4.16 (m, 3H), 3.48-3.43 (dd, J = 14.7, 3.9, 1H), 3.20-3.00 (m, 3H), 1.97- 1.93 (m, 1H), 1.51-1.06 (m, 5H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.57-8.52 (m, 2H), 7.77-7.58 (m, 2H), 7.38-7.17 (m, 10H), 7.26-7.23 (m, 1H), 5.50-5.47 (dd, J = 9.2, 3.9, 1H), 4.50-4.33 (m, 2H), 4.18-4.16 (m, 2H), 3.99-3.96 (m, 1H), 3.36-3.32 (dd, J = 13.0, 3.0, 1H), 3.20-3.00 (m, 2H), 2.60 (td, J = 14.7, 3.9, 1H), 1.70-1.66 (m, 1H), 1.51-1.06 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 170.3, 169.2, 148.5, 147.5, 137.2, 136.0, 134.5, 130.9 (2C), 129.6 (2C), 129.0, 128.9 (2C), 128.8, 128.5 (2C), 127.1, 124.0, 75.4, 59.3, 56.8, 44.0, 40.7, 38.0, 27.1, 24.4, 19.5. 13C NMR (CDCl3, δ [ppm]): 170.1, 169.0, 148.3, 147.5, 137.0, 135.8, 134.5, 130.7 (2C), 129.4 (2C), 129.0, 128.9 (2C), 128.7, 128.5 (2C), 127.1, 123.8, 75.1, 59.0, 56.0, 43.7, 40.7, 38.0, 27.0, 24.6, 19.4. LC/MS (m/z) 522.10 [M+H]+. Purity (HPLC, both diastereomers in total): 99.0%, tR = 8.38 min, 8.55 min. HRMS (m/z) C28H31N3O5S, [M+H]+, calculated 522.20572, found 522.20598, error 0.5 ppm.
Example 2: Synthesis of the Specific Pipecolic Ester Stereo Isomers (R)-2-Hydroxy-4-methyl-N-(pyridin-3-yhnethyl)pentanamide ( A’- 17(1 )
Compound R-17d was prepared according to general procedure A using 1.89 g (R)-2- hydroxy-4-methoxypentanoic acid (14.3 mmol), HOBt (1.93 g, 14.3 mmol), EDC·HCl (2.74 g, 15.2 mmol), and 3 -picolylamine (2.20 mL, 21.5 mmol) in dry DCM (200 mL). The crude oily product was purified by column chromatography (SiO2, DCM/MeOH 9:1 + 0.5% FA) to obtain R-17d as a colorless solid (1.08 g, 4.86 mmol, 34%), Rf: 0.79 (DCSIL; EtOAc/MeOH = 5:1), mp: 47 - 48 °C, IR (ATR), [cm-1]: 3326, 2955, 1639, 1521, 1421,
Figure imgf000121_0001
1281, 1171, 1044, 789, 711. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.53 (br s, 1H), 8.51 (d, J = 4.0, 1H), 7.72-7.68 (m, 1H), 7.32 (dd, J = 7.8, 4.5, 1H), 7.12 (br s, 1H), 4.50 (dd, J = 11.0, 6.2, 2H), 4.25-4.18 (m, 1H), 1.93-1.77 (m, 1H), 1.74-1.68 (m, 1H), 1.60-1.51 (m, 1H), 1.00-0.92 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 174.8, 149.1, 148.9, 135.9, 134.2, 123.8, 71.0, 43.9, 40.6, 24.7, 21.5 (2C).
(S)-2-Hydroxy-4-methyl-N-(pyridin-3-yhnethyl)pentanamide (S- 17(1 )
Compound 5-17d was prepared according to general procedure A using 1.37 g of (S)- 2-hydroxy-4-methoxypentanoic acid (10.4 mmol), HOBt (1.40 g, 10.4 mmol), EDC HCl (1.98 g, 10.4 mmol), and 3-(aminomethyl)pyridine (1.16 mL, 11.4 mmol) in dry DCM (45 mL). After stirring at rt for 21 h and workup according to the general procedure, the crude oily product was purified by column chromatography (SiO2, DCM/MeOH 9:1 + 0.5% FA). Compound 5-17(1 was obtained as a colorless oil (1.64 g, 7.38 mmol, 71%). Rf = 0.79 (DCSIL; DCM/MeOH = 5:1), IR (ATR), [cm-1]: 3319, 2049, 2955, 2870, 1645, 1522, 1427, 1277, 790, 734, 711. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.41-8.37 (m, 2H), 7.60 (d, J = 7.8, 1H), 7.46 (t, J = 6.2, 1H), 7.22 (dd, J = 7.8, 4.9, 1H), 4.99 (br s, 1H), 4.47 (dd, J = 15.2, 6.2, 1H), 4.36 (dd, J = 15.2, 6.2, 1H), 4.18-4.14 (m, 1H), 1.90-1.78 (m, 1H), 1.65-1.62 (m, 1H), 1.54-1.50 (m, 1H), 0.93-0.88 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 175.6, 148.5, 148.4, 136.1, 134.5, 123.9, 70.7, 43.8, 40.5, 24.6, 23.6, 21.4.
(S)-2-Hydroxy-3-phenyl-N-(pyridin-3-ylmethyl)propanamide (S- 17a)
Amidation of 1-phenyl lactic acid (1.00 g, 6.02 mmol) and 3 -picolylamine (410 μL, 6.02 mmol) was carried out following general procedure A using 1.34 g of EDC·HCl (7.22 mmol) and HOBt (406 mg, 3.01 mmol) in dry DCM (240 mL). The product was obtained as a colorless oil (1.18 g, 4.60 mmol, 76%), Rf: 0.69 (DCSIL; EtOAc/MeOH = 10:1). IR (ATR), [cm-1]: 3329, 3154, 2925, 2851, 1737, 1651, 1515, 1071,705. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.95-8.93 (m, 1H), 8.51-8.49 (m, 1H), 8.17-8.13 (m, 2H), 7.67-7.64 (m, 1H), 7.22-7.20 (m, 5H), 4.57-4.42 (m, 3H), 3.19-3.15 (m, 1H), 2.94-2.89 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 171.9, 148.7, 148.2, 136.5, 135.7, 135.1, 129.8 (2C), 128.4 (2C), 126.1, 124.2, 74.1, 42.8, 40.9.
(R)-2-Hydroxy-3-phenyl-N-(pyridin-3-ylmethyl)propanamide (R-17a)
Amidation of d-phenyl lactic acid (1.00 g, 6.02 mmol) with 3 -picolylamine (0.41 mL, 6.02 mmol) was carried out following general procedure A using 1.34 g of EDC·HCl (7.22 mmol) and HOBt (406 mg, 3.01 mmol) in dry DCM (240 mL). Compound R-17a was obtained as a colorless oil (671 mg, 2.61 mmol, 45%), Rf: 0.73 (DCSIL; EtOAc/MeOH = 10:1). IR (ATR), [cm-1]: 3329, 3154, 2925, 2851, 1737, 1651, 1515, 1071, 705. 1H NMR
Figure imgf000122_0001
(CDCl3, δ [ppm], J [Hz]): 8.70-8.68 (m, 1H), 8.46-8.44 (m, 1H), 7.88-7.86 (m, 2H), 7.64- 7.62 (m, 1H), 7.46-7.44 (m, 1H), 7.23-7.21 (m, 5H), 4.50-4.40 (m, 3H), 3.22-3.18 (m, 1H), 2.95-2.90 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 171.9, 148.7, 148.2, 136.3, 134.7, 135.1, 129.8 (2C), 127.9 (2C), 126.1, 124.2, 74.5, 42.8, 40.9.
2-Oxo-4-(pyridin-3-yl)-N-(pyridin-3-yhnethyl)butanamide (18)
Potassium (E)-2-oxo-4-(pyridin-3-yl)but-3-enoate was synthesized following a modified procedure by Stecher et al. (1952). After obtaining the free acid by reaction with hydrochloric acid, (E)-2-oxo-4-(pyridin-3-yl)but-3-enoic acid (3.00 g, 16.9 mmol) was hydrogenated in the microwave (450 W, 15 bar H2) in isopropanol (50 mL) under Pd/C catalysis according to Slavinska et al. (2002). The obtained 2-oxo-4-(pyridin-3-yl)butanoic acid (2.00 g, 11.1 mmol) was further reacted according to general procedure A using EDC HCl (2.55 g, 13.3 mmol), HOBt (75 mg, 0.56 mmol), and DIPEA (3.00 mL, 17.2 mmol) in dry DCM (200 mL). Purification by column chromatography (SiO2, DCM/MeOH 9:1) afforded compound 18 as a yellow oil (2.70 g, 10.0 mmol, 90%). Rf: 0.4 (DCSIL; EtOAc/MeOH = 4:1), IR (ATR), [c -
Figure imgf000123_0002
m 1]: 3183, 2362, 1676, 1425, 1095, 1028, 706. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.54-8.50 (m, 2H), 8.47-8.43 (m, 2H), 7.61-7.52 (m, 2H), 7.47-7.45 (m, 1H), 7.28-7.19 (m, 2H), 4.48 (m, 2H), 3.30 (t, J = 7.3, 2H), 2.94 (t, J = 7.3, 2H), 13C NMR (CDCl3, δ [ppm]): 197.4, 160.0, 149.9, 149.4 (2C), 147.8, 136.2, 135.8 (2C), 132.8, 123.8, 123.6, 41.0, 38.1, 26.4.
( R,S)-2-Hydroxy-4-phenyl-N-(pyridin-3-ylmethyl)butanamide ( R,S-17e)
Amidation of (R/S)-2-hydroxy-4-phenylbutanoic acid (5.40 g, 30.0 mmol) with 3 -picolylamine (3.25 mL, 30.0 mmol) was carried out following general procedure A using 6.90 g of EDC·HCl (36.0 mmol), DIPEA (6.00 mL, 34.4 mmol), and HOBt (2.00 g, 15.0 mmol) in dry DCM (400 mL). Compound R,S-17e was obtained as a white solid (3.71 g, 21.8 mmol, 45%), Rf: 0.29 (DCSIL; EtOAc/MeOH = 10:1). IR (ATR), [cm-1]: 3449, 3026,
Figure imgf000123_0003
2924, 1716, 1645, 1495, 1236, 857, 694. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.50-8.49 (m, 2H), 7.71-7.69 (m, 1H), 7.32-7.24 (m, 7H), 4.59-4.54 (dd, J = 15.2, 6.4, 1H), 4.46 (dd, J = 15.2, 9.9, 1H), 4.26-4.23 (dd, J = 8.4, 3.6, 1H), 2.86-2.82 (m, 2H), 2.28-2.24 (m, 1H), 2.07-1.98 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 174.6, 148.5, 148.3, 141.3, 136.4, 134.5, 128.7 (2C), 128.6 (2C), 126.2, 124.0, 71.6, 40.6, 36.4, 31.5.
(S)-2-Hydroxy-4-phenyl-N-(pyridin-3-ylmethyl)butanamide (S-17e)
Amidation of (S)-2-hydroxy-4-phenylbutanoic acid (1.87 g, 10.4 mmol) with 3- picolylamine (1.16 mL, 11.4 mmol) was carried out following general procedure A using EDC HCl (1.98 g, 10.4 mmol) and HOBt (1.40 g, 10.4 mmol) in dry DCM (300 mL). The product was obtained as a white solid (1.30 g, 4.81 mmol, 46%), Rf: 0.29 (DCSIL; EtOAc/MeOH = 10:1). IR (ATR), [cm-1]: 3449, 3026, 2924, 1716, 1645, 1495, 1236, 857,
Figure imgf000123_0001
694. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.50-8.49 (m, 2H), 7.71-7.69 (m, 1H), 7.32-7.24 (m, 7H), 4.59-4.54 (dd, J = 15.2, 6.4, 1H), 4.48-1.43 (dd, J = 15.2, 9.9, 1H), 4.26-4.23 (dd, J = 8.4, 3.6, 1H), 2.86-2.82 (m, 2H), 2.28-2.24 (m, 1H), 2.07-1.98 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 174.6, 148.5, 148.3, 141.3, 136.4, 134.5, 128.7 (2C), 128.6 (2C), 126.2, 124.0, 71.6, 40.6, 36.4, 31.5.
(R)-2-Hydroxy-4-phenyl-N-(pyridin-3-ylmethyl)butanamide (R-17e)
Amidation of (R)-2-hydroxy-4-phenylbutanoic acid (1.57 g, 8.75 mmol) and 3- picolylamine (0.92 mL, 9.00 mmol) was carried out following general procedure A using EDC HCl (1.67 g, 8.75 mmol) and HOBt (1.07 g, 8.00 mmol) in dry DCM (300 mL). R-17e was obtained as a white solid (1.42 g, 5.25 mmol, 60%), Rf: 0.29 (DCSIL; EtOAc/MeOH = 10:1). IR (ATR), [cm-1]: 3449, 3026, 2924, 1716, 1645, 1495, 1236, 857, 694. 1H NMR
Figure imgf000124_0001
(CDCl3, δ [ppm], J [Hz]): 8.50-8.49 (m, 2H), 7.71-7.69 (m, 1H), 7.32-7.24 (m, 7H), 4.59- 4.54 (dd, J = 15.2, 6.4, 1H), 4.48-4.43 (dd, J = 15.2, 9.9, 1H), 4.26-4.23 (dd, 7 = 8.4, 3.6, 1H), 2.86-2.82 (m, 2H), 2.28-2.24 (m, 1H), 2.07-1.98 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 174.6, 148.5, 148.3, 141.3, 136.4, 134.5, 128.7 (2C), 128.6 (2C), 126.2, 124.0, 71.6, 40.6, 36.4, 31.5.
(R,S)-2-Hydroxy-4-(pyridin-3-yl)-N-(pyridin-3-ylmethyl)butanamide (19)
Following a protocol by Ideguchi et al. (2013), 2-oxo-4-(pyridin-3-yl)-A-(pyridin-3- ylmethyl)butanamide (18, 2.70 g, 10.04 mmol) was dissolved in 50 mL MeOH and NaBH4 (0.75 g, 20.0 mmol) was added in portions under ice cooling. After stirring the reaction mixture at 0°C for 30 min, excess NaBH4 was deactivated with 2M HCl and the solution was neutralized. The solvent was removed in vacuo, the residue was taken up in DCM (50 mL) and washed with water (2 x 50 mL). After separation of the phases, the solvent was removed in vacuo and the crude oily product was purified by column chromatography (SiO2, EtOAc/MeOH = 2:1) to give 19 as a white oily solid (660 mg, 2.43 mmol, 25%). Rf: 0.4 (DCSIL; EtOAc/MeOH = 2:1), IR (ATR), [cm-1]: 3253, 2356, 1645, 1523, 1424, 1098, 709.
Figure imgf000124_0002
1H NMR (CDCl3, δ [ppm], J [Hz]): 8.46-8.41 (m, 2H), 8.31-8.30 (m, 2H), 7.63-7.55 (m, 3H), 7.24-7.18 (m, 2H), 4.53 (dd, 7 = 15.1, 6.5, 1H), 4.39 (dd, 7 = 15.1, 5.8, 1H), 4.11 (dd, 7 = 15.1, 6.5, 1H), 2.79-2.77 (m, 2H), 2.20-2.12 (m, 1H), 2.00-1.92 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 174.8, 149.5, 148.7, 148.5, 147.0, 137.3, 136.9, 136.1, 134.5, 123.9 (2C), 41.0, 38.1, 26.4. 1 -(tert-Butyl) 2-((R)-4-methyl- 1-oxo- 1-((pyridin-3-ylmethyl)amino)pentan-2-yl) (S)- piperidine-1,2-dicarboxylate ( S,R-20d)
Esterification of R-17d (200 mg, 0.90 mmol) and (S)-1-Boc-piperidine-2-carboxylic acid (206 mg, 0.90 mmol) was carried out following general procedure A using EDC·HCl (207 mg, 1.08 mmol) and DMAP (121 mg, 0.99 mmol) in dry DCM (15 mL). After stirring at rt for 24 h and workup according to general procedure A, the crude oily product was purified by flash chromatography (RP 18, A: H2O + 1%FA, B: MeOH + 1% FA, gradient: 5→ 100% B). S,R-20d was obtained as a colorless oil (55 mg, 0.13 mmol, 14%). Rf: 0.41 (DCSIL; DCM/MeOH = 14:1). IR (ATR), -
Figure imgf000125_0001
[cm 1]: 3312, 2956, 2935, 1743, 1686, 1392, 1365, 1154, 773, 711. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.50 (m, 2H), 7.32 (s, 1H), 7.62 (d, J =7.8 Hz, 1H), 7.25-7.20 (m, 1H), 5.27 (dd, 7 = 9.4, 3.9, 1H), 4.55-4.30 (m, 3H), 3.67-3.60 (m, 1H), 3.30 (d, J = 9.9, 1H), 2.02-1.94 (m, 1H), 1.89-1.57 (m, 6H), 1.46-1.42 (m, 2H), 1.35 (s, 9H), 0.96-0.88 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 172.4, 170.7, 157.2, 149.1, 148.7, 135.5, 134.5, 123.7, 80.8, 72.9, 54.7, 43.0, 40.8, 40.4, 28.4 (3C), 26.6, 24.8, 24.4, 23.4, 21.2, 20.1.
1 -(tert-Butyl) 2-((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl) (S)- piperidine- 1,2-dicarboxylate ( S,S-20d )
S,S-20d was prepared following general procedure A using 1.36 g of S-17d (6.10 mmol), (S)-1-Boc-piperidine-2-carboxylic acid (1.54 g, 6.71 mmol), EDC HCl (1.75 g, 9.15 mmol), and DMAP (0.15 g, 1.22 mmol) in dry DCM (200 mL). After stirring at rt for 4 h and workup according to general procedure A, the crude oily product was purified by column chromatography (SiO2, DCM/MeOH 15:1). Compound S,S-20d was obtained as a colorless oil (1.29 g, 2.97 mmol, 49%). IR (ATR), [cm-1]: 3310, 2935, 2865, 1739, 1686,
Figure imgf000125_0002
1364, 1156, 1042, 930, 870, 777, 735. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.51 (m, 2H), 7.74 (br s, 1H), 7.68-7.66 (m, 1H), 7.29-7.26 (m, 1H), 5.37-5.35 (m, 1H), 4.75-4.74 (m, 1H), 4.48-4.46 (m, 1H), 4.40-4.38 (m, 1H), 3.95-3.92 (m, 2H), 3.04-2.86 (m, 2H), 2.80- 2.73 (m, 1H), 2.37-2.22 (m, 3H), 1.80-1.77 (m, 2H), 1.34 (s, 9H), 0.95-0.93 (m, 6H) ppm. 13C NMR (CDCl3, δ [ppm]): 171.2, 170.8, 157.9, 149.6, 148.9, 135.8, 134.0, 123.6, 80.9, 73.5, 54.7, 43.1, 40.9, 40.8, 28.4 (3C), 26.0, 25.0, 24.6, 23.5, 21.4, 21.0. 1 -(tert-Butyl) 2-((S)-1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl) (S)- piperidine- 1,2-dicarboxylate ( S,S- 20a )
Esterification of S-17a (1.38 g, 5.38 mmol) and (S)-1-Boc-piperidine-2-carboxylic acid (1.23 g, 5.38 mmol) was carried out following general procedure A using 1.24 g of EDC·HCl (6.46 mmol) and DMAP (328 mg, 2.69 mmol) in dry DCM (200 mL). Compound S,S-20a was obtained as a colorless oil (1.18 g, 2.52 mmol, 47%), Rf: 0.55 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3318, 2974, 2932, 2862, 1745, 1666, 1364, 1155, 699. 1H NMR
Figure imgf000126_0002
(CDCl3, δ [ppm], J [Hz]): 8.52-8.50 (m, 2H), 7.67-7.60 (m, 2H), 7.28-7.19 (m, 6H), 5.67- 5.65 (m, 1H), 4.71-4.69 (m, 1H), 4.51-4.37 (m, 2H), 3.81-3.78 (m, 1H), 3.42-3.36 (m, 1H), 3.07-3.01 (m, 1H), 2.52 (td, J = 13.0, 2.9, 1H), 2.24-2.22 (m, 1H), 1.56-1.46 (m, 4H), 1.31 (s, 9H), 0.87-0.81 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.9, 169.7, 157.8, 148.9, 148.2, 136.3, 136.1, 134.1, 129.5 (2C), 128.5 (2C), 127.1, 123.7, 80.7, 74.8, 54.4, 42.7, 40.8, 38.1, 28.4 (3C), 25.9, 24.4, 20.5.
1 -(tert-Butyl) 2-((R)- 1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl) (S)- piperidine-1,2 dicarboxylate ( S,R-20a)
Esterification of R-17a (670 mg, 2.61 mmol) and (S)-1-Boc-piperidine-2-carboxylic acid (600 mg, 2.61 mmol) was carried out following general procedure A using 601 mg of EDC·HCl (3.12 mmol) and DMAP (1.31 mmol, 160 mg) in dry DCM (100 mL). Compound S,R-20a was obtained as a colorless oil (860 mg, 1.83 mmol, 70%), Rf: 0.50 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3318, 2974, 2932, 2862, 1745, 1666, 1364, 1155,
Figure imgf000126_0001
699. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.50-8.48 (m, 2H), 7.49-7.47 (m, 1H), 7.25-7.20 (m, 7H), 5.51-5.48 (m, 1H), 4.58-4.28 (m, 3H), 3.63-3.61 (m, 1H), 3.40-3.07 (m, 3H), 1.67- 1.57 (m, 5H), 1.33 (s, 9H), 1.19-1.17 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 171.9, 169.4, 157.2, 149.1, 148.8, 136.4, 135.1, 133.9, 129.5 (2C), 128.5 (2C), 127.0, 123.6, 80.8, 74.3, 54.4, 42.8, 40.8, 37.7, 28.4 (3C), 26.3, 24.4, 19.9.
1 -(/(’//-Butyl) 2-((S)-1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl) (R)- piperidine- 1,2-dicarboxylate (R, S-20a )
Esterification of S-17a (279 mg, 1.09 mmol) and (R)-1-Boc-piperidine-2-carboxylic acid (250 mg, 1.09 mmol) was carried out following general procedure A using EDC·HCl (250 mg, 1.31 mmol) and DMAP (66 mg, 0.55 mmol) in dry DCM (40 mL). Compound R,S- 20a was obtained as a colorless oil (276 mg, 0.59 mmol, 52%), Rf: 0.51 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3337, 3024, 2955, 1745, 1671, 1264, 1154, 711. 1H NMR (CDC13,
Figure imgf000127_0001
δ [ppm], J [Hz]): 8.49-8.46 (m, 2H), 7.50-7.48 (m, 1H), 7.26-7.19 (m, 6H), 5.51-5.48 (m, 1H), 4.58-4.26 (m, 3H), 3.63-3.61 (m, 1H), 3.41-3.39 (m, 1H), 3.23-3.21 (m, 1H), 3.11- 3.09 (m, 1H), 1.67-1.42 (m, 5H), 1.33 (s, 9H), 1.20-1.17 (m, 1H). 13C NMR (CDCl3, 6 [ppm]): 177.6, 175.7, 152.8, 149.3, 148.8, 135.3, 136.1, 134.1, 129.6 (2C), 128.5 (2C), 127.0, 123.6, 81.7, 74.2, 54.5, 42.7, 40.8, 37.8, 28.4 (3C), 25.9, 24.3, 20.5.
1 -(tert-Butyl) 2-((R)-1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl) (R)- piperidine-1,2 dicarboxylate (R,R-20a)
Esterification of R-17a (279 mg, 1.09 mmol) and (R)-1-Boc-piperidine-2-carboxylic acid (250 mg, 1.09 mmol) was carried out following general procedure A using EDC·HCl (250 mg, 1.31 mmol) and DMAP (66 mg, 0.55 mmol) in dry DCM (40 mL). Compound R,R- 20a was obtained as a colorless oil (350 mg, 0.75 mmol, 62%), Rf: 0.51 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3345, 3030, 2955, 1745, 1671, 1413, 1264, 1154, 707. 1H NMR
Figure imgf000127_0002
(CDCl3, δ [ppm], J [Hz]): 8.50-8.48 (m, 2H), 7.64-7.56 (m, 2H), 7.26-7.16 (m, 6H), 5.66- 5.64 (m, 1H), 4.69-4.67 (m, 1H), 4.48-4.34 (m, 2H), 3.79-3.76 (m, 1H), 3.39-3.35 (m, 1H), 3.04-2.99 (m, 1H), 2.53-2.46 (m, 1H), 2.20-2.17 (m, 1H), 1.53-1.44 (m, 4H), 1.29 (s, 9H), 0.89-0.78 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.9, 169.7, 157.8, 149.0, 148.4, 136.2, 136.1, 134.1, 129.5 (2C), 128.5 (2C), 127.1, 123.7, 80.6, 74.7, 54.5, 42.7, 40.7, 38.1, 28.4 (3C), 25.9, 24.4, 20.5.
1 -(tert-Butyl) 2-((R/S)-1-oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl) (2S)- piperidine-1,2-dicarboxylate (S,R/S-20e)
Esterification of R/S-17e (1.27 g, 5.55 mmol) with (S)-1-Boc-piperidine-2-carboxylic acid (1.50 g, 5.55 mmol) was carried out following general procedure A using 1.28 g of EDC·HCl (6.66 mmol) and DMAP (340 mg, 2.78 mmol) in dry DCM (200 mL). Compound S,R/S-20e was obtained as a colorless oil (1.45 g, 3.01 mmol, 54%). Rf: 0.48 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3320, 2931, 2857, 1742, 1671, 1532, 1392, 1154, 700.
Figure imgf000127_0003
Diastereomeric ratio: 55:45. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.54-8.48 (m, 2H), 7.63- 7.61 (m, 1H), 7.35-7.15 (m, 7H), 5.37-5.35 (m, 1H), 4.58-4.33 (m, 3H), 3.68-3.65 (m, 1H), 3.34-3.32 (m, 1H), 2.67-2.65 (m, 2H), 2.38-1.94 (m, 3H), 1.82-1.56 (m, 6H), 1.37-1.35 (m, 9H). 13C NMR (CDCl3, δ [ppm]): 170.8, 169.8, 157.7, 149.0, 148.8, 140.5, 135.6, 133.8, 128.6 (2C), 128.4 (2C), 126.3, 123.5, 80.9, 74.09, 54.7, 43.0, 40.8, 33.5, 31.5, 28.4 (3C), 24.5, 20.9, 20.2. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.54-8.48 (m, 2H), 7.62 (m, 1H), 7.35- 7.15 (m, 7H), 5.26-5.24 (m, 1H), 4.80-4.78 (m, 1H), 4.58-4.33 (m, 2H), 4.00-3.96 (m, 1H), 2.80 (td, J = 19.4, 3.0, 1H), 2.67-2.65 (m, 2H), 2.38-1.94 (m, 3H), 1.82-1.56 (m, 6H), 1.37- 1.35 (m, 9H). 13C NMR (CDCl3, δ [ppm]): 170.8, 169.8, 157.7, 149.0, 148.8, 140.5, 135.6, 133.8, 128.6 (2C), 128.4 (2C), 126.3, 123.5, 80.9, 73.67, 54.7, 43.0, 40.8, 33.5, 31.5, 28.4 (3C), 24.5, 20.9, 20.2.
1 -(tert-Butyl) 2-((R/S)-1-oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl) (2R)- piperidine-1,2 dicarboxylate (R,R/S-20e)
Esterification of R/S-17e (1.27 g, 5.55 mmol) with (R)-1-Boc-piperidine-2-carboxylic acid (1.50 g, 5.55 mmol) was carried out following general procedure A using 1.28 g of EDC·HCl (6.66 mmol) and DMAP (340 mg, 2.78 mmol) in dry DCM (200 mL). Compound R,R/S-20e was obtained as a white oily solid (1.46 g, 21.8 mmol, 55%). Rf: 0.50 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3319, 2932, 2857, 1742, 1676, 1532, 1393, 1154, 1041,
Figure imgf000128_0001
700. Diastereomeric ratio: 57:43. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.48 (m, 2H), 7.64-7.62 (m, 1H), 7.29-7.14 (m, 7H), 5.37-5.35 (m, 1H), 4.81-4.79 (m, 1H), 4.53-4.37 (m, 2H), 4.01-3.99 (m, 1H), 3.34-3.32 (m, 1H), 2.69-2.65 (m, 2H), 2.38-1.96 (m, 3H), 1.81- 1.46 (m, 6H), 1.37-1.35 (m, 9H). 13C NMR (CDCl3, δ [ppm]): 170.1, 157.2, 149.2, 148.9,
140.6, 135.8, 135.3, 128.8 (2C), 128.5 (2C), 126.4, 123.7, 80.9, 74.1, 54.7, 43.0, 40.8, 34.0,
31.6, 28.4 (3C), 24.5, 21.2, 20.2. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.48 (m, 2H), 7.64-7.62 (m, 1H), 7.29-7.14 (m, 7H), 5.28-5.26 (m, 1H), 4.53-4.37 (m, 3H), 3.70-3.68 (m, 1H), 2.84-2.77 (m, 1H), 2.69-2.65 (m, 2H), 2.38-1.96 (m, 3H), 1.81-1.46 (m, 6H), 1.37- 1.35 (m, 9H). 13C NMR (CDCl3, δ [ppm]): 170.1, 157.2, 149.2, 148.9, 140.6, 135.8, 135.3, 128.6 (2C), 128.4 (2C), 126.4, 123.7, 80.9, 73.7, 54.7, 43.0, 40.8, 33.3, 31.4, 28.4 (3C), 24.5, 20.9, 20.2.
1 -(tert-Butyl) 2-((R/S)-1-oxo-4-(pyridin-3-yl)-1-((pyridin-3-ylmethyl)amino)butan-2-yl) (S)-piperidine-1,2 dicarboxylate (S,R/S-20f)
S,R/S-20f was synthesized according to general procedure A using 660 mg of 19 (2.43 mmol), EDC·HCl (560 mg, 2.91 mmol), and DMAP (148 mg, 1.22 mmol) in dry DCM (50 mL). After purification by column chromatography (SiO2, EtOAc/MeOH = 4:1), compound S,R/S-20f was obtained as a yellowish oil (700 mg, 1.45 mmol, 60%). Rf: 0.5 (DCSIL; EtOAc/MeOH = 4:1). IR (ATR),
Figure imgf000129_0003
[cm-1]: 3323, 2933, 2360, 1743, 1671, 1155, 719. Diastereomeric ratio: 53:47. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.43 (m, 4H), 7.67- 7.54 (m, 2H), 7.28-7.25 (m, 2H), 5.36-5.35 (m, 1H), 4.81-4.79 (m, 1H), 4.58-4.36 (m, 2H), 4.01-3.97 (m, 1H), 3.34-3.29 (m, 1H), 2.74-2.58 (m, 3H), 2.35-1.47 (m, 7H), 1.37-1.35 (m, 9H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.43 (m, 4H), 7.67-7.54 (m, 2H), 7.28-7.25 (m, 2H), 5.27-5.25 (m, 1H), 4.58-4.36 (m, 3H), 3.68-3.64 (m, 1H), 2.84-2.82 (m, 1H), 2.74- 2.58 (m, 2H), 2.35-1.47 (m, 8H), 1.37-1.35 (m, 9H). 13C NMR (CDCl3, δ [ppm]): 172.1, 169.7, 157.2, 149.4, 148.7, 148.4, 147.6, 136.8, 136.5, 136.2, 134.0, 123.8 (2C), 81.0, 73.7, 54.8, 43.2, 40.8, 33.6, 28.7, 28.4, 26.7, 24.5, 20.9. 13C NMR (CDCl3, δ [ppm]): 170.9, 169.7,
157.2, 149.2, 148.7, 148.4, 147.1, 136.8, 136.5, 136.2, 134.0, 123.8 (2C), 81.0, 73.2, 54.8,
43.2, 40.8, 32.8, 28.7, 28.4, 26.0, 24.5, 20.2.
(R)-4-Methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate ( S,R- 10d )
S,R-20d (50 mg, 0.12 mmol) was Boc-deprotected according to general procedure B using 2 mL of TFA in dry DCM (5 mL). After stirring at rt for 18 h, the reaction was worked up according to the general procedure. Subsequently, the intermediate product (29.7 mg, 89 μmol) was further reacted according to general procedure C using NMM (69 μL, 0.62 mmol) and phenylmethane sulfonyl chloride (41 mg, 0.21 mmol) in dry DCM (20 mL). After stirring at rt for 4 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (SiO2, A: DCM, B: MeOH, gradient: 0
Figure imgf000129_0001
30% B) giving the product as a colorless oily solid (26 mg, 53 μmol, 60%). Rf = 0.53 (DCSIL; DCM/MeOH = 15:1). IR (ATR), [cm-1]: 3356, 3063, 3032, 2956, 1740, 1670, 1540, 826,
Figure imgf000129_0002
790, 731, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.56 (s, 1H), 8.50 (d, J = 3.9, 1H), 7.65 (d, J = 7.8, 1H), 7.42-7.34 (m, 5H), 7.27 (br s, 1H), 7.25-7.22 (m, 1H), 5.30-5.23 (m, 1H), 4.49-4.38 (m, 2H), 4.21-4.19 (m, 3H), 3.25-3.17 (m, 1H), 3.10-3.02 (m, 1H), 2.01-1.93 (m, 1H), 1.90-1.73 (m, 3H), 1.71-1.30 (m, 5H), 0.96-0.88 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 170.5, 170.2, 149.5, 148.8, 136.0, 134.1, 131.0 (2C), 129.03, 129.01, 128.8 (2C), 123.6, 73.9, 59.1, 56.2, 44.2, 41.0, 40.8, 27.1, 24.7 (2C), 23.2, 21.9, 19.7. LC/MS (m/z) 488.50 [M+H]+. Purity (HPLC): 95.1%, tR = 8.47 min. HRMS (m/z) C25H33N3O5S, [M+H]+, calculated 488.22137, found 488.22146, error 0.2 ppm.
(S)-4-Methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate ( S,S- 10d)
Following general procedure B, S,S-20d (500 mg, 1.15 mmol) was first deprotected using TFA (1.0 mL, 13.0 mmol) in dry DCM (4.0 mL) stirring at rt for 1 d. After workup according to general procedure B, 208 mg of the amine intermediate (623 μmol) was reacted according to general procedure C using NMM (0.21 mL, 1.87 mmol) and phenylmethanesulfonyl chloride (220 mg, 1.15 mmol) in dry DCM (3 mL). After stirring at rt for 3 d, the reaction was worked up according to the general procedure and purified by flash chromatography (run 1: SiO2, 2 x 12 g, A: CHCl3 B: MeOH, gradient: 0
Figure imgf000130_0001
20% B; run 2:
RP 18, A: H2O, B: MeOH, gradient: 5
Figure imgf000130_0002
70% B). S,S-10d was obtained as a colorless oily solid (135 mg, 277 μmol, 44%). Rf = 0.55 (DCSIL; DCM/MeOH = 15:1). IR (ATR), [cm-1]:
Figure imgf000130_0003
3354, 3061, 3036, 2952, 1739, 1671, 1536, 825, 792, 741, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.54 (br s, 2H), 7.70-7.58 (m, 2H), 7.46-7.30 (m, 5H), 7.25 (br s, 1H), 5.41-5.31 (m, 1H), 4.49-4.35 (m, 2H), 4.21-4.19 (m, 2H), 4.08 (d, J = 4.3 Hz, 1H), 3.36 (d, J = 12.9, 1H), 3.02 (ddd, J = 13.1, 13.1, 2.7, 1H), 2.20-2.00 (m, 2H), 1.83-1.73 (m, 2H), 1.70-1.60 (m, 2H), 1.41-1.22 (m, 2H), 1.19-0.97 (m, 1H), 0.95-0.88 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 170.4, 170.3, 149.5, 148.6, 135.8, 134.1, 130.9 (2C), 129.2, 128.9 (2C), 128.8, 123.6, 74.1, 59.5, 57.0, 44.3, 41.2, 40.7, 27.1, 25.0, 24.6, 23.3, 21.5, 20.3. LC/MS (m/z) 488.45 [M+H]+. Purity (HPLC): 98.9%, tR = 8.46 min. HRMS (m/z) C25H33N3O5S, [M+H]+, calculated 488.22137, found 488.22235, error 2.0 ppm.
(S)-1-Oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate ( S,S- 10a)
S,S-20a (860 mg, 1.84 mmol) was Boc-deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (650 mg, 1.77 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (337 mg, 1.77 mmol) and NMM (210 μL, 1.95 mmol) in dry DCM (35 mL). Compound S,S-10a was obtained as a colorless oil (300 mg, 0.57 mmol, 32%), Rf: 0.41 (DCSIL; EtOAc/PE = 95:5). IR (ATR), v [cm-1]: 3349, 3050, 2943, 1740, 1671, 1536, 1321, 1124, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.54 (d, J = 1.8, 1H), 8.51 (dd, 7 = 4.8, 1.7, 1H), 7.70 (t, 7 = 5.7, 1H), 7.58 (dt, 7 = 7.8, 1.9, 1H), 7.37- 7.17 (m, 11H), 5.66 (dd, 7 = 9.2, 3.9, 1H), 4.48-4.37 (m, 2H), 4.14 (s, 2H), 3.96-3.94 (m, 1H), 3.45 (dd, 7 = 14.7, 4.0, 1H), 3.11 (dd, 7 = 14.7, 9.2, 1H), 3.13-3.11 (m, 1H), 2.59 (td, 7 = 13.0, 2.9, 1H), 1.96-1.92 (m, 1H), 1.34-1.08 (m, 4H), 0.45-0.35 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.1, 169.2, 149.7, 148.8, 136.1, 135.8, 133.8, 130.8 (2C), 129.5 (2C), 129.2 (2C), 128.9 (2C), 128.6, 127.1 (2C), 123.5, 75.4, 59.4, 56.8, 43.7, 40.8, 38.0, 27.1, 24.3, 19.4. LC/MS (m/z) 522.10 [M+H]+. Purity (HPLC): 96.5%, tR = 8.55 min. HRMS (m/z) C28H31N3O5S, [M+H]+, calculated 522.20572, found 522.20657, error 1.6 ppm.
(R)-1-Oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate ( S,R-10a)
S,R-20a (860 mg, 1.84 mmol) was Boc-deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (650 mg, 1.77 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (337 mg, 1.77 mmol) and NMM (210 μL, 1.95 mmol) in dry DCM (30 mL). Compound S,R-10a was obtained as a colorless oil (320 mg, 0.61 mmol, 35%), Rf: 0.42 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3349,
Figure imgf000131_0001
3039, 2943, 1744, 1684, 1536, 1323, 1124, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.50- 8.48 (m, 2H), 7.50-7.48 (m, 1H), 7.38-7.33 (m, 5H), 7.24-7.15 (m, 6H), 7.09 (t, 7 = 5.8, 1H), 5.50-5.47 (dd, 7 = 8.4, 4.0, 1H), 4.49-4.43 (dd, 7 = 14.9, 6.4, 1H), 4.36-4.31 (dd, 7 = 14.9, 5.8, 1H), 4.16 (m, 2H), 4.12 (m, 1H), 3.36-3.31 (dd, 7 = 14.4, 4.0, 1H), 3.17-3.13 (m, 1H), 3.09-3.03 (dd, 7 = 14.4, 8.4, 1H), 3.03-2.98 (m, 1H), 1.69-1.65 (m, 1H), 1.50-1.25 (m, 4H), 1.09-1.06 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.2, 168.9, 149.5, 148.8, 135.9, 135.8, 133.7, 130.9 (2C), 129.6 (2C), 129.0 (2C), 128.7 (2C), 128.5, 127.1 (2C), 123.6, 75.1, 59.1, 56.0, 44.1, 40.8, 38.0, 26.9, 24.6, 19.5. LC/MS (m/z) 522.10 [M+H]+. Purity (HPLC): 99.5%, tR = 8.41 min. HRMS (m/z) C28H31N3O5S, [M+H]+, calculated 522.20572, found 522.20601, error 0.6 ppm.
(R)-1-Oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl (R)-1-
(benzylsulfonyl)piperidine-2-carboxylate (R,R- 10a) R,R-20a (350 mg, 0.75 mmol) was Boc-deprotected following general procedure B using 3.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (284 mg, 0.78 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (150 mg, 0.78 mmol) and NMM (100 μL, 0.94 mmol) in dry DCM (20 mL). R,R-10a was obtained as a colorless oil (120 mg, 0.23 mmol, 31%), Rf: 0.40 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3345, 3062, 2933,
Figure imgf000132_0001
1739, 1671, 1537, 1322, 1192, 1124, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.51 (m, 2H), 7.73 (t, 1H, 7 = 5.6), 7.65-7.63 (m, 1H), 7.37-7.25 (m, 5H), 7.23-7.15 (m, 6H), 5.68- 5.64 (dd, 1H, J= 9.2, 3.9), 4.50-4.38 (m, 2H), 4.16 (m, 2H), 3.96 (m, 1H), 3.47-3.43 (dd, 1H, 7 = 14.7, 3.9), 3.15-3.10 (m, 1H), 3.13-3.07 (dd, 1H, 7 = 14.7, 9.2), 2.63-2.56 (td, 7 = 13.0, 2.9, 1H), 1.96-1.93 (m, 1H), 1.34-1.07 (m, 4H), 0.45-0.35 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.1, 169.2, 148.9, 147.9, 136.6, 136.0, 134.3, 130.8 (2C), 129.5 (2C), 129.2 (2C), 128.9 (2C), 128.6, 127.1 (2C), 123.8, 75.4, 59.4, 56.8, 43.7, 40.7, 38.0, 27.1, 24.3, 19.4. LC/MS (m/z) 522.50 [M+H]+, tR = 8.52 min. Purity (HPLC): 95.6%. HRMS (m/z) C28H31N3O5S, [M+H]+, calculated 522.20572, found 522.20569, error 0.1 ppm.
(S)-1-Oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2-yl (R)-1- (benzylsulfonyl)piperidine-2-carboxylate (R, S- 10a) R,S-20a (276 mg, 0.60 mmol) was Boc-deprotected following general procedure B using 3.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (200 mg, 0.55 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (104 mg, 0.55 mmol) and NMM (70 μL, 0.66 mmol) in dry DCM (10 mL). R,S-10a was obtained as a colorless oil (80 mg; 0.15 mmol, 28%), Rf: 0.41 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3362, 3034, 1736,
Figure imgf000132_0002
1696, 1540, 1299, 1179, 778, 693. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.53-8.50 (m, 2H), 7.67-7.65 (m, 1H), 7.38-7.16 (m, 12H), 5.48 (dd, 7 = 8.4, 3.9, 1H), 4.54 (dd, 7 = 15.0, 6.3, 1H), 4.38 (dd, 7 = 15.0, 5.8, 1H), 4.20-4.16 (m, 3H), 3.34 (dd, 7 = 14.4, 4.0, 1H), 3.21-3.18 (m, 1H), 3.07 (dd, 7 = 14.4, 8.4, 1H), 3.05-2.99 (m, 1H), 1.72-1.68 (m, 1H), 1.52-1.23 (m, 4H), 1.12-1.06 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.3, 169.0, 147.3, 146.5, 135.8, 135.2, 130.9, 129.7 (2C), 129.6 (2C), 129.0 (2C), 128.9 (2C), 128.5, 127.2 (2C), 124.4, 75.1, 59.1, 56.0, 44.0, 40.6, 37.9, 26.9, 24.6, 19.5. LC/MS (m/z) 522.45 [M+H]+. Purity (HPLC): 98.5%, tR = 8.38 min. HRMS (m/z) C28H31N3O5S, [M+H]+, calculated 522.20572, found 522.20608, error 0.7 ppm.
(R/S)-1-Oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate (S,R/S-10e)
S,R/S-20e (1.45 g, 3.00 mmol) was Boc-deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (440 mg, 1.15 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (260 mg, 1.38 mmol) and NMM (150 μL,
1.38 mmol) in dry DCM (20 mL). S,R/S-10e was obtained as a colorless oil (90 mg; 0.16 mmol, 15%), Rf: 0.60 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3308, 3046, 2921,
Figure imgf000133_0001
2852, 1741, 1671, 1536, 1322, 1125, 695. Diastereomeric ratio: 55:45. 1H NMR (CDCl3, 6 [ppm], J [Hz]): 8.63-8.61 (m, 1H), 8.54-8.52 (m, 1H), 7.83-7.79 (m, 2H), 7.41-7.36 (m, 6H), 7.26-7.12 (m, 5H), 5.38 (t, J = 5.6, 1H), 4.50-4.44 (m, 2H), 4.27-4.25 (m, 3H), 3.42-
3.39 (m, 1H), 3.11-3.04 (m, 1H), 2.70-2.62 (m, 2H), 2.24-2.21 (m, 3H), 1.65-1.59 (m, 2H), 1.39-1.09 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.2, 169.6, 147.6, 146.7, 140.6, 138.1,
135.2, 130.9 (2C), 129.2, 128.8 (2C), 128.7 (2C), 128.5 (2C), 126.4 (2C), 124.3, 74.8, 59.5, 57.O, 44.5, 40.6, 34.0, 31.4, 27.1, 24.6, 20.4. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.63-8.61 (m, 1H), 8.54-8.52 (m, 1H), 7.83-7.79 (m, 2H), 7.41-7.36 (m, 6H), 7.26-7.12 (m, 5H), 5.25 (t, J = 5.6, 1H), 4.50-4.44 (m, 2H), 4.27-4.25 (m, 2H), 4.16-4.14 (m, 1H), 3.29-3.25 (m, 1H), 3.11-3.04 (m, 1H), 2.70-2.62 (m, 2H), 2.24-2.21 (m, 2H), 2.01-1.99 (m, 1H), 1.65- 1.59 (m, 2H), 1.39-1.09 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.2, 169.6, 147.6, 146.7, 140.6, 138.1, 135.2, 130.9 (2C), 129.0, 128.8 (2C), 128.7 (2C), 128.5 (2C), 126.2 (2C),
124.3, 74.8, 59.0, 56.1, 44.2, 40.6, 33.5, 30.7, 27.1, 24.6, 19.7. LC/MS (m/z) 536.45 [M + H]+. Purity (HPLC, both diastereomers in total): 99.3%, tR = 8.69 min, 8.89 min. HRMS (m/z) C29H33N3O5S, [M+H]+, calculated 536.22137, found 536.22152, error 0.3 ppm.
(R/S)-1-Oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl (R)-1- (benzylsulfonyl)piperidine-2-carboxylate (R,R/S-10e)
R,R/S-20e (1.45 g, 3.00 mmol) was Boc-deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (230 mg, 0.60 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (137 mg, 0.72 mmol) and NMM (80 μL, 0.72 mmol) in dry DCM (12 mL). R,R/S-10e was obtained as a colorless oil (102 mg, 0.19 mmol, 31%), Rf: 0.60 (DCSIL; EtOAc/PE = 95:5). IR (ATR), [cm-1]: 3349, 3044, 2921,
Figure imgf000134_0001
2852, 1741, 1671, 1536, 1322, 1125, 695. Diastereomeric ratio: 54:46. 1H NMR (CDCl3, 6 [ppm], J [Hz]): 8.59-8.58 (m, 1H), 8.51-8.49 (m, 1H), 7.78-1.76 (m, 1H), 7.70-7.66 (m, 1H), 7.41-7.34 (m, 6H), 7.27-7.11 (m, 5H), 5.38 (t, J = 5.6, 1H), 4.45-4.43 (m, 2H), 4.26- 4.23 (m, 3H), 3.42-3.39 (m, 1H), 3.10-2.99 (m, 1H), 2.72-2.60 (m, 2H), 2.24-2.22 (m, 3H), 1.64-1.56 (m, 2H), 1.39-1.09 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.2, 169.6, 147.6, 146.7, 140.6, 138.1, 135.2, 130.9 (2C), 129.2, 128.8 (2C), 128.7 (2C), 128.5 (2C), 126.4 (2C), 124.3, 74.8, 59.5, 57.0, 44.5, 40.6, 34.0, 31.4, 27.1, 24.6, 20.4. 1H NMR (CDCl3, 6 [ppm], J [Hz]): 8.59-8.58 (m, 1H), 8.51-8.49 (m, 1H), 7.56-7.54 (m, 1H), 7.70-7.66 (m, 1H), 7.41-7.34 (m, 6H), 7.27-7.11 (m, 5H), 5.25 (t, J = 5.6, 1H), 4.45-4.43 (m, 2H), 4.26- 4.23 (m, 2H), 4.14-4.12 (m, 1H), 3.26-3.23 (m, 1H), 3.10-2.99 (m, 1H), 2.72-2.60 (m, 2H), 2.24-2.22 (m, 2H), 2.01-1.99 (m, 1H), 1.64-1.56 (m, 2H), 1.39-1.09 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.2, 169.6, 147.6, 146.7, 140.6, 138.1, 135.2, 130.9 (2C), 129.0, 128.8 (2C), 128.7 (2C), 128.5 (2C), 126.2 (2C), 124.3, 74.8, 59.0, 56.1, 44.2, 40.6, 33.5, 30.7, 27.1, 24.6, 19.7. LC/MS (m/z) 536.50 [M+H]+. Purity (HPLC, both diastereomers in total): 95.2%, tR = 8.68 min, 8.87 min. HRMS (m/z) C29H33N3O5S, [M+H]+, calculated 536.22137, found 536.22097, error 0.7 ppm.
(R)-1-Oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate (S,R-10e)
Esterification of R-17e (1.27 g, 5.55 mmol) and (S)-1-Boc-piperidine-2-carboxylic acid (1.50 g, 5.55 mmol) was conducted following general procedure A using 1.28 g of EDC·HCl (6.66 mmol) and DMAP (340 mg, 2.78 mmol) in dry DCM (200 mL). The intermediate product was Boc -deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (233 mg, 0.61 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (133 mg, 0.70 mmol) and NMM (80 μL, 0.72 mmol) in dry DCM (12 mL). S,R-10e was obtained as a colorless oil (102 mg, 0.19 mmol, 31%), Rf: 0.58 (DCSIL; EtOAc/PE = 95:5), IR (ATR), v [cm-1]: 2921, 2852, 1741, 1671, 1536, 1322, 1125, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.63-8.53 (m, 2H), 7.77-7.75 (m, 1H), 7.55- 7.52 (m, 1H), 7.39-7.37 (m, 5H), 7.34-7.32 (m, 1H), 7.26-7.12 (m, 5H), 5.26 (t, J = 5.6, 1H), 4.52-4.40 (m, 2H), 4.27-4.23 (m, 3H), 3.27-3.24 (m, 1H), 3.06-3.03 (m, 1H), 2.73-2.59 (m, 2H), 2.24-2.20 (m, 2H), 2.01-1.99 (m, 1H), 1.61-1.58 (m, 2H), 1.42-1.31 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.1, 169.6, 148.1, 147.6, 140.7, 137.3, 134.8, 130.9 (2C), 129.1, 129.0, 128.8 (2C), 128.6 (2C), 128.5 (2C), 126.3, 124.1, 74.8, 59.0, 56.2, 44.2, 40.7, 33.5, 30.8, 26.9, 24.6, 19.9. LC/MS (m/z) 536.50 [M+H]+. Purity (HPLC): 95.1%, tR = 8.68 min. HRMS (m/z) C29H33N3O5S, [M+H]+, calculated 536.22137, found 536.22245, error 2.0 ppm.
(S)-1-Oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2-yl (S)-1- (benzylsulfonyl)piperidine-2-carboxylate ( S,S- lOe)
Esterification of S-17e (0.92 g, 4.00 mmol) and (S)-1-Boc-piperidine-2-carboxylic acid (1.08 g, 5.55 mmol) was conducted following general procedure A using 0.92 g of EDC HCl (4.80 mmol) and DMAP (245 mg, 2.00 mmol) in dry DCM (160 mL). The intermediate product was Boc -deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (233 mg, 0.61 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (140 mg, 0.74 mmol) and NMM (80 μL, 0.81 mmol) in dry DCM (15 mL). S,S-10e was obtained as a colorless oil (102 mg, 31%), Rf: 0.58 (DCSIL; EtOAc/PE = 95:5), IR (ATR),
Figure imgf000135_0001
[cm-1]: 2921, 2852, 1741, 1671, 1536, 1322, 1125, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.58 (s, 1H), 8.52 (d, J = 4.5, 1H), 7.75 (t, J = 5.8, 1H), 7.67 (d, J = 7.8, 1H), 7.42-7.37 (m, 5H), 7.27-7.12 (m, 6H), 5.38 (t, J = 5.6, 1H), 4.45-4.43 (m, 2H), 4.23 (s, 2H), 4.13-4.11 (m, 1H), 3.42-3.39 (m, 1H), 3.05 (td, J = 13.0, 2.7, 1H), 2.75- 2.60 (m, 2H), 2.26-2.15 (m, 3H), 1.68-1.62 (m, 2H), 1.36-1.29 (m, 2H), 1.15-1.09 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.1, 169.4, 149.1, 148.2, 140.6, 136.3, 134.2, 130.9 (2C), 129.2, 128.9 (2C), 128.8, 128.6 (2C), 128.5 (2C), 126.3, 123.7, 74.8, 59.5, 57.0, 44.7, 40.7, 34.0, 31.3, 27.1, 24.6, 20.4. LC/MS (m/z) 536.50 [M+H]+. Purity (HPLC, both diastereomers in total): 95.4%, tR = 8.89 min. HRMS (m/z) C29H33N3O5S, [M+H]+, calculated 536.22137, found 536.22170, error 0.6 ppm.
(R/S)-1-Oxo-4-(pyridin-3-yl)-1-((pyridin-3-ylmethyl)amino)butan-2-yl (S)-1-
(benzylsulfonyl)piperidine-2-carboxylate (S,R/S-10f) S,R/S-20f was Boc-deprotected following general procedure B using 2.00 mL TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (555 mg, 1.45 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (276 mg, 1.45 mmol) and NMM (175 μL, 1.60 mmol) in dry DCM (30 mL). After purification by flash chromatography (SiO2, A: DCM, B: MeOH, gradient: 0→ 30% B), S,R/S-10f was obtained as a colorless oil (380 mg, 0.71 mmol, 48%). Rf: 0.3 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3345, 3033, 2933, 1739, 1672, 1322,
Figure imgf000136_0002
1125, 696. Diastereomeric ratio: 53:47. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.59-8.57 (m, 1H), 8.52-8.50 (m, 1H), 8.43-8.39 (m, 2H), 7.82-7.64 (m, 2H), 7.52-7.50 (m, 1H), 7.41- 7.36 (m, 5H), 7.26-7.23 (m, 2H), 5.38-5.37 (m, 1H), 4.46-4.37 (m, 2H), 4.24-4.22 (m, 3H), 3.42-3.40 (m, 1H), 3.06-2.93 (m, 1H), 2.79-2.56 (m, 3H), 2.27-2.15 (m, 3H), 1.69-1.57 (m, 2H), 1.40-1.27 (m, 2H). 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.59-8.57 (m, 1H), 8.52-8.50 (m, 1H), 8.43-8.39 (m, 2H), 7.82-7.64 (m, 2H), 7.52-7.50 (m, 1H), 7.41-7.36 (m, 5H), 7.26- 7.23 (m, 2H), 5.27-5.25 (m, 1H), 4.46-4.37 (m, 2H), 4.24-4.22 (m, 2H), 4.13-4.11 (m, 1H), 3.25-3.21 (m, 1H), 3.06-2.93 (m, 1H), 2.79-2.56 (m, 3H), 2.27-2.15 (m, 2H), 2.05-2.02 (m, 1H), 1.69-1.57 (m, 2H), 1.40-1.27 (m, 2H). 13C NMR (CDCl3, δ [ppm]): 170.0, 169.1, 149.6, 149.4, 148.6, 147.6, 136.6, 136.3, 136.1, 134.1, 130.9 (2C), 129.3, 128.94 (2C), 128.90, 123.7 (2C), 74.5, 59.6, 57.0, 44.6, 40.8, 33.6, 28.3, 27.0, 24.6, 20.4. 13C NMR (CDCl3, δ [ppm]): 169.8, 169.1, 149.5, 149.3, 148.5, 147.3, 136.4, 136.2, 136.1, 134.0, 130.9 (2C), 129.1, 128.86 (2C), 128.8, 123.7 (2C), 74.3, 59.0, 56.3, 44.3, 40.8, 33.1, 27.7, 26.8, 24.6, 19.8. LC/MS (m/z) 537.10 [M+H]+, 269.20 [M+2H]2+. Purity (HPLC, both diastereomers in total): 98.8%, tR = 6.52 min, 6.59 min. HRMS (m/z) C28H32N4O5S, [M+H]+, calculated 537.21662, found 537.21764, error 1.9 ppm.
Example 3: Synthesis of the Pipecolic Amide Derivatives
Allyl-l-phenylalaninate hydrochloride (S-23a)
S-23a was synthesized according to general procedure G using 1.00 g of L- phenylalanine (6.05 mmol) and thionyl chloride (1.00 mL, 13.3 mmol) in allyl alcohol (12 mL, 181.6 mmol). The product was obtained as a white oily solid (957 mg, 3.96 mmol, 66%). Rf: 0.1 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3032, 2875, 1731,
Figure imgf000136_0001
1482, 1224, 823, 730. 1H NMR (DMSO-d6, δ [ppm], J [Hz]): 8.60 (s, 3H), 7.34-7.27 (m, 5H), 5.95-5.90 (m, 1H), 5.39-5.27 (m, 2H), 4.64-4.62 (m, 2H), 4.08 (t, J = 6.3, 1H), 3.14 (d, J = 6.3, 2H). 13C NMR (DMSO-d6, δ [ppm]): 170.1, 135.2, 131.7, 129.5 (2C), 128.5 (2C), 127.2, 118.2, 66.1, 52.5, 33.8.
Allyl-d-phenylalaninate hydrochloride ( R-23a) R-23a was synthesized according to general procedure G using 1.00 g of D- phenylalanine (6.05 mmol) and thionyl chloride (1.00 mL, 13.3 mmol) in allyl alcohol (12 mL, 181.6 mmol). Compound R-23a was obtained as a white oily solid (930 mg; 64%), Rf: 0.1 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3032, 2875, 1731, 1482,
Figure imgf000137_0003
1224, 823, 730. 1H NMR (DMSO-d6, δ [ppm], J [Hz]): 8.60 (s, 3H), 7.33-7.26 (m, 5H), 5.95-5.89 (m, 1H), 5.37-5.28 (m, 2H), 4.67-4.65 (m, 2H), 4.11 (t, J = 6.3, 1H), 3.10 (d, J = 6.3, 2H). 13C NMR (DMSO-d6, δ [ppm]): 170.3, 135.1, 131.5, 129.5 (2C), 128.5 (2C), 127.1, 118.5, 65.8, 53.3, 35.8.
Allyl-l-homophenylalaninate hydrochloride (S-23e)
S-23e was synthesized according to general procedure G using 500 mg of L- homophenylalanine (2.79 mmol) and thionyl chloride (0.50 mL, 6.14 mmol) in allyl alcohol (5.70 mL, 83.7 mmol). Compound S-23e was obtained as a white oily solid (420 mg; 60%), Rf: 0.1 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3052, 2857, 1741, 1582,
Figure imgf000137_0001
1519, 1263, 1192, 956, 700. 1H NMR (DMSO-d6, δ [ppm], J [Hz]): 8.76 (s, 3H), 7.32-7.21 (m, 5H), 5.98-5.91 (m, 1H), 5.39 (dd, J = 17.2, 1.4, 1H), 5.29 (dd, J = 10.5, 1.4, 1H), 4.70- 4.66 (m, 2H), 4.05-4.03 (m, 1H), 2.80-2.62 (m, 2H), 2.13-2.08 (m, 2H). 13C NMR (DMSO- d6, δ [ppm]): 169.0, 140.2, 131.7, 128.5 (2C), 128.3 (2C), 126.2, 118.8, 65.9, 51.5, 31.8, 30.2.
Allyl-d-homophenylalaninate hydrochloride (R-23e)
D-Homophenylalanine (500 mg, 2.79 mmol) was treated with allyl alcohol (5.70 mL, 83.7 mmol) and thionyl chloride (0.50 mL, 6.14 mmol) following general procedure G to obtain compound R-23e as a white oily solid (462 mg, 1.81 mmol, 65%), Rf: 0.1 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3045, 2853, 1741, 1581, 1518, 1263, 1096,
Figure imgf000137_0002
956, 700. 1H NMR (DMSO-d6, δ [ppm], J [Hz]): 8.85 (s, 3H), 7.32-7.18 (m, 5H), 6.00-5.90 (m, 1H), 5.43-5.37 (m, 1H), 5.30-5.27 (m, 1H), 4.69-4.67 (m, 2H), 4.04-4.02 (m, 1H), 2.83- 2.76 (m, 1H), 2.69-2.61 (m, 1H), 2.18-2.08 (m, 2H). 13C NMR (DMSO-d6, δ [ppm]): 169.0, 140.3, 131.8, 128.5 (2C), 128.3 (2C), 126.2, 118.7, 65.9, 51.5, 31.8, 30.3. (S)-1-(Allyloxy)-3-(4-fluorophenyl)-1-oxopropan-2-aminium chloride (S-23g)
S-23g was synthesized following general procedure G using 2450 mg of (S)-2-amino- 3-(4-fluorophenyl)propanoic acid (13.4 mmol), allyl alcohol (29.1 mL, 428 mmol), thionyl chloride (6.05 mL, 82.9 mmol) and additional DMF (1.0 mL, 12.9 mmol). After purification according to the general procedure, compound S-23g was obtained as a white oily solid (2300 mg, 8.86 mmol, 66%), Rf: 0.6 (DCSIL; DCM/MeOH = 20:1; KMnO4). IR (ATR), v [cm-1]: 3151, 2996, 2791, 1738, 1602, 1509, 1489, 1219, 1192, 991, 938, 822. 1H NMR (CD3OD, δ [ppm], J [Hz]): 7.31-7.26 (m, 2H), 7.13-7.07 (m, 2H), 5.97-5.85 (m, 1H), 5.37- 5.26 (m, 2H), 4.71-4.69 (m, 2H), 4.34-4.32 (m, 1H), 3.29-3.13 (m, 2H). 13C NMR (CD3OD, δ [ppm], J [Hz]): 169.7, 164.9 (d, JCF = 245.0, 1C), 132.4 (d, JCF = 8.2, 2C), 132.4, 131.3 (d, JCF = 3.3, 1C), 120.0, 116.8 (d, JCF = 21.8, 2C), 68.0, 55.2, 36.6.
Allyl-l-isoleudnate hydrochloride (S- 23d)
L-Leucine (2.00 g, 15.3 mmol) was treated with allyl alcohol (37.0 mL, 544 mmol) and thionyl chloride (4.00 mL, 54.8 mmol) in additional dry DCM (2 mL) following general procedure G to give S-23d as a colorless oil (2200 mg, 13.0 mmol, 84%), Rf: 0.66 (DCALOX; DCM/MeOH = 10:1; KMnO4). Data was consistent with the literature.10 tert-Butyl (S)-2-(((S)-1-(allyloxy)-1-oxo-3-phenylpropan-2-yl)carbamoyl)piperidine-1- carboxylate (S, S- 24a)
For the amidation of S-23a with (S)- 1 -Boc-pipcridinc-2-carboxylic acid (837 mg, 3.64 mmol), general procedure A was followed using 762 mg of EDC·HCl (3.97 mmol) and HOBt (223 mg, 1.66 mmol) in dry DCM (130 mL). S,S-24a was obtained as a yellow oil (915 mg, 2.20 mmol, 65%), Rf: 0.36 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR),
Figure imgf000138_0001
[cm -1]: 3340, 3004, 2935, 2859, 1741, 1677, 1509, 1455, 1158, 1031, 699. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.29-7.22 (m, 3H), 7.14-7.12 (m, 2H), 6.93-6.91 (m, 1H), 5.92-5.82 (m, 1H), 5.33-5.28 (dd, J = 17.2, 1.3, 1H), 5.25 (dd, J = 10.5, 1.1, 1H), 4.92-4.90 (m, 1H), 4.70- 4.61 (m, 1H), 4.62 (d, J = 5.8, 2H), 4.01-3.84 (m, 1H), 3.24 (dd, J = 14.0, 5.7, 1H), 3.05 (dd, J = 14.0, 7.2, 1H), 2.37-2.21 (m, 2H), 1.43 (s, 9H), 1.59-1.31 (m, 5H). 13C NMR (CDCl3, 8 [ppm]): 171.2, 170.9, 154.5, 136.0, 131.6, 129.4 (2C), 128.7 (2C), 127.3, 119.2, 80.7, 66.2, 53.1, 48.9, 42.3, 38.2, 28.4, 25.6, 24.9, 20.5. tert-Butyl (S)-2-(((R)- 1 -(allyloxy )-1-oxo-3-phenylpropan-2-yl)carbamoyl)piperidine-1- carboxylate (S,R-24a)
For the amidation of R-23a (800 mg, 3.31 mmol) with (S)-1-Boc-piperidine-2- carboxylic acid (837 mg, 3.64 mmol) general procedure A was followed using 762 mg of EDC HCl (3.97 mmol) and 223 mg of HOBt (1.66 mmol) in dry DCM (130 mL). S,R-24a was obtained as a yellow oil (688 mg, 1.65 mmol, 51%), Rf: 0.36 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR),
Figure imgf000139_0001
[cm-1]: 3340, 3008, 2935, 2859, 1741, 1677, 1509, 1455, 1158, 1031, 699. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.28-7.24 (m, 3H), 7.12-7.10 (m, 2H), 6.57-6.55 (m, 1H), 5.91-5.82 (m, 1H), 5.33-5.24 (m, 2H), 4.93-4.88 (m, 1H), 4.79-4.65 (m, 1H), 4.61 (d, 7 = 5.8, 2H), 4.04-3.94 (m, 1H), 3.12-3.10 (m, 2H), 2.77-2.75 (m, 1H), 2.29-2.27 (m, 1H), 1.44 (s, 9H), 1.62-1.26 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.3, 171.0, 154.6, 135.8, 131.6, 129.3 (2C), 128.8 (2C), 127.3, 119.3, 80.7, 66.2, 53.1, 48.9, 42.6, 38.0, 28.5, 25.0, 24.9, 20.7. tert-Butyl (S)-2-(((S)-1-(allylamino)-1-oxo-4-phenylbutan-2-yl)carbamoyl)piperidine-1- carboxylate (S,S-24e)
For the amidation of S-23e (370 mg, 1.45 mmol) with (S)-1-Boc-piperidine-2- carboxylic acid (366 mg, 1.60 mmol), general procedure A was followed using 334 mg of EDC·HCl (1.74 mmol), HOBt (100 mg, 0.73 mmol) and NMM (200 μL, 1.74 mmol) in dry DCM (60 mL). S,S-24e was obtained as a yellow oil (520 mg, 1.21 mmol, 83%), Rf: 0.5 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3341, 3012, 2935, 2862, 1737, 1684,
Figure imgf000139_0002
1365, 1249, 1158, 927, 699. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.30-7.14 (m, 5H), 6.93- 6.91 (m, 1H), 5.94-5.85 (m, 1H), 5.32 (dq, J= 17.3, 1.5, 1H), 5.26 (dq, J= 10.5, 1.2, 1H), 4.77-4.63 (m, 2H), 4.60 (dt, 7 = 5.9, 1.5, 2H), 4.15-4.02 (m, 1H), 3.80-3.78 (m, 1H), 2.67- 2.61 (m, 2H), 2.31-2.17 (m, 2H), 2.06-1.96 (m, 1H), 1.50 (s, 9H), 1.65-1.41 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.7, 171.3, 165.3, 140.7, 131.6, 128.7 (2C), 128.5 (2C), 126.3, 119.2, 80.9, 66.1, 53.2, 42.6, 34.4, 31.9, 28.5, 25.6, 25.0, 20.5. tert-Butyl (S)-2-(((R)- 1- (allylamino)-1-oxo-4-phenylbutan-2-yl)carbamoyl)piperidine- 1- carboxylate (S,R-24e) For the amidation of R-23e (400 mg, 1.56 mmol) with (S)-1-Boc-piperidine-2- carboxylic acid (428 mg, 1.87 mmol), general procedure A was followed using 300 mg of EDC·HCl (1.56 mmol), HOBt (42 mg, 0.31 mmol), and NMM (210 μL, 1.87 mmol) in dry DCM (60 mL). S,R-24e was obtained as a colorless oil (468 mg, 1.09 mmol, 70%), Rf: 0.5 (DCSIL; PE/EtOAc = 4:1; KMnO4). IR (ATR), [cm-1]: 3341, 3009, 2935, 2862, 1737, 1684,
Figure imgf000140_0001
1365, 1249, 1158, 927, 699. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.30-7.14 (m, 5H), 6.66- 6.64 (m, 1H), 5.94-5.84 (m, 1H), 5.33 (dq, J = 17.3, 1.5, 1H), 5.26 (dq, J = 10.5, 1.2, 1H), 4.82-4.80 (m, 1H), 4.70-4.64 (m, 1H), 4.61-4.58 (m, 2H), 4.12-4.02 (m, 1H), 2.93-2.86 (m, 1H), 2.67-2.62 (m, 2H), 2.32-2.17 (m, 2H), 2.04-1.96 (m, 1H), 1.50 (s, 9H), 1.65-1.41 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.9, 171.4, 162.5, 140.6, 131.6, 128.7 (2C), 128.5 (2C), 126.4, 119.2, 80.9, 66.1, 53.2, 42.3, 34.0, 31.8, 28.5, 25.5, 25.0, 20.6. tert-Butyl (S)-2-(((S)-1-(allyloxy)-3-(4-fluorophenyl)-1-oxopropan-2- yl)carbamoyl)piperidine-1-carboxylate ( S,S-24g)
S-23g (1950 mg, 7.51 mmol) was reacted according to general procedure A using (S)- l-Boc-piperidine-2-carboxylic acid (1.81 g, 7.88 mmol), HBTU (3.28 g, 8.64 mmol), and NEt3 (1.10 mL, 15.0 mmol) in dry DCM (200 mL). After stirring at rt for 4 d and workup according to general procedure A, the crude oily product was purified by column chromatography (aloxide basic III, PE/EtOAc/MeOH 90:30:4). S,S-24g was obtained as a colorless oil (3100 mg, 7.13 mmol, 95%). Rf: 0.66 (DCALOX; PE/EtOAc/MeOH = 9:3:0.4; KMnO4). IR (ATR), [cm-1]: 3330, 2974, 2937, 1740, 1671, 1508, 1158, 988, 928, 822. 1H
Figure imgf000140_0002
NMR (CDCl3, δ [ppm], J [Hz]): 7.13-7.05 (m, 2H), 7.01-6.92 (m, 2H), 6.61-6.32 (m, 1H), 5.92-5.82 (m, 1H), 5.31 (d, J = 17.2, 1H), 5.26 (d, J = 10.5, 1H), 4.87 (br s, 1H), 4.70 (br s, 1H), 4.61 (d, J = 5.9, 2H), 4.10-3.78 (m, 1H), 3.24-2.98 (m, 2H), 2.44 (br s, 1H), 2.26-2.18 (m, 1H), 1.80-1.20 (m, 5H), 1.44 (s, 9H). 13C NMR (CDCl3, δ [ppm]): 171.1, 162.2 (d, 1JCF = 245.5, 1C), 131.8, 131.5, 131.0 (d, 3JCF = 7.8, 2C), 119.4, 115.6 (d, 2JCF = 21.7, 2C), 80.9, 66.3, 55.5 & 54.1 (br s, 1C, rotamers), 53.2, 42.4 & 41.1 (br s, 1C, rotamers), 28.4 (3C), 25.6, 24.9, 20.5. tert-Butyl (S)-2-(((S)-1-(allyloxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)piperidine-1- carboxylate ( S,S-24d) For the amidation of S-23d (2.20 g, 12.9 mmol) with (S)-1-Boc-piperidine-2- carboxylic acid (3.77 g, 16.4 mmol), general procedure A was followed using HBTU (5.73 g, 15.1 mmol) and NEt3 (3.00 mL, 21.5 mmol) in DMF (due to solubility, 50 mL). After stirring at rt for 3 d and workup following general procedure A, the crude oily product was purified by column chromatography (aloxide basic III, Cy/EtOAc/MeOH = 15:4:1) and flash chromatography (SiO2, ELSD, A: CyH, B: EtOAc/MeOH 95:5, gradient:
Figure imgf000141_0001
100% B).
Compound S,S-24d was obtained as a colorless solid (1.90 g, 4.97 mmol, 39%). Rf: 0.51 (DCSIL; CyH/EtOAc/MeOH = 15:4:1; KMnO4). IR (ATR), -
Figure imgf000141_0003
[cm 1]: 3315, 2956, 2936, 2869, 2160, 1737, 1687, 1666, 1544, 1383, 1277, 930, 780.1H NMR (CDCl3, δ [ppm], J [Hz]): 6.65-6.20 (m, 1H), 5.95-5.83 (m, 1H), 5.32 (dd, J = 17.2, 1.3, 1H), 5.24 (d, J = 10.5, 1H), 4.74 (s, 1H), 4.61 (d, J = 5.7, 2H), 4.59 (s, 1H), 4.20-3.90 (m, 1H), 2.75 (s, 1H), 2.26 (s, 1H), 1.70-1.35 (m, 8H), 1.48 (s, 9H), 0.94 (d, J = 5.9, 6H). 13C NMR (CDCl3, δ [ppm], J [Hz]): 172.5, 171.2, 155.6 (br s, 1C), 131.8, 118.8, 80.9, 65.9, 55.7 & 53.8 (br s, 1C, retainers), 50.9, 42.6 (br s, 1C), 41.7, 28.5 (s, 3C), 25.5 (br s, 1C), 25.1 (s, 2C), 23.0, 21.9, 20.6.
Allyl ((S)-1-(benzylsulfonyl)piperidine-2-carbonyl)-l-phenylalaninate ( S,S-25a)
S,S-24a was Boc -deprotected following general procedure B using 5.00 mL of TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (736 mg, 2.33 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (530 mg, 2.79 mmol) and NMM (300 μL, 2.56 mmol) in dry DCM (50 mL). S,S-25a was obtained as a yellow oil (720 mg, 1.53 mmol, 66%). Rf: 0.45 (DCSIL; PE/EtOAc = 2:1; KMnO4). IR (ATR), [cm-1]: 3344, 3033, 2953, 2938, 1732,
Figure imgf000141_0002
1666, 1534, 1455, 1259, 694. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.43-7.36 (m, 5H), 7.28- 7.14 (m, 5H), 6.66 (d, 7 = 8.0, 1H), 5.94-5.84 (m, 1H), 5.35-5.25 (m, 2H), 4.88-4.83 (m, 1H), 4.66-4.64 (m, 2H), 4.30-4.28 (m, 1H), 4.23 (s, 2H), 3.40-3.37 (m, 1H), 3.27 (dd, J = 14.0, 5.4, 1H), 3.28 (dd, J = 14.0, 8.0, 1H), 2.59-2.52 (m, 1H), 2.13-2.09 (m, 1H), 1.58- 1.19 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.1, 169.9, 136.0, 131.6, 130.9 (2C), 129.4 (2C), 129.1, 129.0, 128.9 (2C), 128.8 (2C), 127.3, 119.2, 66.2, 59.0, 56.4, 53.5, 42.3, 37.9, 26.0, 24.6, 19.8.
Allyl ((S)-1-(benzylsulfonyl)piperidine-2-carbonyl)-D-phenylalaninate ( S,R-25a) S,R-24a (600 mg, 1.28 mmol) was Boc-deprotected following general procedure B using 3.00 mL of TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (500 mg, 1.58 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (360 mg, 1.90 mmol) and NMM (170 μL, 1.58 mmol) in dry DCM (30 mL). S,R-25a was obtained as a yellow oil (505 mg, 1.07 mmol, 68%), Rf: 0.45 (DCSIL; PE/EtOAc = 2:1; KMnO4). IR (ATR), -
Figure imgf000142_0001
[cm 1]: 3344, 3033, 2953, 2938, 1732, 1666, 1534, 1455, 1259, 916, 694. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.41-7.36 (m, 5H), 7.29-7.13 (m, 5H), 6.68 (d, J = 8.0, 1H), 5.91-5.82 (m, 1H), 5.33- 5.24 (m, 2H), 4.85-4.83 (m, 1H), 4.63-4.60 (m, 2H), 4.31-4.29 (m, 1H), 4.24-4.22 (m, 2H), 3.54-3.50 (m, 1H), 3.18-3.00 (m, 3H), 2.17-2.14 (m, 1H), 1.60-1.23 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.1, 169.9, 135.7, 131.6, 130.9 (2C), 129.4 (2C), 129.1, 129.0, 128.9 (2C), 128.8 (2C), 127.3, 119.2, 66.2, 59.0, 56.4, 53.4, 43.8, 37.9, 26.0, 24.5, 19.8.
(S)-N-((S)-1-(Allylamino)-1-oxo-4-phenylbutan-2-yl)-1-(benzylsulfonyl)piperidine-2- carboxamide ( S,S-25e)
S,S-24e (520 mg, 1.20 mmol) was Boc-deprotected following general procedure B using 3.00 mL of TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (328 mg, 0.99 mmol) was further reacted according to general procedure C using phenylmethane sulfonyl chloride (227 mg, 1.20 mmol) and NMM (120 μL, 1.09 mmol) in dry DCM (20 mL). S,S-25e was obtained as a yellow oil (345 mg, 0.71 mmol, 72%), Rf: 0.55 (DCSIL; PE/EtOAc = 2:1; KMnO4). IR (ATR), [cm-1]: 3345,
Figure imgf000142_0002
3030, 2955, 2938, 1731, 1670, 1530, 1455, 1254, 917, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.48-7.36 (m, 5H), 7.28-7.16 (m, 5H), 6.80 (d, 7 = 7.7, 1H), 5.94-5.87 (m, 1H), 5.35- 5.24 (m, 2H), 4.62-4.56 (m, 3H), 4.33-4.31 (m, 3H), 3.60-3.62 (m, 1H), 3.07-2.99 (m, 1H), 2.67 (t, J= 8.0, 2H), 2.25-2.01 (m, 3H), 1.63-1.21 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.6, 170.2, 140.6, 131.6, 130.9 (2C), 129.1, 129.0, 128.8 (2C), 128.7 (2C), 128.6 (2C), 126.4, 119.2, 66.2, 59.1, 56.4, 52.5, 44.3, 33.9, 31.9, 26.2, 24.6, 19.9.
(R)-V-((S)-1-(Allylamino)-1-oxo-4-phenylbutan-2-yl)- 1-( benzylsulfonyl )piperidine-2- carboxamide (S,R-25e)
S,R-24e (400 mg, 0.93 mmol) was Boc-deprotected following general procedure B using 3.00 mL of TFA in dry DCM (20 mL). After workup according to the general procedure, the free amine intermediate (297 mg, 0.90 mmol) was further reacted according to general procedure C using phenylmethanesulfonyl chloride (205 mg, 1.08 mmol) and NMM (110 μL, 0.99 mmol) in dry DCM (20 mL). S,R-25e was obtained as a colorless oil (266 mg, 0.55 mmol, 61%), Rf: 0.55 (DCSIL; PE/EtOAc = 2:1; KMnO4). IR (ATR), v [cm-1]: 3345, 3030, 2955, 2938, 1731, 1670, 1533, 1454, 1264, 1147, 917, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.48-7.37 (m, 5H), 7.29-7.16 (m, 5H), 6.79 (d, J = 8.02, 1H), 5.93-5.86 (m, 1H), 5.35-5.24 (m, 2H), 4.64-4.57 (m, 3H), 4.33-4.31 (m, 3H), 3.66-3.64 (m, 1H), 3.24-3.14 (m, 1H), 2.65 (t, J = 8.0, 2H), 2.25-2.17 (m, 2H), 2.00-1.95 (m, 1H), 1.63-1.28 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 171.7, 170.0, 140.6, 131.7, 130.9 (2C), 129.1, 129.0, 128.9 (2C), 128.6 (2C), 128.4 (2C), 126.3, 119.2, 66.2, 58.7, 56.5, 52.2, 44.0, 33.8, 31.7, 25.8, 24.5, 19.9.
Allyl-(S)-2-((S)-1-((4-fluorobenzyl)sulfonyl)piperidine-2-carboxamido)-3-(4- fluorophenyl)propanoate ( S,S-25g)
S,S-24g (3060 mg, 7.04 mmol) was Boc-deprotected following general procedure B using TFA (10 mL) in dry DCM (10 mL). After stirring at rt for 3 d and workup according to the general procedure, the amine intermediate (1.80 g, 5.38 mmol) was reacted according to general procedure C, using (4-fluorophenyl)methanesulfonyl chloride (1.36 g, 6.51 mmol) and DIPEA (3.00 mL, 17.2 mmol) in dry DCM (60 mL). After stirring at rt for 3 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (run 1: SiO2, A: DCM, B: MeOH, gradient: 0→ 30% B; run 2: RP 18, A: H2O + 0.1% FA, B: MeOH + 0.1% FA, gradient: 5→ 100% B). Compound S,S-25g was obtained as a colorless solid (448 mg, 0.95 mmol, 16%). Rf: 0.44 (DCALOX; PE/EtOAc/MeOH = 15:5:0.5; KMnO4). IR (ATR), v [cm-1]: 3356, 2939, 1739, 1667, 1603, 1508, 1327, 1215, 1147, 1061, 835, 715. Mp:104-106 °C. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.44-7.38 (m, 2H), 7.16-7.10 (m, 2H), 7.09-7.03 (m, 2H), 7.00-6.93 (m, 2H), 6.65 (d, J= 7.9, 1H), 5.89 (ddt, J = 17.0, 10.3, 5.9, 1H), 5.32 (d, J = 17.0, 1H), 5.27 (d, J = 10.3, 1H), 4.86-4.79 (m, 1H), 4.63 (d, J = 5.9, 2H), 4.30 (br d, J = 4.2, 1H), 4.20 (br s, 2H), 3.43 (d, J =
13.4, 1H), 3.23 (dd, J = 14.1, 5.4, 1H), 3.01 (dd, J = 14.1, J =7.6 , 1H), 2.69 (td, J = 13.4, 2.7, 1H), 2.12 (d, J = 13.3, 1H), 1.63-1.54 (m, 1H), 1.48-1.18 (m, 4H). 13C NMR (CDCl3, 6 [ppm]): 170.9, 170.0, 163.2 (d, 1JCF = 248.7, 1C), 162.2 (d, 1JCF = 245.6, 1C), 132.7 (d, 3JCF =
8.4, 2C), 131.7 (d, 4JCF = 3.3, 1C), 131.5, 131.0 (d, 3JCF = 8.0, 2C), 124.9 (d, 4JCF = 3.3, 1C),
119.4, 115.9 (d, 2JCF = 21.7, 2C), 115.7 (d, 2JCF = 21.4, 2C), 66.4, 58.2, 56.3, 53.6, 43.7, 37.2, 26.4, 24.8, 19.9. LC/MS (m/z) 507.25 [M+H]+. Purity (HPLC): 95.0%, tR = 9.58 min. HRMS (m/z) C25H28F2N2O5S, [M+H]+, calculated 507.17598, found 507.17627, error 0.6 ppm.
Allyl-(S)-1-((4-fluorobenzyl)sulfonyl)piperidine-2-carbonyl)-L-leucinate ( S,S-25d)
S,S-24d (200 mg, 462 μmol) was Boc-deprotected according to general procedure B using TFA (3.00 mL) in dry DCM (10 mL). After stirring at rt for 1 d and workup according to the general procedure, the amine intermediate (1.40 g, 4.96 mmol) was reacted according to general procedure C using (4-fluorophenyl)methanesulfonyl chloride (1.35 g, 6.45 mmol) and NMM (2.50 mL, 22.7 mmol) in dry DCM (100 mL). After stirring for 3 d and workup according to the general procedure, the crude oily product was purified consecutively by column chromatography (SiO2, EtOAc/PE/MeOH 1:3:0.05) and flash chromatography (run 1: SiO2, A: CyH, B: EtOAc/MeOH/FA (95:5:0.05); gradient:
Figure imgf000144_0001
run 2: RP 18, A: H2O + 0.1% FA, B: MeOH + 0.1% FA, gradient: 5→ 100% B). Compound S,S-25d was obtained as a colorless oil (1.39 g, 3.06 mmol, 62%). Rf: 0.71 (DCALOX; PE/EtOAc/MeOH = 3:1:0.05; KMnO4). IR (ATR), -
Figure imgf000144_0002
[cm 1]: 3532, 3359, 3078, 2953, 2870, 1739, 1674, 1604, 1508, 1327, 1292, 1126, 1058, 840, 711. Mp: 89 °C. 1H NMR (CDCl3, δ [ppm], J [Hz]): 7.47-7.41 (m, 2H), 7.10-7.03 (m, 2H), 6.51 (d, J = 8.3, 1H), 5.96-5.85 (m, 1H), 5.33 (d, J =
17.1, 1H), 5.25 (d, J = 10.5, 1H), 4.68-4.56 (m, 3H), 4.38 (s, 1H), 4.30-4.21 (m, 2H), 3.60- 3.52 (m, 1H), 3.11-3.02 (m, 1H), 2.21-2.16 (m, 1H), 1.73-1.46 (m, 7H), 1.39-1.24 (m, 1H), 0.97-0.94 (m, 6H). 13C NMR (CDCl3, δ [ppm]): 172.3, 170.3, 163.2 (d, 1JCF = 248, 1C), 132.7 (d, 3JCF = 8.3, 2C), 131.7, 125.0 (d, 4JCF = 3.5, 1C), 118.9, 115.9 (d, 2JCF = 21.6, 2C),
66.1, 58.1, 56.3 (br s, 1C), 51.2 (br s, 1C), 44.0, 41.3, 26.7, 25.1, 25.0, 23.0 (2C), 21.9, 20.0.
(S)-1-(Benzylsulfonyl)-N-((S)-1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2- yl)piperidine-2-carboxamide ( S,S-21 a )
S,S-25a was allyl-deprotected according to general procedure D using Pd(PPh3)4 (25 mg, 0.02 mmol) and morpholine (20 μL, 0.22 mmol) in dry THF (10 mL). After workup according to the general procedure, the carboxylic acid intermediate (90 mg, 0.21 mmol) was further reacted according to general procedure A using 3 -picolylamine (30 μL, 0.25 mmol), EDC·HCl (48 mg, 0.25 mmol), and HOBt (15 mg, 0.11 mmol) in dry DCM (10 mL). S,S-21a was obtained as a white oily solid (89 mg, 0.17 mmol, 82%), Rf: 0.2 (DCSIL; EtOAc = 100%). IR (ATR), v [cm-1]: 3268, 3030, 2936, 1731, 1650, 1540, 1495, 1322, 1146, 1056, 1029, 697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.60-8.58 (m, 2H), 7.60-7.58 (m, 1H), 7.37 (s, 5H), 7.29-7.14 (m, 6H), 6.92 (t, 7 = 5.6, 1H), 6.65 (d, 7 = 8.3, 1H), 4.79-4.73 (m, 1H), 4.41-4.39 (m, 2H), 4.23 (s, 2H), 4.10-4.08 (m, 1H), 3.31-3.29 (m, 2H), 3.03-2.97 (m, 1H), 2.68-2.66 (m, 1H), 2.44-2.36 (m, 1H), 2.11-2.09 (m, 1H), 1.45-0.87 (m, 4H). 13C NMR (CDCl3, δ [ppm]): 170.9, 170.6, 148.5, 147.9, 136.6 (2C), 134.8, 130.7 (2C), 129.2 (2C), 129.0 (2C), 128.9 (2C), 128.5, 127.2 (2C), 123.9, 58.7, 57.0, 54.5, 43.7, 41.0, 37.5, 25.6, 24.3, 19.5. LC/MS (m/z) 521.45 [M+H]+. Purity (HPLC): 97.9%, tR = 7.89 min. HRMS (m/z) C28H32N4O4S, [M+H]+, calculated 521.22170, found 521.22088, error 1.6 ppm.
(S)-1-(Benzylsulfonyl)-N-((R)-1-oxo-3-phenyl-1-((pyridin-3-ylmethyl)amino)propan-2- yl)piperidine-2-carboxamide ( S,R-21a)
S,R-25a was allyl-deprotected according to general procedure D using Pd(PPh3)4 (50 mg, 0.04 mmol) and morpholine (40 μL, 0.44 mmol) in dry THF (10 mL). After workup according to the general procedure, the carboxylic acid intermediate (150 mg, 0.40 mmol) was further reacted according to general procedure A using 3 -picolylamine (50 μL, 0.44 mmol), EDC·HCl (85 mg, 0.44 mmol), and HOBt (27 mg, 0.20 mmol) in dry DCM (20 mL). S,R-21a was obtained as a white oily solid (100 mg, 0.20 mmol, 49%), Rf: 0.2 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3296, 3032, 2939, 1649, 1534, 1322, 1125,
Figure imgf000145_0001
697. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.46-8.44 (m, 2H), 7.55-7.53 (m, 1H), 7.41-7.35 (m, 5H), 7.25-7.20 (m, 6H), 6.94 (t, 7 = 5.9, 1H), 6.82 (d, 7 = 7.8, 1H), 4.67-4.65 (m, 1H), 4.36 (d, 7 = 6.0, 2H), 4.27-4.18 (m, 2H), 4.13-4.11 (m, 1H), 3.35-3.33 (m, 1H), 3.14 (dd, 7 = 13.9, 7.9, 1H), 3.05-2.96 (m, 2H), 2.67 (m, 1H), 1.89-1.86 (m, 1H), 1.45-1.19 (m, 4H). 13C NMR (CDCl3, δ [ppm]): 171.0, 170.9, 148.6, 148.0, 136.6, 136.5, 134.3, 130.9 (2C), 129.3 (2C), 129.0 (2C), 128.9 (2C), 128.8, 127.1 (2C), 123.9, 58.8, 56.7, 54.8, 44.2, 41.0, 37.8, 26.9, 24.5, 19.8. LC/MS (m/z) 521.45 [M+H]+. Purity (HPLC): 99.3%, tR = 7.90 min. HRMS (m/z) C28H32N4O4S, [M+H]+, calculated 521.22170, found 521.22135, error: 0.7 ppm.
(S)-1-(Benzylsulfonyl)-N-((S)-1-oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2- yl)piperidine-2-carboxamide ( S,S-21 e )
S,S-25e (345 mg, 0.71 mmol) was allyl-deprotected according to general procedure D using Pd(PPh3)4 (82 mg, 0.07 mmol) and morpholine (60 μL, 0.75 mmol) in dry THF (10 mL). After workup according to the general procedure, the carboxylic acid intermediate (315 mg, 0.71 mmol) was further reacted according to general procedure A using 3- picolylamine (90 μL, 0.85 mmol), EDC·HCl (163 mg, 0.85 mmol), and HOBt (50 mg, 0.36 mmol) in dry DCM (30 mL). S,S-21e was obtained as a colorless oil (102 mg, 0.19 mmol, 31%), Rf: 0.35 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3287, 3029, 2921,
Figure imgf000146_0002
2852, 1741, 1671, 1536, 1322, 1125, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.52-8.47 (m, 2H), 7.65-7.63 (m, 1H), 7.43-7.36 (m, 5H), 7.25-7.11 (m, 7H), 6.85 (d, J = 8.1, 1H), 4.49- 4.40 (m, 3H), 4.29 (s, 2H), 4.19-4.17 (m, 1H), 3.49-3.46 (m, 1H), 2.99-2.93 (m, 1H), 2.64 (t, 7 = 7.9, 2H), 2.27-2.22 (m, 1H), 2.10-2.07 (m, 1H), 2.01-1.95 (m, 1H), 1.57-1.49 (m, 2H), 1.32-1.25 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 171.4, 170.9, 148.6, 148.1, 140.7, 136.4,
134.4, 130.9 (2C), 129.1 (2C), 129.0 , 128.7 (3C), 128.5, 126.3 (2C), 123.9, 58.8, 56.9, 53.4, 44.1, 41.0, 33.5, 32.1, 26.1, 24.6, 19.8. LC/MS (m/z) 535.50 [M+H]+. Purity (HPLC): 99.2%, tR = 8.19 min. HRMS (m/z) C29H34N4O4S, [M+H]+, calculated 535.23735, found 535.23729, error: 0.1 ppm.
(S)-1-(Benzylsulfonyl)-N-((R)-1-oxo-4-phenyl-1-((pyridin-3-ylmethyl)amino)butan-2- yl)piperidine-2-carboxamide ( S,R-21e)
S,R-25e (150 mg, 0.31 mmol) was allyl-deprotected according to general procedure D using Pd(PPh3)4 (35 mg, 0.03 mmol) and morpholine (30 μL, 0.33 mmol) in dry THF (10 mL). After workup according to the general procedure, the carboxylic acid intermediate (137 mg, 0.31 mmol) was further reacted according to general procedure A using 3- picolylamine (45 μL, 0.37 mmol), EDC·HCl (70 mg, 0.37 mmol), and HOBt (22 mg, 0.16 mmol) in dry DCM (10 mL). S,R-21e was obtained as a colorless oil (58 mg, 0.11 mmol, 33%), Rf: 0.35 (DCSIL; EtOAc = 100%). IR (ATR), [cm-1]: 3035, 2921, 2852, 1741, 1671,
Figure imgf000146_0001
1536, 1322, 1125, 695. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.63-8.53 (m, 2H), 7.76-7.74 (m, 1H), 7.55-7.52 (m, 1H), 7.38 (s, 5H), 7.33-7.30 (m, 1H), 7.27-7.12 (m, 6H), 5.25 (t, J = 5.6, 1H), 4.49 (dd, J = 14.9, 6.2, 1H), 4.42 (dd, J = 14.9, 5.9, 1H), 4.27-4.25 (m, 1H), 4.23 (s, 2H), 3.27-3.24 (m, 1H), 3.06-3.03 (m, 1H), 2.73-2.59 (m, 2H), 2.24-2.20 (m, 2H), 2.03- 2.00 (m, 1H), 1.61-1.58 (m, 2H), 1.42-1.31 (m, 3H). 13C NMR (CDCl3, δ [ppm]): 170.1,
169.5, 148.5, 147.6, 140.7, 137.2, 134.8, 130.9 (2C), 129.1, 129.0, 128.8 (2C), 128.6, 128.5 (2C), 126.3 (2C), 124.1, 74.8, 59.0, 56.2, 44.1, 40.7, 33.5, 30.8, 27.0, 24.6, 19.8. LC/MS (m/z) 535.45 [M+H]+. Purity (HPLC): 97.9%, tR = 8.20 min, HRMS (m/z) C29H34N4O4S, [M+H]+, calculated 535.23735, found 535.23660, error: 1.4 ppm. (S)-1-((4-Fluorobenzyl)sulfonyl)-N-((S)-3-(4-fluorophenyl)-1-oxo-1-((pyridin-3- ylmethyl)amino)propan-2-yl)piperidine-2-carboxamide ( S, S-21g)
S,S-25g (375 mg, 740 μmol) was allyl-deprotected according to general procedure D using Pd(PPh3)4 (86 mg, 74 μmol) and morpholine (70 μL, 0.80 mmol) in dry THF (30 mL). After stirring at rt for 21 h, the reaction mixture was worked up according to the general procedure. The crude oily intermediate product was purified by flash chromatography (SiO2, A: PE, B: EtOAc/MeOH 9:1 + 0.1% FA, gradient: 0→ 100% B) to obtain the carboxylic acid intermediate as a colorless solid (290 mg, 622 μmol, 84%). Subsequently, the intermediate product (277 mg, 590 μmol) was reacted according to general procedure A using HBTU (262 mg, 690 μmol), 3 -picolylamine (90 μL, 0.88 mmol), and DIPEA (0.21 mL, 1.21 mmol) in dry DCM (30 mL). After stirring at rt for 4 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (RP 18, A: H2O + 0.2% FA, B: MeOH + 0.2% FA, gradient: 5→ 100% B). Compound S,S-21g was obtained as a colorless solid (205 mg, 0.37 mmol, 62%). Rf: 0.56 (DCSIL; DCM/MeOH = 10:1). IR (ATR),
Figure imgf000147_0001
[cm-1]: 3290, 3068, 2941, 1650, 1603, 1508, 1326, 1220, 1126, 839, 773, 711. Mp:78 °C. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.55-8.44 (m, 2H), 7.56-7.52 (m, 1H), 7.38-7.32 (m, 2H), 7.26-7.21 (m, 1H), 7.16-7.02 (m, 5H), 6.94-6.80 (m, 3H), 4.75-4.66 (m, 1H), 4.45-4.30 (m, 2H), 4.22-4.11 (m, 3H), 3.34 (d, J = 13.5, 1H), 3.20 (dd, J = 14.2, 5.9, 1H), 2.97 (dd, J = 14.2, 8.6, 1H), 2.58 (ddd, J = 13.5, 13.5, 2.9, 1H), 2.07 (d, J = 13.9, 1H), 1.54-1.44 (m, 1H), 1.43-1.35 (m, 1H), 1.33-1.22 (m, 1H), 1.20-1.09 (m, 1H), 1.09-0.96 (m, 1H). 13C NMR (CDCl3, δ [ppm]): 170.6, 170.4, 163.3 (d, 1JCF = 249.3, 1C), 162.1 (d, 1JCF = 245.8, 1C), 149.4, 149.0, 135.7, 133.6, 132.6 (d, 3JCF = 8.3, 2C), 132.3 (d, 4JCF = 3.2, 1C), 130.8 (d, 3JCF = 7.9, 2C), 124.4 (d, 4JCF = 3.4, 1C), 123.7, 116.1 (d, 2JCF = 21.8, 2C), 115.8 (d, 2JCF = 21.3, 2C), 57.9, 57.0, 54.6, 43.8, 41.2, 36.9, 25.9, 24.5, 19.6. LC/MS (m/z) 557.45 [M+H]+. Purity (HPLC): 97.0%, tR = 8.10 min, HRMS C28H30F2N4O4S, [M+H]+, calculated 557.20286, found 557.20306, error: 0.4 ppm.
(S)-1-((4-Fluorobenzyl)sulfonyl)-N-((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)-amino)- pentan-2-yl)piperidine-2-carboxamide ( S,S-21d)
S,S-25d (1.30 g, 2.87 mmol) was allyl-deprotected according to general procedure D using Pd(PPh3)4 (332 mg, 287 μmol) and morpholine (260 μL, 3.01 mmol) in dry THF (30 mL). After stirring at rt for 4 d, the reaction mixture was worked up according to the general procedure. The crude oily product was purified by flash chromatography (run 1: SiO2, A: PE, B: EtOAc/MeOH 9:1 + 0.1% FA, gradient:
Figure imgf000148_0001
; run 2: RP 18, A: H2O
+ 0.1% FA, B: MeOH + 0.1% FA, gradient:
Figure imgf000148_0002
100% B) to obtain the carboxylic acid intermediate as a colorless oily solid (740 mg, 1.79 mmol, 62%). 681 mg of the intermediate product (1.64 mmol) were further treated according to general procedure A using DIPEA (180 pF, 1.80 mmol), 3 -picolylamine (260 pF, 2.55 mmol), and HBTU (917 mg, 2.42 mmol) in dry DCM (45 mF). After stirring at rt for 5 d and workup according to the general procedure, the crude oily product was purified by column chromatography (aloxide basic III, Cy/EtOAc/MeOH/FA = 50:48:1.9:0.1) and flash chromatography (run 1: RP 18, A: H2O + 0.1% FA, B: MeOH + 0.1% FA, gradient: 5→ 100% B; run 2: RP 18, A: H2O + 0.1% FA, B: ACN + 0.1% FA, gradient: 5→ 100% B). Compound S,S-21d was obtained as a colorless solid (479 mg, 0.95 mmol, 58%). Rf: 0.35 (DCALOX; CyH/EtOAc/MeOH/FA = 10:10:1:0.1). IR (ATR), [cm-1]: 3286, 3053, 2953, 2869, 1648, 1604, 1541, 1508, 1327,
Figure imgf000148_0003
1222, 1126, 975, 840, 772, mp: 70 °C. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.52-8.49 (m, 2H), 7.61-7.57 (m, 1H), 7.42-7.37 (m, 2H), 7.25-7.21 (m, 1H), 7.12-7.05 (m, 2H), 6.79 (t, J = 5.6, 1H), 6.57-6.54 (m, 1H), 4.50-4.44 (m, 1H), 4.42 (d, J = 6.0 , 2H), 4.24 (br s, 2H), 4.22-4.18 (m, 1H), 3.52 (d, J = 13.5, 1H), 3.03-2.93 (br s, 1H), 2.22-2.15 (m, 1H), 1.83-1.73 (m, 1H), 1.69-1.53 (m, 4H), 1.46-1.24 (m, 3H), 0.94 (d, J = 6.3, 3H), 0.91 (d, J = 6.3, 3H). 13C NMR (CDCl3, δ [ppm]): 171.8, 170.6, 163.3 (d, 1JCF = 249, 1C), 149.3, 149.0, 135.6,
133.9, 132.7 (d. 3JCF = 8.4, 2C), 124.5 (d, 4JCF = 3.4, 1C), 123.7, 116.1 (d, 2JCF = 21.7, 2C),
57.9, 57.0, 52.3, 44.2, 41.1, 40.5, 26.3, 25.2, 24.8, 23.2, 21.7, 19.9. EC/MS (m/z) 505.10 [M+H]+. Purity (HPFC): 99.0%, tR = 7.74 min, HRMS (m/z) C25H33FN4O4S, [M+H]+, calculated 505.22793, found 505.22772, error: 0.4 ppm.
3-(((S)-2-((R/S)-1-(Benzylsulfonyl)piperidine-2-carboxamido)-3-phenylpropanamido)- methyl)pyridine 1 -oxide (R/S,S-22a)
S,S-21a (75 mg, 0.14 mmol) was reacted with m-CPBA (50 mg, 0.28 mmol) in EtOAc (5 mF) following general procedure E. After stirring at rt for 2 h, additional EtOAc (20 mF) was added and the solution was washed with sat. NaHCO3 solution (3 x 20 mF) and water (3 x 20 mF). After separation of the phases, the crude oily product was purified by column chromatography (SiO2, EtOAc/PE/MeOH = 4:2:1). R/S,S-22a was obtained as a white oily solid (60 mg, 0.11 mmol, 78%), Rf: 0.38 (DCSIL; DCM/MeOH = 10:1). IR (ATR), [cm-1]: 3262, 3038, 2933, 1656, 1519, 1325, 1135, 786, 691. During basic purification, isomerization of the pipecolic acid stereocenter occurred. Diastereomeric ratio: 58:42. 1H NMR (ACN-d3, δ [ppm], J [Hz]): 8.03 (s, 1H), 7.96 (d, 7 = 6.4, 1H), 7.51 (t, J = 5.9, 1H), 7.42-7.33 (m, 5H), 7.29-7.01 (m, 8H), 4.72-4.58 (m, 1H), 4.35-4.16 (m, 5H), 3.46-3.44 (m, 1H), 3.22-3.12 (m, 1H), 2.95-2.85 (m, 2H), 2.03-2.01 (m, 1H), 1.58-1.26 (m, 4H), 1.16- 1.00 (m, 1H). 1H NMR (ACN-d3, δ [ppm], J [Hz]): 8.04 (s, 1H), 7.92 (d, J = 6.2, 1H), 7.58 (t, J = 5.9, 1H), 7.42-7.33 (m, 5H), 7.29-7.01 (m, 8H), 4.72-4.58 (m, 1H), 4.35-4.16 (m, 5H), 3.45 (d, J= 12.5, 1H), 3.26 (td, J= 12.7, 3.1, 1H), 2.95-2.85 (m, 2H), 1.83-1.81 (m, 1H), 1.58-1.26 (m, 4H), 1.16-1.00 (m, 1H). 13C NMR (ACN-d3, δ [ppm]): 171.8, 171.2, 140.6, 139.3, 138.6, 137.8, 131.5 (2C), 129.9 (2C), 129.0-128.8 (5C), 127.3, 126.6, 125.6, 58.3,
56.4, 55.1, 43.8, 40.2, 37.9, 27.0, 24.8, 19.9. 13C NMR (ACN-d3, δ [ppm]): 171.8, 171.2, 140.6, 139.4, 138.4, 138.0, 131.5 (2C), 129.9 (2C), 129.0-128.8 (5C), 127.2, 126.6, 125.5, 58.1, 56.3, 55.1, 43.9, 40.2, 37.9, 28.0, 25.0, 19.9. LC/MS (m/z) 537.10 [M+H]+. Purity (HPLC, both diastereomers in total): 99.5%, tR = 9.08 min; HRMS (m/z) C28H32N4O5S, [M+H]+, calculated 537.21662, found 537.21776, error: 2.1 ppm.
3-(((S)-2-((S)-1-((4-Fluorobenzyl)sulfonyl)piperidine-2-carboxamido)-4-methyl- pentanamido)methyl)pyridine 1-oxide (.S..S-22d)
S,S-21d (25 mg, 50 μmol) was reacted according to general procedure E using 21 mg of m-CPBA (99 μmol) in EtOAc (4 mL). After stirring at rt for 2 h, the crude oily product was purified by flash chromatography (run 1: SiO2, A: DCM, B: MeOH, gradient: 0→ 30% B; run 2: RP 18, A: H2O, B: ACN, gradient: 5→ 60% B). Compound S,S-22d was obtained as a white solid (20 mg, 38 μmol, 77%). Rf: 0.39 (DCSIL; DCM/MeOH = 9:1). IR (ATR),
Figure imgf000149_0001
[cm-1]: 3400-3200, 3070, 2952, 2871, 1654, 1541, 1508, 1223, 772, 761, 754, 716. Mp: 87 °C. 1H NMR (CD3OD, δ [ppm], J [Hz]): 8.31 (br s, 1H), 8.23-8.20 (m, 1H), 7.57-7.53 (m, 1H), 7.51-7.44 (m, 3H), 7.14-7.06 (m, 2H), 4.48-4.26 (m, 6H), 3.51-3.41 (m, 2H), 2.11- 2.03 (m, 1H), 1.72-1.54 (m, 6H), 1.47-1.32 (m, 2H), 0.98 (d, J = 6.4, 3H), 0.95 (d, J = 6.4, 3H). 13C NMR (CD3OD, δ [ppm], J [Hz]): 175.3, 173.8, 164.3 (d, 1JCF = 246.3, 1C), 140.9,
139.4, 139.0, 134.1 (d, 3JCF = 8.4, 2C), 130.0, 127.7, 127.2 (d, 4JCF = 3.3, 1C), 116.3 (d, 2JCF = 21.8, 2C), 57.9, 57.0, 53.4, 44.8, 41.6, 40.9, 29.0, 26.00, 25.95, 23.3, 22.0, 20.7. LC/MS (m/z) 521.15 [M+H]+. Purity (HPLC): 99.4%, tR = 8.74 min, HRMS (m/z) C25H33FN4O5S, [M+H]+, calculated 521.22285, found 521.22230, error: 1.1 ppm.
3-(((S)-2-((S)-1-((4-Fluorobenzyl)sulfonyl)piperidine-2-carboxamido)-3-(4-fhioro- phenyl)propanamido)methyl)pyridine 1-oxide (S,S-22g)
S,S-21g (10 mg, 18 μmol) was reacted according to general procedure E using 6.00 mg of m-CPBA (34.8 μmol) in 4 mL of EtOAc. After stirring overnight, the reaction mixture was loaded directly onto silica gel and purified by flash chromatography (SiO2, 2 • 4 g, A: DCM, B: MeOH, gradient: 0→ 30% B). Compound S,S-22g was obtained as a colorless solid (6.0 mg, 11 μmol, 58%). Rf: 0.48 (DCSIL; DCM/MeOH = 9:1). IR (ATR),
Figure imgf000150_0001
[cm-1]: 3292, 3073, 2968, 1656, 1508, 1222, 1157, 841, 817, 774. Mp: 98 °C. 1H NMR (CD3OD, δ [ppm], J [Hz]):8.26 (br s, 1H), 8.21 (d, J = 6.0, 1H), 7.48-7.39 (m, 4H), 7.27- 7.21 (m, 2H), 7.13-7.06 (m, 2H), 7.02-6.95 (m, 2H), 4.62-4.56 (m, 1H), 4.36-4.18 (m, 5H), 3.47-3.42 (m, 1H), 3.20 (ddd, J = 12.7, 12.7, 2.5, 1H), 3.15-2.92 (m, 2H), 2.03 (br d, J = 13.7, 1H), 1.65-1.47 (m, 3H), 1.40-1.19 (m, 2H). 13C NMR (CD3OD, δ [ppm], J [Hz]): 173.6, 173.3, 164.35 (d, 1JCF = 246.2, 1C), 163.29 (d, 1JCF = 243.8, 1C), 140.6, 139.5, 139.0, 134.14 (d, 3JCF = 8.3, 2C), 134.14 (d, 4JCF = 3.4, 1C), 132.1 (d, 3JCF = 8.0, 2C), 130.1, 127.7, 127.0 (d, 4JCF = 3.3, 1C), 116.26 (d, 2JCF = 21.9, 2C), 116.22 (d, 2JCF = 21.5, 2C), 58.0, 57.1, 56.3, 44.7, 40.9, 37.8, 28.6, 25.8, 20.6. LC/MS (m/z) 573.10 [M+H]+. Purity (HPLC): 97.4%, tR = 9.00 min, HRMS (m/z) C28H30F2N4O5S, [M+H]+, calculated 573.19777, found 573.19779, error: 0.0 ppm. tert-Butyl (S)-(l-oxo-3-(4-(prop-2-yn-1-yloxy)phenyl)-1-((pyridin-3-ylmethyl)amino)- propan-2-yl)carbamate ( S 26h )
(S)-2-((tert-Butoxycarbonyl)amino)-3-(4-(prop-2-yn-1-yloxy)phenyl)propanoic acid (639 mg, 2.00 mmol) was amide-coupled to 3 -picolylamine (244 μL, 2.40 mmol) according to general procedure A using DIPEA (696 μL, 4.00 mmol) and HBTU (833 g, 2.20 mmol) in dry DCM (80 mL). After stirring at rt for 5 d, the reaction was worked up according to the general procedure and purified by column chromatography (aloxide basic III, Cy/EtOAc/MeOH = 10:10:1) and flash chromatography (RP 18, A: H2O, B: MeOH, gradient: 5→ 100% B). Compound S-26h was obtained as a white solid (640 mg, 1.56 mmol, 78%). Rf: 0.56 (DCSIL; DCM/MeOH = 10:1). Mp: 164 °C. IR (ATR), [cm-1]: 3303, 3030, 2989, 1680,
Figure imgf000150_0002
1666, 1507, 1288, 827, 811, 751, 715. 1H NMR (CD3OD, δ [ppm], J [Hz]): 8.50-8.36 (m, 2H), 7.58 (d, J = 7.5, 1H), 7.40-7.31 (m, 1H), 7.12 (d, J = 8.5, 2H), 6.87 (d, J = 8.5, 2H), 4.70-4.68 (m, 2H), 4.40 (d, J = 15.3, 1H), 4.31 (d, J = 15.2, 1H), 4.24 (t, J = 7.2, 1H), 2.98 (dd, J = 13.7, 7.0, 1H), 2.92 (t, J = 2.4, 1H), 2.82 (dd, J = 13.6, 8.0, 1H), 1.39 (br s, 9H). 13C NMR (CD3OD, δ [ppm]): 174.5, 158.1, 158.0, 149.4, 148.7, 137.6, 136.4, 131.4 (2C), 131.2, 125.2, 116.0 (2C), 80.7, 79.9, 76.7, 57.8, 56.6, 41.4, 38.4, 28.7. tert-Butyl (S)-(1-(4-fluorophenyl)-2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)carbamate (5-26i)
(S)-A-Boc-4-Fluorophenylglycine (275 mg, 1.02 mmol) was amide-coupled to 3- picolylamine (146 μL, 1.43 mmol) according to general procedure A using DIPEA (0.4 mL, 2.3 mmol) and HBTU (426 mg, 1.12 mmol) in dry DCM (40 mL). After stirring at rt for 3 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (aloxide (neutral), A: DCM, B: MeOH, gradient: 0
Figure imgf000151_0001
25% B). S-26i was obtained as a colorless oil (340 mg, 946 μmol, 93%). Rf: 0.61 (DCSIL, DCM/MeOH = 9:1). IR (ATR), [cm-1]: 3300, 3062, 2975, 2929, 1701, 1662, 1604, 1507, 795, 745, 711. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.42 (dd, J = 4.8, 1.7, 1H), 8.35 (d, J = 1.7, 1H), 7.44 (d, J = 7.8, 1H), 7.34-7.29 (m, 2H), 7.16 (dd, J = 7.8, 4.8, 1H), 7.04 (br s, 1H), 7.01-6.94 (m, 2H), 5.87 (br s, 1H), 5.23 (br s, 1H), 4.45-4.32 (m, 2H), 1.36 (s, 9H). 13C NMR (CDCl3, δ [ppm]):
170.6, 162.7 (d, 1JCF = 247.4, 1C), 155.3, 149.0, 148.9, 135.4, 134.1 (d, 4JCF = 2.9, 1C),
133.7, 129.0 (d, 3JCF = 8.3, 2C), 123.7, 116.0 (d, 2JCF = 21.7, 2C), 80.5, 57.9, 41.2, 28.4 (3C). tert-Butyl (S)-2-(((S)-1-oxo-3-(4-(prop-2-yn-1-yloxy)phenyl)-1-((pyridin-3-ylmethyl)- amino)propan-2-yl)carbamoyl)piperidine-1-carboxylate ( S,S-27h)
S-26h (310 mg, 757 μmol) was Boc-deprotected according to general procedure B using TFA (9.00 mL) and dry DCM (10 mL). After stirring at rt for 1 d, the reaction was worked up according to the general procedure. The intermediate product (318 mg, 757 μmol) was amide-coupled to (S)-1-Boc-piperidine-2-carboxylic acid (196 mg, 856 μmol) following general procedure A using HBTU (282 mg, 744 μmol) and DIPEA (1.04 mL, 5.95 mmol) in dry DCM (40 mL). After stirring at rt for 2 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (aloxide (neutral), A: DCM, B: MeOH, gradient: 0→ 30% B). Compound S,S-27h was obtained as a slightly yellowish solid (380 mg, 730 μmol, 98%). Rf: 0.50 (DCSIL; DCM/MeOH = 10:1). Mp: 77 °C. IR (ATR),
Figure imgf000152_0002
[cm-1]: 3286, 3061, 2968, 2940, 1648, 1509, 869, 785, 773, 707. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.48 (dd, J = 4.8, 1.8, 1H), 8.43 (d, J = 1.8, 1H), 7.46 (d, J = 7.8, 1H), 7.21 (dd, J = 7.8, 4.8, 1H), 7.11-7.05 (m, 2H), 6.89-6.84 (m, 2H), 6.67 (br s, 1H), 6.47-6.43 (m, 1H), 4.71-4.65 (m, 1H), 4.67-4.63 (m, 2H), 4.63-4.59 (m, 1H), 4.44-4.30 (m, 2H), 3.82 (br s, 1H), 3.06 (d, 7 = 7.1, 2H), 2.50 (t, 7 = 2.4, 1H), 2.43 (br s, 1H), 2.18-2.10 (m, 1H), 1.59-1.43 (m, 3H), 1.40 (s, 9H), 1.37-1.22 (m, 2H). 13C NMR (CDCl3, δ [ppm]): 171.8, 170.9, 156.8, 155.7, 149.4, 149.0, 135.6, 133.7, 130.4 (2C), 129.4, 123.7, 115.4 (2C), 81.0, 78.6, 75.7, 56.0, 55.2, 54.4, 41.1 (2C), 37.1, 28.4 (3C), 25.5, 24.7, 20.4. tert-Butyl (S)-2-(((S)-1-(4-fluorophenyl)-2-oxo-2-((pyridin-3-ylmethyl)amino)ethyl)- carbamoyl)piperidine-1-carboxylate ( S,S-27 i )
S-26i (450 mg, 1.25 mmol) was Boc-deprotected according to general procedure B using TFA (5.0 mL, 66.0 mmol) in dry DCM (20 mL). After stirring at rt for 1 d, the reaction was worked up according to the general procedure. The intermediate product (245 mg, 946 μmol) was further reacted with (S)-1-Boc-piperidine-2-carboxylic acid (317 mg, 1.38 mmol) according to general procedure A using HBTU (395 mg, 1.04 mmol) and DIPEA (1.00 mL) in dry DCM (15 mL). After stirring at rt for 1 d, the reaction was worked up according to the general procedure and the crude oily product was purified by flash chromatography (aloxide (neutral), A: CHCl3 B: MeOH, 0→ 30% B). S,S-27i was obtained as a white solid (440 mg, 935 μmol, 99%). Rf: 0.78 (DCALOX; DCM/MeOH = 10:1). Mp: 147 °C. IR (ATR), [cm-1]: 3318, 3042, 2941, 1646, 1507, 1364, 1158, 837, 712. 1H NMR
Figure imgf000152_0001
(CDCl3, δ [ppm], J [Hz]): 8.50-8.46 (m, 2H), 7.62-7.56 (m, 1H), 7.39-7.27 (m, 3H), 7.01 (t, 7 = 8.6, 2H), 6.83 (br s, 1H), 5.49 (d, 7 = 6.7, 1H), 4.72 (br s, 1H), 4.51-4.39 (m, 2H), 3.96 (br s, 1H), 3.01 (br s, 1H), 2.68 (br s, 1H), 2.16-2.09 (m, 1H), 1.62-1.30 (m, 5H), 1.45 (s, 9H). 13C NMR (CDCl3, δ [ppm]): 171.5, 169.9, 162.8 (d, 1JCF = 247.6, 1C), 156.0, 149.0, 148.9, 135.5, 134.0 (d, 4JCF = 3.3, 1C), 133.7, 129.0 (d, 3JCF = 8.3, 2C), 123.7, 116.1 (d, 2JCF = 21.7, 2C), 81.1, 56.6, 54.8, 42.9, 41.3, 28.4 (3C), 25.7, 24.8, 20.5.
(S)-1-(Benzylsulfonyl)-N-((S)-1-oxo-3-(4-(prop-2-yn-1-yloxy)phenyl)-1-((pyridin-3- ylmethyl)amino)propan-2-yl)piperidine-2-carboxamide ( S,S-21 h ) S,S-27h (450 mg, 864 pmol) was Boc-deprotected according to General Procedure B using TFA (5 mL) and dry DCM (5 mL). After stirring at rt for 1 d, workup was conducted according to the general procedure. The intermediate product (363 mg, 863 μmol) was subsequently treated with phenylmethanesulfonyl chloride (330 mg, 1.73 mmol) and NMM (960 μL, 8.63 mmol) in dry DCM (45 mL) according to general procedure C. After stirring at rt for 5 d, the reaction was worked up according to the general procedure. Purification by flash chromatography (RP 18, A: H2O, B: MeOH, gradient: 40→ 80% B) afforded 5, 5-21h as a white solid (240 mg, 418 μmol, 48%), Rf: 0.51 (DCSIL; DCM/MeOH = 9:1). Mp: 123 °C. IR (ATR), [cm-1]: 3289, 2938, 2120, 1650, 1508, 1322, 1180, 823, 780, 739. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.51 (dd, J = 4.8, 1.7, 1H), 8.48 (d, J = 1.7, 1H), 7.53-7.49 (m, 1H), 7.40-7.35 (m, 5H), 7.25-7.21 (m, 1H), 7.10-7.06 (m, 2H), 6.89-6.83 (m, 2H), 6.71 (t, J = 5.8, 1H), 6.57 (d, J = 8.3, 1H), 4.73-4.65 (m, 1H), 4.65-4.60 (m, 2H), 4.45-4.33 (m, 2H), 4.22 (s, 2H), 4.07 (br d, J = 4.3, 1H), 3.32 (br d, J = 13.6, 1H), 3.22 (dd, J = 14.4, 5.9, 1H), 2.96 (dd, J = 14.4, 8.9, 1H), 2.48-2.38 (m, 2H), 2.10 (br d, J = 13.8, 1H), 1.50-0.87 (m, 5H). 13C NMR (CDCl3, δ [ppm]): 170.8, 170.5, 156.8, 149.4, 149.0, 135.6, 133.7, 130.8 (2C), 130.3 (2C), 129.5, 129.3, 129.0 (2C), 128.6, 123.7, 115.4 (2C), 78.6, 75.6, 58.8, 57.1, 56.0, 54.6, 43.8, 41.1, 36.7, 25.6, 24.3, 19.6. LC/MS (m/z) 575.20 [M+H]+. Purity (HPLC): 99.2%, tR = 8.18 min. HRMS (m/z) C31H34N4O5S, [M+H]+, calculated 575.23227, found 575.23197, error: 0.5 ppm. (S)-1-((4-Fluorobenzyl)sulfonyl)-N-((S)-1-(4-fluorophenyl)-2-oxo-2-((pyridin-3- ylmethyl)amino)ethyl)piperidine-2-carboxamide ( S,S-21 i )
S,S-27i (200 mg, 425 μmol) was first Boc-deprotected according to general procedure
B using 4 mL of TFA in dry DCM (20 mL). After stirring at rt for 1 d, the reaction was worked up according to the general procedure. Subsequently, the intermediate product (TFA salt, 254 mg, 425 μmol) was reacted according to general procedure C using (4- fluorophenyl)methanesulfonyl chloride (160 mg, 765 μmol) and DIPEA (740 μL, 4.25 mmol) in dry DCM (20 mL). After stirring at rt for 4 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (run 1: RP 18, A: H2O, B: MeOH, gradient: 5 → 70% B; run 2: RP 18, A: H2O + 0.1% FA, B: MeOH +
0.1% FA, gradient: 5 → 60% B). Compound S,S-21i was obtained as a white solid (40 mg,
74 μmol, 17%). Rf : 0.47 (DCSIL; DCM/MeOH = 10:1), Mp: 105 °C. IR (ATR), [cm-1]: 3289, 3061, 2939, 1652, 1604, 1507, 1326, 1223, 837, 773. 1H NMR (CDCl3, δ [ppm], J [Hz]): 8.58-8.38 (m, 2H), 7.54 (d, J = 7.7, 1H), 7.46 (d, J = 6.7, 1H), 7.38-7.31 (m, 4H), 7.25-7.22 (br s, 1H), 7.07-6.97 (m, 4H), 6.77 (br s, 1H), 5.48 (d, J = 6.7, 1H), 4.49-4.39 (m, 2H), 4.34-4.29 (m, 1H), 4.23-4.11 (m, 2H), 3.45 (d, J = 13.0, 1H), 3.03-2.92 (m, 1H), 2.12 (d, J = 13.3, 1H), 1.62-1.42 (m, 3H), 1.36-1.26 (m, 2H). 13C NMR (CDCl3, δ [ppm], J [Hz]): 170.3, 169.8, 163.2 (d, 1JCF = 248.6, 1C), 162.9 (d, 1JCF = 248.2, 1C), 148.6, 148.4, 136.2, 133.4 (d, 4JCF = 3.3, 1C), 132.7 (d, 3JCF = 8.4, 2C), 131.5, 129.3 (d, 3JCF = 8.3, 2C), 124.7 (d,4JCF = 3.3, 1C), 123.9, 116.3 (d, 2JCF = 21.7, 2C), 115.9 (d, 2JCF = 21.7, 2C), 58.0, 57.0, 56.6, 44.1, 41.4, 26.8, 24.8, 19.8. LC/MS (m/z) 543.10 [M+H]+. Purity (HPLC): 99.0%, tR = 7.74 min. HRMS (m/z) C27H28F2N4O4S, [M+H]+, calculated 543.18721, found 543.18764, error: 0.8 ppm.
(S)-1-(Benzylsulfonyl)-N-((S)-1-(4-fluorophenyl)-2-oxo-2-((pyridin-3- ylmethyl)amino)ethyl)piperidine-2-carboxamide ( S,S-28 i )
S,S-27i (200 mg, 425 μmol) was Boc -deprotected according to general procedure B using 4 mL of TFA in dry DCM (20 mL). After stirring at rt for 1 d, the reaction was worked up according to the general procedure. Subsequently, the intermediate product (85 mg, 229 μmol) was reacted according to general procedure C using phenylmethane sulfonyl chloride (101 mg, 482 μmol) and DIPEA (120 μL, 688 μmol) in dry DCM (15 mL). After stirring at rt for 2 d and workup according to the general procedure, the crude oily product was purified by flash chromatography (RP 18, A: H2O + 0.1% FA, B: MeOH + 0.1% FA, gradient: 5→ 100% B). Compound S,S-28i was obtained as a white, slightly yellowish oily solid (40 mg, 76 μmol, 33%). Rf: 0.50 (DCSIL; DCM/MeOH = 10:1). Mp: 143 °C. IR (ATR), [cm-1]: 3300, 3063, 3037, 2935, 1650, 1507, 837, 784, 737, 698. 1H NMR (CDCl3, 6 [ppm], J [Hz]): 8.47-8.33 (m, 2H), 7.59 (d, J = 7.0, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.38-7.29 (m, 8H), 7.21-7.15 (br s, 1H), 6.94 (t, J = 8.5, 2H), 5.54 (d, J = 7.0, 1H), 4.44-4.31 (m, 2H), 4.29-4.25 (m, 1H), 4.22-4.18 (m, 2H), 3.42 (d, J = 12.6, 1H), 3.01-2.91 (m, 1H), 2.02 (d, J = 13.0, 1H), 1.55-1.20 (m, 5H). 13C NMR (CDCl3, δ [ppm], J [Hz]): 170.4, 169.9, 162.7 (d, 1JCF = 247.7, 1C), 148.4, 148.1, 136.2, 134.2, 133.5 (d, 1JCF = 3.2, 1C), 129.10 (d, 3JCF = 8.3, 2C), 129.08, 128.9 (2C), 128.80 (2C), 128.78, 123.9, 116.0 (d, 2JCF = 21.7, 2C), 58.8, 56.7, 56.5, 44.0, 41.1, 26.5, 24.6, 19.7. LC/MS (m/z) 525.35 [M+H]+. Purity (HPLC): 96.9%, tR = 7.65 min. HRMS (m/z) C27H29FN4O4S, [M+H]+, calculated 525.19663, found 525.19591, error: 1.4 ppm.
Example 4: Synthesis of MIPS-0052721, MIPS-0052756, MIPS-0052488, MIPS-0052581, MIPS-0052658, MIPS-0052695, MIPS-005275
General Experimental: Anhydrous DCM was purchased in an anhydrous form and stored under nitrogen. PS refers to commercial petroleum spirits with a boiling point range of 60-80 °C. All column (flash) chromatography was performed on silica gel SiO2 (40-63 μm, normal phase liquid chromatogrphay) or C18 spherical (20-35 pm, 100 Å, reverse phase liquid chromatography) using automated chromatography systems (Biotage Isolera/Selekt) unless otherwise indicated. Analytical TLC was performed using aluminium backed 0.2 mm thick silica gel 60 GF254 plates. The TLC plates were visualised using a 254 nm UV lamp, otherwise stained with 10% phosphomolybdic Acid (PMA) in ethanol. 1H NMR spectra were recorded at 400 MHz, 13C NMR at 101 MHz, 19F NMR at 377 MHz, unless otherwise indicated. For selected compounds, the number of attached hydrogens to each carbon atom was determined using Distortionless Enhancement by Polarization Transfer with detection of quaternary carbons (DEPTQ-135), as indicated. Chemical shifts were calibrated using residual nondeuterated solvent as an internal reference and are reported in parts per million (δ) relative to trimethylsilane (δ = 0). High-resolution mass spectra (HR-ESI) were recorded on a time of flight mass spectrometer fitted with an electrospray (ESI) ion source, the capillary voltage was 2400 V. Melting points were measured using an electrothermal melting point apparatus. Liquid chromatography-Mass spectrometry (LC-MS) was performed using an UHPLC/MS 1260/6120, detection at 254 nm and 214 nm. Liquid chromatography-Mass spectrometry (LC-MS) was performed using ESI and APCI LC-MS. Each method used 254 nm detector and a reverse phase C8(2) 5 μ 50 × 4.6 mm 100A column. The column temperature was 30 °C and the injection volume, 5 μL. The eluent system used was solvent A (H2O 0.1% formic acid) and solvent B (MeCN 0.1% formic acid). The gradient for ESI was 5 to 100% B over A over 4 min then eluted 100% B for 6 min. The gradient for APCI was 5 to 100% B over A over 2 min then eluted 100% B for 2 min.
(2,4,6-Trifluorophenyl)methanesulfonyl chloride
Figure imgf000156_0001
A solution of thiourea (0.51 g, 6.67 mmol), 2,4,6-trifluorobenzylbromide (1.5 g, 6.67 mmol) in absolute EtOH (8 mL) was heated to reflux for 2 h. After this time, the reaction was concentrated under reduced pressure to give 2-(2,4,6-trifluorobenzyl)isothiouronium bromide (quant.) as a white solid, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) δ 9.19 (s, 4H), 7.32 (t, J = 8.7 Hz, 2H), 4.51 (s, 2H). LCMS Rf (min) = 2.803. MS m/z = 221.0 [M]+. aq. 2M HCl (0.5 mL) Was added to a suspension of 2- (2,4,6-trifluorobenzyl)isothiouronium bromide in MeCN (10 mL) at 0 °C and let stir for 10 min, where it became an orange solution. After this time, NCS (3.56 g, 26.68 mmol) was added at 0 °C, let stir for 10 min then let stir for 72 h at rt. EtOAc (10 mL) and aq. 2M HCl (2 mL) were added to the reaction, and the organic layer was separated. The aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were dried (MgSO4) and concentrated to give a yellow semi- solid crude residue. This crude material was loaded onto a short-plug of SiO2 as a suspension in 10% EtOAc in PS, and flushed through with 10% EtOAc in PS (150 mL). The collected filtrate were concentrated to give (2,4,6- trifluorophenyl)methanesulfonyl chloride (0.46 g, 52%, purity@~80%) as a colourless liquid. 1H NMR (400 MHz, CDCl3) 6 6.83 (t, J = 8.2 Hz, 2H), 4.96 (s, 2H); 19F NMR (376 MHz, CDCl3) δ -101.5, -107.8.
(S)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxylic add
Figure imgf000156_0002
A solution of (2,4,6-trifluorophenyl)methanesulfonyl chloride (0.46 g, 1.87 mmol, purity @90%) in anhydrous DCM (1.5 mL), was added to a clear solution of benzyl (S)- piperidine-2-carboxylate hydrochloride (0.40 g, 1.56 mmol), DIPEA (0.82 mL, 4.68 mmol) in anhydrous DCM (8 mL) at 0 °C, then warm to rt over 3 h. After this time, the reaction was concentrated onto SiO2 and chromatographed (SiO2, 10% EtOAc in PS) to give (S)- 1 -((2,4,6- trifluorobenzyl)sulfonyl)piperidine-2-carboxylic acid (0.63 g, 94%) as a white solid. 1 H NMR (400 MHz, CDCl3) 6 7.42 - 7.30 (m, 5H), 6.72 (t, J = 8.1 Hz, 2H), 5.25 (d, J = 12.2 Hz, 1H), 5.18 (d, J = 12.2 Hz, 1H), 4.68 (d, J = 4.3 Hz, 1H), 4.34 (s, 2H), 3.69 (d, J = 10.6 Hz, 1H), 3.31 (td, J = 12.5, 2.4 Hz, 1H), 2.25 (d, J = 13.5 Hz, 1H), 1.84 - 1.63 (m, 3H), 1.63 - 1.47 (m, 1H), 1.23 - 1.14 (m, 1H). HRMS calcd. for C20H20F3NO4SNa (M+Na)+ = 450.0957, found 450.0960.
(S)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxylic add
Figure imgf000157_0001
A solution of benzyl (S)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxylate (0.63 g, 1.46 mmol) in MeOH (7 mL) and THF (7 mL) was evacuated and backfilled with N2 x 4. Pd/C (Pd/C 10%, contains 67% water, 0.31 g) was added, and the mixture was evacuated and backfilled with H2, then let stir for 24 h at rt. After this time, the reaction was evacuated and backfilled with N2 x 1, then filtered through Celite, rinsed with MeOH and concentrated. The crude residue was concentrated onto SiO2 and chromatographed (SiO2, 0 - 15% MeOH in DCM) to give (S)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxylic acid (0.41 mg, 84%) as a grey solid.1H NMR (400 MHz, CDCl3) 6 6.73 (t, J = 8.1 Hz, 2H), 4.66 (d, J = 3.2 Hz, 1H), 4.37 (s, 2H), 3.68 (d, J = 11.6 Hz, 1H), 3.30 (t, J = 12.6 Hz, 1H), 2.26 (d, J = 12.7 Hz, 1H), 1.75 (t, J = 16.4 Hz, 3H), 1.63 - 1.50 (m, 1H), 1.44 - 1.30 (m, 1H); 19F NMR (376 MHz, CDCl3) 6 -105.8, -109.4; LCMS Rf (min)= 3.159. MS m/z = 336.1 [M-H]-.
Tert-butyl (S)-(4-methyl- 1-((( 1 -methyl- 1H-pyrazol-4-yl)methyl)amino)-1 -oxopentan-2- yl)carbamate
Figure imgf000157_0002
EDCI.HCl (0.62 g, 3.24 mmol), HOBt (0.44 g, 3.24 mmol) and DIPEA (1.2 mL, 6.75 mmol) were added to a solution of Boc-Leu-OH (0.63 g, 2.70 mmol) in anhydrous DMF (14 mL) at 0 °C and let stir for 10 min. (1 -Methyl- 1H-pyrazol-4-yl)methanamine (0.30 g, 2.70 mmol) was then added and let stir for 16 h at rt. After this time, the reaction was poured into water (10 mL) and EtOAc (10 mL) was added. The organic layer was washed with H2O (20 mL) and brine (20 mL), dried (MgSO4) and concentrated to give a crude residue, which was taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM) to give tert-butyl (S)-(4-methyl-1-(((1 -methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2-yl)carbamate (0.77 g, 89%) as a clear syrup. 1H NMR (400 MHz, DMSO) δ 8.03 (t, J = 5.3 Hz, 1H), 7.50 (s, 1H), 7.26 (s, 1H), 6.79 (d, J = 8.1 Hz, 1H), 4.07 (d, J = 5.5 Hz, 2H), 3.96 - 3.87 (m, 1H), 3.76 (s, 3H), 1.62 - 1.48 (s, 1H), 1.45 - 1.28 (m, 11H), 0.86 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H); LCMS Rf (min) = 3.327. MS m/z = 269.2 [M-tBu+]+.
4-(((S)-2-Ammonio-4-methylpentanamido)methyl)-1-methyl-1H-pyrazol-1-ium 2,2,2- trifluoroacetate
Figure imgf000158_0001
A solution of tert-butyl (S)-(4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)- 1-oxopentan-2-yl)carbamate (0.77 g, 2.37 mmol) in TFA (5 mL) was let stir for 5 h. After this time, the reaction was concentrated under reduced pressure to give 4-(((S)-2-ammonio-4- methylpentanamido)methyl)-1-methyl-1H-pyrazol- 1 -ium 2,2,2-trifluoroacetate (quant.) as a yellow syrup, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) δ 8.73 (t, J = 5.3 Hz, 1H), 8.10 (br s, 4H), 7.59 (s, 1H), 7.34 (s, 1H), 4.17 (d, J = 5.4 Hz, 2H), 3.80 (s, 3H), 3.69 (dd, J = 11.7, 6.4 Hz, 1H), 1.68 - 1.41 (m, 3H), 0.89 (t, J = 6.2 Hz, 6H). 19F NMR (376 MHz, DMSO) δ -74.7; LCMS Rf (min) = 2.522. MS m/z = 225.2 [M]+.
(S)-N-((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2-yl)-1-
((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxamide (MIPS-0052721)
Figure imgf000159_0001
EDCI.HCl (35 mg, 0.18 mmol), HOBt (24 mg, 0.18 mmol) and DIPEA (0.07 mL, 0.38 mmol) were added to a solution of (S)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2- carboxylic acid (54 mg, 0.15 mmol) in DMF (1 mL) at 0 °C and let stir for 10 min. A solution of 1 -methyl-4-(((S)-4- methyl-2-(methylammonio)pentanamido) methyl)-1H-pyrazol- 1-ium 2,2,2-trifluoroacetate (0.10 g, 0.23 mmol), DIPEA (0.07 mL, 0.38 mmol) in DMF (1 mL) was then added at 0 °C and let stir for 16 h at rt. After this time, the reaction mixture was diluted with H2O (~1.5 mL) and chromatographed (Cl 8, 5 - 100% MeCN in H2O) to give the titled compound (70.3 mg, 86%) as a white solid upon freeze-drying. 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 7.32 (s, 1H), 6.77 (t, J = 8.0 Hz, 2H), 6.45 (d, J = 8.5 Hz, 1H), 6.37 (t, J = 5.0 Hz, 1H), 4.49 - 4.32 (m, 4H), 4.25 (dd, J = 5.5, 1.4 Hz, 2H), 3.86 (s, 3H), 3.75 - 3.59 (m, 1H), 3.16 (td, J = 13.0, 2.6 Hz, 1H), 2.25 (d, J = 14.1 Hz, 1H), 1.80 - 1.50 (m, 7H), 1.45 - 1.28 (m, 1H), 0.95 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H); 19F NMR (376 MHz, CDCl3) δ -104.7, -109.3; LCMS Rf (min) = 3.270. MS m/z = 544.3 [M+H]+; HRMS calcd. for C24H33F3N5O4S (M+H)+ = 544.2200, found 544.2208. Tert-butyl (R)-3-(((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1- oxopentan-2-yl)carbamoyl)thiomorpholine-4-carboxylate
Figure imgf000159_0002
EDCI.HCl (46 mg, 0.24 mmol), HOBt (32 g, 0.24 mmol) and DIPEA (80 μL, 0.5 mmol) were added to a solution of (R)-4-(tert-butoxycarbonyl)thiomorpholine-3-carboxylic acid (50 mg, 0.20 mmol) in anhydrous DMF (1 mL) at 0 °C and let stir for 10 min. 4-(((S)-2- ammonio-4-methylpentanamido)methyl)- 1-methyl-1H-pyrazol- 1-ium 2,2,2-trifluoroacetate (0.14 g, 0.30 mmol) was then added and let stir for 16 h at rt. After this time, the reaction was poured into water (10 mL) and extracted with EtOAc (10 mL). The organic layer was washed with H2O (20 mL) and brine (20 mL). The combined aq. layer was extracted again with EtOAc (2 x 10 mL) and the combined organic layer was dried (MgSO4) and concentrated to give a crude residue, which was taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM) to give Tert-butyl (R)-3-(((S)-4-incthyl- 1 -((( 1-methyl-1H-pyrazol-4- yl)methyl)amino)-1-oxopentan-2-yl)carbamoyl)thiomorpholine-4-carboxylate (90.7 mg, quant.) as a clear syrup. 1 H NMR (400 MHz, CDCl3) 6 7.39 (s, 1H), 7.33 (s, 1H), 6.45 - 6.20 (m, 2H), 5.00 - 4.86 (m, 1H), 4.43 (td, J = 8.5, 6.0 Hz, 1H), 4.35 - 4.19 (m, 3H), 3.87 (s, 3H), 3.13 (ddd, J = 13.6, 2.9, 1.6 Hz, 2H), 2.82 (dd, J = 13.6, 4.0 Hz, 1H), 2.75 - 2.65 (m, 1H), 2.47 - 2.38 (m, 1H), 1.76 - 1.53 (m, 3H), 1.48 (s, 9H), 0.94 (dd, J = 6.2, 4.3 Hz, 6H). LCMS Rf (min) = 3.445. MS m/z = 454.2 [M+H]+.
(3R)-3-(((2S)-4-methyl-1-(((1-methyl-1H-pyrazol-1-ium-4-yl)methyl)amino)-1- oxopentan-2-yl)carbamoyl)thiomorpholin-4-ium 2,2,2-trifluoroacetate
Figure imgf000160_0001
A solution of tert-butyl (R)-3-(((S)-4-methyl-1-(((l-methyl-1H-pyrazol-4- yl)methyl)amino)-1-oxopentan-2-yl)carbamoyl)thiomorpholine-4-carboxylate (90.7 mg, 0.20 mmol) in TFA (3 mL) was let stir for 5 h. After this time, the reaction was concentrated under reduced pressure to give 4-(((S)-2-ammonio-4-methylpentanamido)methyl)- 1 -methyl- 1H- pyrazol- 1-ium 2,2,2-trifluoroacetate (0.14 g, quant.) as a yellow syrup, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) 6 9.15 (d, J = 10.9 Hz, 1H), 9.04 - 8.92 (m, 1H), 8.67 (d, J = 7.9 Hz, 1H), 8.31 (t, J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.27 (s, 1H), 4.34 - 4.25 (m, 1H), 4.15 - 3.85 (m, 2H, masked under H2O peak), 3.78 (s, 3H), 3.54 (d, J = 12.6 Hz, 1H), 3.14 (d, J = 11.5 Hz, 1H), 3.06 - 2.81 (m, 4H), 2.75 - 2.66 (m, 1H), 1.66 - 1.56 (m, 1H), 1.51 - 1.37 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.5 Hz, 3H); 19F NMR (376 MHz, DMSO) δ -74.7; LCMS Rf (min) = 2.751. MS m/z = 354.2 [M]+.
(R)-N-((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2-yl)-4-
((2,4,6-trifluorobenzyl)sulfonyl)thiomorpholine-3-carboxamide (MIPS-0052756)
Figure imgf000161_0001
A solution of (2,4,6-Trifluorophenyl)methanesulfonyl chloride (96 mg, 0.33 mmol, purity @~85%) in anhydrous DCM (1 mL) was slowly added into a mixture of (3R)-3-(((2S)- 4-methyl- 1-(((1 -methyl-1H-pyrazol-1-ium-4-yl)methyl)amino)-1-oxopentan-2- yl)carbamoyl)thiomorpholin-4-ium 2,2,2-trifluoroacetate (0.14 g, 0.20 mmol), DIPEA (0.29 mL, 1.68 mmol) in anhydrous DCM (2 mL) at 0 °C, then let stir for 3 h at rt. After this time, the reaction was concentrated under reduced pressure to give a crude residue, then taken up in DMSO and chromatographed (C18, 5 - 100% MeCN in H2O) to give the titled compound (64.3 mg, 55%) as a white solid upon freeze-drying. 1H NMR (400 MHz, CDCl3) 6 7.38 (s, 1H), 7.31 (s, 1H), 6.77 (t, J = 8.0 Hz, 2H), 6.49 (d, J = 8.1 Hz, 1H), 6.40 - 6.35 (m, 1H), 4.70 (t, J = 3.3 Hz, 1H), 4.53 - 4.37 (m, 3H), 4.27 (d, J = 5.5 Hz, 2H), 3.97 (dt, J = 5.7, 3.2 Hz, 1H), 3.85 (s, 3H), 3.48 - 3.37 (m, 1H), 3.11 - 3.04 (m, 1H), 2.97 (dd, J = 14.2, 3.9 Hz, 1H), 2.83 (td, J = 13.3, 3.1 Hz, 1H), 2.47 (dd, J = 13.8, 1.6 Hz, 1H), 1.79 (ddd, J = 13.5, 8.2, 5.1 Hz, 1H) 1.71 - 1.59 (m, 2H), 0.95 (t, J = 6.6 Hz, 6H); 19F NMR (376 MHz, CDCl3) 6 -104.5, -109.4; LCMS Rf (min) = 3.573. MS m/z = 562.1 [M+H]+; HRMS calcd. for C23H30F3N5O4S2Na (M+Na)+ = 584.1584, found 584.1600. tert- butyl (S)-(4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)carbamate
Figure imgf000161_0002
EDCI.HCl (1.06 g, 5.54 mmol), HOBt (0.75 g, 5.54 mmol) and DIPEA (2 mL, 11.55 mmol) were added to a solution of (tert-butoxycarbonyl)-L-leucine (1.07 g, 4.62 mmol) in DMF (23 mL) at 0 °C and let stir for 10 min. Pyridin-3-ylmethanamine (0.5 g, 4.62 mmol) was then added and let stir for 16 h at rt. After this time, the reaction was diluted with H2O, extracted with EtOAc (3 x 20 mL). The organic layer was washed with brine (20 mL), dried (MgSO4) and concentrated to give tert-butyl (S)-(4-methyl-1-oxo-1-((pyridin-3- ylmethyl)amino)pentan-2-yl)carbamate (1.42 g, 96%) as an orange gum. 1H NMR (400 MHz, DMSO) δ 8.52 - 8.28 (m, 3H), 7.62 (d, J = 7.6 Hz, 1H), 7.35 - 7.29 (m, 1H), 6.92 (d, J = 7.9 Hz, 1H), 4.36 - 4.21 (m, 2H), 3.96 (dd, J = 14.0, 8.5 Hz, 1H), 1.63 - 1.52 (m, 1H), 1.52 - 1.28 (m, 11H), 0.87 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H). LCMS Rf (min) = 2.870. MS m/z = 322.3 [M+H]+.
(S)-2-amino-4-methyl-N-(pyridin-3-ylmethyl)pentanamide
Figure imgf000162_0001
A solution of tert-butyl (S)-(4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2- yl)carbamate (1.4 g, 4.36 mmol) in TFA (5 mL) was let stir for 5 h. After this time, the reaction was poured into ice water, and neutralised using sat. aq. NaHCOi. The aq. solution was then extracted with DCM (3 x 15 mL). The combined organic layers were dried (MgSO4), and concentrated to give (S)-2-amino-4-methyl-A-(pyridin-3- ylmethyl)pentanamide (0.22 g, 22%) as a pink syrup. 1H NMR (400 MHz, DMSO) 6 8.55 - 8.40 (m, 3H), 7.64 (d, J = 7.8 Hz, 1H), 7.34 (dd, J = 7.5, 4.9 Hz, 1H), 4.29 (d, J = 5.5 Hz, 2H), 3.19 (dd, 7 = 8.6, 5.5 Hz, 1H), 1.75 - 1.64 (m, 1H), 1.40 (ddd, J = 13.4, 8.1, 5.5 Hz, 1H), 1.24 (dt, J = 13.6, 7.8 Hz, 1H), 0.87 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 5.8 Hz, 3H). LCMS Rf (min) = 0.603. MS m/z = 222.1 [M+H]+.
(S)-3-((2-ammonio-4-methylpentanamido)methyl)pyridin-1-ium 2,2,2-trifluoroacetate
Figure imgf000162_0002
A solution of tert-butyl (S)-(4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2- yl)carbamate (1.25 g, 4.36 mmol) in TFA (5 mL) was let stir for 5 h at rt. The reaction was then concentrated under reduced pressure to give (S)-3-((2-ammonio-4- methylpentanamido)methyl)pyndin-1-ium 2,2,2-trifluoroacetate (2.5 g, quant.) as an orange syrup. 1H NMR (400 MHz, DMSO) δ 9.23 (t, J = 5.6 Hz, 1H), 8.76 (s, 2H), 8.28 - 8.18 (m, 4H), 7.85 (dd, 7 = 7.7, 5.6 Hz, 1H), 4.50 (ddd, J = 33.8, 15.6, 5.7 Hz, 2H), 3.81 (dd, J = 11.6, 6.6 Hz, 1H), 1.67 - 1.53 (m, 3H), 0.89 (t, J = 5.4 Hz, 6H). LCMS Rf (min) = 0.518. MS m/z = 222.2 [M]+.
Tert-butyl (R)-(4-methyl-1-oxo-1-((1-(pyridin-3-yl)cydopropyl)amino)pentan-2- yl)carbamate
Figure imgf000163_0001
EDCI.HCl (0.17 g, 0.89 mmol), HOBt (0.12 g, 0.89 mmol) and DIPEA (0.32 mL, 1.9 mmol) were added to a solution of (tert-butoxycarbonyl)-D-leucine (0.17 g, 0.75 mmol) in DMF (4 mL) at 0 °C and let stir for 10 min. A solution of 1-(pyridin-3-yl)cyclopropan-1- amine (0.17 g, 0.75 mmol) in DMF (1 mL) was then added and let stir for 16 h at rt. The reaction was diluted with H2O (5 mL), extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine (10 mL), dried (MgSO4) and concentrated to give a crude residue, which was taken up in DCM and chromatographed (0 - 20% MeOH in DCM) to give tert-butyl (R)-(4-methyl-1-oxo-1-((l-(pyridin-3-yl)cyclopropyl)amino)pentan-2- yl)carbamate (0.27 g, purity@90%) as a yellow syrup. 1H NMR (400 MHz, DMSO) 6 8.66 (s, 1H), 8.40 - 8.27 (m, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.23 (dd, J = 7.7, 4.7 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 3.92 (dd, J = 14.3, 8.4 Hz, 1H), 1.67 - 1.10 (m, 16H), 0.88 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). LCMS Rf (min) = 2.993. MS m/z = 348.3 [M+H]+.
(R)-3-(1-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)cydopropyl)pyridine 1- oxide
Figure imgf000163_0002
m-CPBA (75% containing m-Chlorobenzoic acid and H2O, 0.22 g, 1.25 mmol) was added to a solution of tert-butyl (R)-(4-methyl-1-oxo-1-((l-(pyridin-3- yl)cyclopropyl)amino)pentan-2-yl)carbamate (0.22 g, 0.62 mmol) in DCM at 0 °C, then let warm to rt and stir for 3 h. After this time, the reaction mixture was loaded onto S iO2 and chromatographed (0 - 20% MeOH in DCM) to give (R)-3-(1-(2-((tert- butoxycarbonyl)amino)-4-methylpentanamido)cyclopropyl)pyridine 1 -oxide (0.2 g, 88%) as a white solid. 1H NMR (401 MHz, DMSO) δ 8.70 (s, 1H), 8.00 (d, J = 6.4 Hz, 1H), 7.97 (s, 1H), 7.26 (dd, J = 7.6, 6.7 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 3.89 (dd, J = 14.6, 7.8 Hz, 1H), 1.62 - 1.22 (m, 16H), 0.87 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H).; LCMS Rf (min) = 3.037. MS m/z = 364.3 [M+H]+.
(R)-3-(1-(2-ammonio-4-methylpentanamido)cyclopropyl)-1-hydroxypyridin-1-ium 2,2,2- trifluoroacetate
Figure imgf000164_0001
A solution of (R)-3-(1-(2-((tert-butoxycarbonyl)amino)-4- methylpentanamido)cyclopropyl)pyridine 1 -oxide (0.24 g, 0.66 mmol) in TFA (3 mL) was left to stir for 3 h at rt. After this time, the reaction was concentrated to give (R)-3-(1-(2- ammonio-4-methylpentanamido)cyclopropyl)-1-hydroxypyridin-1-ium 2,2,2-trifluoroacetate (0.26 g, 81%) as a light-yellow gum. 1H NMR (400 MHz, DMSO) δ 9.23 (s, 1H), 8.18 - 8.00 (m, 5H), 7.34 (dd, J = 8.0, 6.5 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 1.66 - 1.50 (m, 3H), 1.40 (ddd, J = 10.0, 7.0, 4.9 Hz, 1H), 1.31 (ddd, J = 9.8, 6.8, 5.0 Hz, 1H), 1.22 (ddd, J = 12.0, 6.7, 5.2 Hz, 1H), 1.13 (ddd, 7 = 8.8, 7.0, 3.7 Hz, 1H), 0.91 (d, J = 3.3 Hz, 3H), 0.90 (d, J = 3.3 Hz, 3H); LCMS Rf (min) = 0.529/ 1.921. MS m/z = 264.2 [M + H]+.
Tert-butyl (S)-(4-methyl- 1-((( 1 -methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2- yl)carbamate
Figure imgf000164_0002
EDCI.HCl (0.62 g, 3.24 mmol), HOBt (0.44 g, 3.24 mmol) and DIPEA (1.2 mL, 6.75 mmol) were added to a solution of Boc-Leu-OH (0.63 g, 2.70 mmol) in anhydrous DMF (14 mL) at 0 °C and let stir for 10 min. (1 -Methyl-1H-pyrazol-4-yl)methanamine (0.30 g, 2.70 mmol) was then added and let stir for 16 h at rt. After this time, the reaction was poured into water (10 mL) and EtOAc (10 mL) was added. The organic layer was washed with H2O (20 mL) and brine (20 mL), dried (MgSO4) and concentrated to give a crude residue, which was taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM) to give tert-butyl (S)-(4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2-yl)carbamate (0.77 g, 89%) as a clear syrup. 1H NMR (400 MHz, DMSO) δ 8.03 (t, J = 5.3 Hz, 1H), 7.50 (s, 1H), 7.26 (s, 1H), 6.79 (d, J = 8.1 Hz, 1H), 4.07 (d, J = 5.5 Hz, 2H), 3.96 - 3.87 (m, 1H), 3.76 (s, 3H), 1.62 - 1.48 (s, 1H), 1.45 - 1.28 (m, 11H), 0.86 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H); LCMS Rf (min) = 3.327. MS m/z = 269.2 [M-tBu+]+.
4-(((S)-2-Ammonio-4-methylpentanamido)methyl)-1-methyl-1H-pyrazol-1-ium 2,2,2- trifluoroacetate
Figure imgf000165_0001
A solution of tert-butyl (S)-(4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)- l-oxopentan-2-yl)carbamate (0.77 g, 2.37 mmol) in TFA (5 mL) was let stir for 5 h. After this time, the reaction was concentrated under reduced pressure to give 4-(((S)-2-ammonio-4- methylpentanamido)methyl)-1-methyl-1H-pyrazol- 1 -ium 2,2,2-trifluoroacetate (quant.) as a yellow syrup, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) δ 8.73 (t, J = 5.3 Hz, 1H), 8.10 (br s, 4H), 7.59 (s, 1H), 7.34 (s, 1H), 4.17 (d, J = 5.4 Hz, 2H), 3.80 (s, 3H), 3.69 (dd, J = 11.7, 6.4 Hz, 1H), 1.68 - 1.41 (m, 3H), 0.89 (t, J = 6.2 Hz, 6H). 19F NMR (376 MHz, DMSO) δ -74.7; LCMS Rf (min) = 2.522. MS m/z = 225.2 [M]+.
Tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2- yl)carbamoyl)morpholine-4-carboxylate
Figure imgf000165_0002
EDCI.HCl (94 mg, 0.49 mmol), HOBt (70 mg, 0.49 mmol) and DIPEA (0.18 mL, 1.03 mmol) were added to a solution of (S)-4-(tert-butoxycarbonyl)morpholine-3-carboxylic acid (94 mg, 0.41 mmol) in DMF (2 mL) at 0 °C and let stir for 10 min. A solution of (S)-2- amino-4-methyl-A-(pyridin-3-ylmethyl)pentanamide (0.14 g, 0.61 mmol) in DMF (1 mL) was then added and let stir for 16 h at rt. After this time, the reaction was diluted with H2O, extracted with DCM (3 x 10 mL). The organic layer was washed with H2O (10 mL) and brine (10 mL), dried (MgSO4) and concentrated to give a crude residue, which was then taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM) to give tert-butyl (S)-3-(((S)-4- methyl- 1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)carbamoyl)morpholine-4- carboxylate (0.15 g, 84%) as a transparent gum. 1H NMR (400 MHz, CDCl3) 6 8.55 (br s, 2H), 7.67 (d, J = 7.5 Hz, 1H), 7.33 (br s, 1H), 6.81 (br s, 1H), 6.39 (d, J = 7.5 Hz, 1H), 4.57 - 4.21 (m, 5H), 3.84 (apparent d, J = 10.3 Hz, 2H), 3.57 (dd, J = 11.6, 3.3 Hz, 1H), 3.52 - 3.43 (m, 1H), 3.20 - 3.05 (m, 1H), 1.79 - 1.67 (m, 1H), 1.65 - 1.53 (m, 2H), 1.46 (s, 9H), 0.94 (t, J = 7.0 Hz, 6H); LCMS Rf (min) = 3.059. MS m/z = 435.3 [M + H]+.
(S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-1-ium-3-ylmethyl)amino)pentan-2- yl)carbamoyl)morpholin-4-ium 2,2,2-trifluoroacetate
Figure imgf000166_0001
A solution of tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3- ylmethyl)amino)pentan-2-yl)carbamoyl)morpholine-4-carboxylate (0.15 g, 0.35 mmol) in TFA (3 mL) was let stir for 5 h. After this time, the reaction was concentrated under reduced pressure to give (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-1-ium-3-ylmethyl)amino)pentan-2- yl)carbamoyl)morpholin-4-ium 2,2,2-trifluoroacetate (quant.) as a yellow gum, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) 6 9.37 (br s, 1H), 9.19 (br s, 1H), 8.80 - 8.72 (m, 2H), 8.69 (d, J = 4.7 Hz, 1H), 8.65 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.76 (dd, J = 7.2, 5.8 Hz, 1H), 4.39 (d, J = 5.8 Hz, 2H), 4.37 - 4.30 (m, 1H), 4.20 - 3.89 (m, 3H, masked by H2O resonance), 3.71 - 3.62 (m, 1H), 3.57 - 3.49 (m, 1H), 3.22 (d, J = 12.2 Hz, 1H), 3.17 - 3.07 (m, 1H), 1.67 - 1.41 (m, 3H), 0.90 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H); LCMS Rf (min) = 0.519. MS m/z = 335.2 [M]+. (S)-N-((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)-4-((2,4,6- trifluorobenzyl)sulfonyl)morpholine-3-carboxamide (MIPS-0052488)
Figure imgf000167_0001
A solution of (2,4,6-trifluorophenyl)methanesulfonyl chloride (84 mg, 0.34 mmol) in anhydrous DCM (1 mL) was slowly added into a stirring solution of (S)-3-(((S)-4-methyl-1- oxo-1-((pyridin-1-ium-3-ylmethyl)amino)pentan-2-yl)carbamoyl)morpholin-4-ium 2,2,2- trifluoroacetate (0.16 g, 0.28 mmol) in dry DCM (2 mL) at 0 °C, then let stir for 3 h at rt. After this time, the reaction was concentrated, taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM). The obtained product (purity @~90%) was purified through reverse phase chromatography (DMSO load, Cl 8, 5 - 100% MeCN in H2O) to give (S)-N- ((S)-4-methyl- 1-oxo-1-((pyridin-3-ylmethyl)amino )pentan-2-yl)-4-((2, 4,6- trifluorobenzyl)sulfonyl)morpholine-3-carboxamide (29.2 mg, 15%, purity@94% by 1H NMR) as a white solid upon freeze-drying. 1H NMR (401 MHz, DMSO) 6 8.61 (t, J = 5.8 Hz, 1H), 8.46 (d, J = 1.8 Hz, 1H), 8.44 (dd, J = 4.8, 1.5 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.40 - 7.25 (m, 3H), 4.52 (d, J = 14.2 Hz, 1H), 4.42 (dd, J = 11.4, 6.0 Hz, 1H), 4.36 (d, J = 14.2 Hz, 1H), 4.30 (t, J = 5.5 Hz, 2H), 4.19 (d, J = 3.1 Hz, 1H), 4.16 (d, J = 12.0 Hz, 1H), 3.86 (dd, J = 11.1, 3.0 Hz, 1H), 3.75 (dd, J = 11.7, 3.6 Hz, 1H), 3.60 (dd, J = 11.9, 3.7 Hz, 1H), 3.49 - 3.39 (m, 2H), 1.63 - 1.54 (m, 1H), 1.54 - 1.42 (m, 2H), 0.90 (d, J = 6.5 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H). 19F NMR (376 MHz, CDCl3) 6 -104.42 (s), -109.26 (s). LCMS Rf (min) = 3.080. MS m/z = 543.3 [M+H]+. HRMS calcd. for C24H30F3N4O5S (M+H)+ = 543.1884, found 543.1873.
1-Benzyl 4-( tert- butyl) (S)-2-(((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-
2-yl)carbamoyl)piperazine-l,4-dicarboxylate
Figure imgf000168_0001
EDCI.HCl (0.13 g, 0.66 mmol), HOBt (89 mg, 0.66 mmol) and DIPEA (0.24 mL, 1.38 mmol) were added to a solution of (S)-1-((benzyloxy)carbonyl)-4-(tert- butoxycarbonyl)piperazine-2-carboxylic acid (0.20 g, 0.55 mmol) in DMF (3 mL) at 0 °C and let stir for 10 min. A solution of (S)-3-((2-ammonio-4-methylpentanamido)methyl)pyridin-1- ium 2,2,2-trifluoroacetate (0.37 g, 0.83 mmol), DIPEA (0.24 mL, 1.38 mmol) in DMF (1.5 mL) was then added and let stir for 16 h at rt. After this time, the reaction was poured into H2O, and EtOAc (5 mL) was added. The organic layer was separated, washed with brine x 2. The aq. layer was re-extracted with EtOAc (2 x 5 mL). The combined organic layer was dried (MgSO4), concentrated, to give the resultant crude residue, which was then taken up in DCM and chromatographed (SiO2, 0 - 10% MeOH in DCM) to give 1 -benzyl 4-(tert-butyl) (5)-2- (((S)-4-methyl- 1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)carbamoyl)piperazine- 1,4- dicarboxylate (0.30 g, 96%) as a clear solid. 1H NMR (401 MHz, CDCl3) 6 8.55 (s, 1H), 8.48 (d, J = 3.7 Hz, 1H), 7.66 (s, 1H), 7.52 (br s, 1H), 7.40 - 7.31 (m, 1H), 7.29 - 7.22 (m, 3H), 6.49 - 6.30 (m, 1H), 5.14 (s, 2H), 4.71 - 4.29 (m, 5H), 3.88 (br s, 2H), 3.49 - 3.08 (m, 1H), 3.00 (t, J = 10.9 Hz, 1H), 1.96 - 1.73 (m, 3H), 1.41 (s, 9H), 0.95 - 0.81 (m, 6H); LCMS Rf (min) = 3.217. MS m/z = 568.4 [M+H]+.
Tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2- yl)carbamoyl)piperazine-1-carboxylate
Figure imgf000168_0002
A solution of 1-benzyl 4-(tert-butyl) (S)-2-(((S)-4-methyl-1-oxo-1-((pyridin-3- ylmethyl)amino)pentan-2-yl)carbamoyl)piperazine-l,4-dicarboxylate (0.27 g, 0.47 mmol) in MeOH (5 mL) and THF (5 mL) was evacuated and backfilled with N2 x 4. Pd/C (0.13 g, containing 20% H2O) was added and the suspension was evacuated and backfilled with H2 x 4, then let stir for 1 h. After this time, the reaction was evacuated and backfilled with N2 x 1, filtered through Celite and rinsed with MeOH. The resultant organic layer was concentrated to give the titled compound (0.20 g, 96%) as a dark syrup, which was used in the next step without further purification. 1H NMR (401 MHz, DMSO) 6 8.54 (t, J = 6.0 Hz, 1H), 8.48 - 8.42 (m, 2H), 7.93 (d, J = 17.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.34 (dd, J = 7.6, 4.5 Hz, 1H), 4.39 - 4.23 (m, 3H), 3.84 - 3.73 (m, 1H), 3.58 (d, J = 12.4 Hz, 1H), 2.83 (d, J = 12.8 Hz, 2H), 2.57 - 2.52 (m, 1H), 1.58 - 1.47 (m, 3H), 1.40 - 1.35 (m, 1H), 0.87 (d, J = 6.2 Hz, 3H), 0.83 (d, J = 6.2 Hz, 3H). LCMS Rf (min) = 2.796, MS m/z = 434.4 [M+H]+.
Tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2- yl)carbamoyl)-4-((2,4,6-trifluorobenzyl)sulfonyl)piperazine-1-carboxylate
Figure imgf000169_0001
A solution of (2,4,6-trifluorophenyl)methanesulfonyl chloride (0.28 g, 1.12 mmol, purity @80%) in anhydrous DCM (1 mL) was added into a solution of tert-butyl (S)-3-(((S)-4- methyl- 1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)carbamoyl)piperazine-1-carboxylate (0.20, 0.45 mmol) and DIPEA (0.39 mL, 2.25 mmol) in anhydrous DCM (2.5 mL) at 0 °C then let warm stir for 16 h at rt. The reaction was then concentrated, taken up again in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM) to give tert-butyl (S)-3-(((S)-4-methyl- 1-oxo-1-((pyridin-3-ylmethyl)amino )pentan-2-yl)carbamoyl)-4-((2, 4,6- trifluorobenzyl)sulfonyl)piperazine-1-carboxylate (0.24 g, purity@80%), which was then taken up in DMSO and chromatographed (Cl 8, 5 - 100% MeCN in H2O), giving tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)carbamoyl)-4-((2,4,6- trifluorobenzyl)sulfonyl)piperazine-1-carboxylate (0.17 g, 58%) as a clear glass. 1H NMR (400 MHz, CDCl3) 6 8.54 (s, 1H), 8.47 (d, J = 3.6 Hz, 1H), 7.64 (d, J = 7.0 Hz, 1H), 7.45 (br s, 1H), 7.23 (dd, J = 7.7, 4.9 Hz, 1H), 6.74 (t, J = 8.1 Hz, 3H), 6.67 - 6.61 (m, 1H), 4.70 - 4.26 (m, 6H), 4.04 (d, J = 12.6 Hz, 1H), 3.65 - 3.45 (m, 2H), 3.16 (dd, J = 14.1, 4.1 Hz, 1H), 3.11 - 2.99 (m, 1H), 1.96 - 1.78 (m, 1H), 1.67 - 1.02 (m, 12H), 0.95 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H); 19F NMR (376 MHz, CDCl3) 6 -105.5, -109.7; LCMS Rf (min) = 3.037.
MS m/z = 364.3 [M+H]+.
(S)-N-((S)-4-methyl-1-oxo-1-((pyridin-3-ylmethyl)amino)pentan-2-yl)-1-((2,4,6- trifluorobenzyl)sulfonyl)piperazine-2-carboxamide (MIPS-0052581)
Figure imgf000170_0001
A solution of tert-butyl (S)-3-(((S)-4-methyl-1-oxo-1-((pyridin-3- ylmethyl)amino)pentan-2-yl)carbamoyl)-4-((2,4,6-trifluorobenzyl)sulfonyl)piperazine-1- carboxylate (53.2 mg, 0.08 mmol) in TFA (3 mL) was let stir for 3 h at rt. After this time, the reaction was concentration, re-dissolved in DCM and to that solution was added NEt3. This mixture was then concentrated again, and purified through reverse phase chromatography (C18, 5 - 100% MeCN in H2O) to give (S)-N-((S)-4-mcthyl- l -oxo- l -((pyridin-3- ylmethyl)amino)pentan-2-yl)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperazine-2-carboxamide (31 mg, 70%) as an off-white solid upon freeze-drying. 1H NMR (400 MHz, CDCl3) 6 8.52 (d, J = 1.4 Hz, 1H), 8.49 (dd, J = 4.8, 1.2 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.23 (dd, J = 8.0, 5.0 Hz, 1H), 6.74 (t, J = 7.9 Hz, 3H), 4.50 - 4.38 (m, 4H), 4.31 (d, J = 14.0 Hz, 1H), 4.24 (s, 1H), 3.54 (d, J = 11.3 Hz, 1H), 3.40 (d, J = 12.7 Hz, 1H), 3.26 (td, J = 12.5, 3.2 Hz, 1H), 2.99 (d, J = 11.7 Hz, 1H), 2.93 (dd, J = 12.9, 3.9 Hz, 1H), 2.82 (td, J = 12.0, 3.6 Hz, 1H), 1.83 - 1.72 (m, 1H), 1.70 - 1.50 (m, 3H), 0.96 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H). 19F NMR (376 MHz, CDCl3) 6 -105.0, -109.2. LCMS Rf (min) = 3.238. MS m/z = 542.1 [M+H]+. HRMS calcd. for C24H30F3N5O4SNa (M+Na)+ = 564.1863, found 564.1871.
Benzyl (S)-1-(N-(2,4,6-trifluorophenyl)sulfamoyl)piperidine-2-carboxylate
Figure imgf000171_0001
SO2CI2 (60 μL, 0.68 mmol) was slowly added to a solution of imidazole (0.14 g, 2.04 mmol) and 2,4,6-trifluoroaniline (0.10 g, 0.68 mmol) in anhydrous DCM (2.5 mL) at -78 °C, then let warm to rt and stir for 30 min, where LCMS indicated the formation of intermediate A-(2, 4, 6-trifluorophenyl)-1H- imidazole- 1 -sulfonamide (-50% conversion, with -20% of bis- trifluorophenylsulfamide as by-product). After this time, (S)-2- ((benzyloxy)carbonyl)piperidin-1-ium chloride (0.17 g, 0.68 mmol) was added and let stir for 16 h at rt. EtOAc (5 mL) and H2O (5 mL) were added to the reaction mixture and the organic layer was separated. The organic layer was then washed with H2O (5 mL) and brine (5 mL), dried (MgSO4) and concentrated onto SiO2, which was then chromatographed (0 - 50% EtOAc in PS) to give the titled compound (0.16 g, 56%) as an orange gum. 1H NMR (401 MHz, DMSO) δ 9.39 (s, 1H), 7.40 - 7.31 (m, 5H), 7.27 (td, J = 9.7, 1.7 Hz, 2H), 5.21 - 5.09 (m, 4H), 4.39 (d, J = 3.8 Hz, 1H), 3.50 (d, J = 10.4 Hz, 1H), 3.23 (td, J = 12.7, 2.7 Hz, 1H), 2.00 (d, J = 13.0 Hz, 1H), 1.70 - 1.53 (m, 3H), 1.38 (dt, J = 16.2, 12.6 Hz, 1H), 1.25 - 1.05 (m 1H). LCMS Rf (min) = 3.581. MS m/z = 429.2 [M+H]+.
Benzyl (S)-1-(A-methyl-A-(2,4,6-trifluorophenyl)sulfamoyl)piperidine-2-carboxylate
Figure imgf000171_0002
Mel (0.2 mL, 3.0 mmol) was added to a solution of benzyl (S)-1-(A-(2,4,6- trifluorophenyl)sulfamoyl)piperidine-2-carboxylate (0.16 g, 0.37 mmol), DIPEA (0.52 mL, 2.96 mmol) in anhydrous THF at 0 °C, then let warm to rt and left to stir for 16 h. After this time, the reaction was diluted with H2O, extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine (10 mL), dried (MgSO4), concentrated onto SiO2 and chromatographed (0 - 50% EtOAc in PS) to give Benzyl (S)- 1 -(N-methyl-A-(2,4,6- trifluorophenyl)sulfamoyl)piperidine-2-carboxylate (0.11 g, 67%) as a yellow gum. 1H NMR (400 MHz, CDCl3) 6 7.40 - 7.29 (m, 5H), 6.71 (dd, J = 14.5, 7.5 Hz, 2H), 5.23 (d, J = 12.3 Hz, 1H), 5.19 (d, J = 12.3 Hz, 1H), 4.68 (d, J = 5.3 Hz, 1H), 3.86 - 3.74 (m, 1H), 3.39 (td, J = 13.0, 3.1 Hz, 1H), 3.08 (s, 3H), 2.22 (d, J = 14.0 Hz, 1H), 1.91 - 1.49 (m, 5H); 19F NMR (376 MHz, CDCl3) 6 -106.6, -112.7, -113.3. LCMS Rf (min) = 3.698. MS m/z = 443.2 [M+H]+.
(S)-1-(N-methyl-N-(2,4,6-trifluorophenyl)sulfamoyl)piperidine-2-carboxylic add
Figure imgf000172_0001
A solution of benzyl (S)-1-(A-methyl-A-(2,4,6-trifluorophenyl)sulfamoyl)piperidine- 2-carboxylate (0.11 g, 0.25 mmol) in MeOH (2.5 mL) and THF (2.5 mL) was evacuated and backfilled with N2 x 4. Pd/C (55 mg, containing 20% H2O) was added and the suspension was evacuated and backfilled with H2 x 4, then let stir for 4 h. After this time, the reaction was evacuated and backfilled with N2 x 1, filtered through Celite and rinsed with MeOH. The resultant organic layer was concentrated to give (S)-1-(A-methyl-A-(2,4,6- trifluorophenyl)sulfamoyl)piperidine-2-carboxylic acid (74 mg, 84%) as a dark syrup. 1H NMR (400 MHz, CDCl3) 6 6.72 (td, J = 7.7, 3.5 Hz, 1H), 4.68 - 4.63 (m, 1H), 3.76 (d, J = 12.0 Hz, 1H), 3.35 (td, J = 12.7, 1.7 Hz, 1H), 3.13 (s, 2H), 2.26 (d, J = 12.6 Hz, 1H), 1.87 - 1.31 (m, 5H); 19F NMR (376 MHz, CDCl3) 6 -106.5, -112.7, -113.2; LCMS Rf (min) = 3.521. MS m/z = 353.0 [M+H]+.
(S)-N-((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-
2-yl)-1-(A-methyl-A-(2,4,6-trifluorophenyl)sulfamoyl)piperidine-2-carboxamide (MIPS- 0052658)
Figure imgf000173_0001
EDCI.HCl (48 mg, 0.25 mmol), HOBt (34 mg, 0.25 mmol) and DIPEA (0.09 mL, 0.53 mmol) were added to a solution of (S)-1-(A-methyl-A-(2,4,6- trifluorophenyl)sulfamoyl)piperidine-2-carboxylic acid (74 mg, 0.21 mmol) in DMF (1 mL) at 0 °C and let stir for 10 min. A solution of 4-(((S)-2-ammonio-4- methylpentanamido)methyl)-1-methyl- 1/7-pyrazol-l-ium 2,2,2-trifluoroacetate (0.17 g, 0.32 mmol), DIPEA (0.09 mL, 0.53 mmol) in DMF (1 mL) was then added and let stir for 16 h at rt. The reaction was diluted with H2O (~3 mL) and directly purified through reverse phase chromatography (C18, 5 - 100% MeCN in H2O) to give (S)-A-((S)-4-methyl-1-(((l-methyl- 1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2-yl)-1-(A- methyl- A-(2, 4,6- trifluorophenyl)sulfamoyl)piperidine-2-carboxamide (69 mg, 59%) as a white solid upon freeze-drying. 1H NMR (400 MHz, CDCl3) 6 7.35 (s, 1H), 7.28 (s, 1H), 6.76 (t, J = 8.2 Hz, 2H), 6.71 (d, J = 8.2 Hz, 1H), 6.34 (t, J = 4.8 Hz, 1H), 4.49 - 4.37 (m, 2H), 4.24 (d, J = 5.6 Hz, 2H), 3.85 (d, J = 9.8 Hz, 1H), 3.84 (s, 3H), 3.17 (s, 3H), 3.05 (td, J = 13.4, 2.8 Hz, 1H), 2.37 (d, J = 13.9 Hz, 1H), 1.83 - 1.37 (m, 8H), 0.94 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H); 19F NMR (376 MHz, CDCl3) 6 -105.3, -113.2; LCMS Rf (min) = 3.557. MS m/z = 559.2 [M+H]+. HRMS calcd. for C24H34F3N6O4S (M+H)+ = 559.2309, found 559.2320.
3-(1-((R)-2-((R)-1-(Terf-butoxycarbonyl)piperidine-2-carboxamido)-4- methylpentanamido)cydopropyl)pyridine 1-oxide
Figure imgf000173_0002
EDCI.HCl (51 mg, 0.26 mmol), HOBt (35 mg, 0.26 mmol) and DIPEA (0.1 mL, 0.55 mmol) were added to a solution of (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (50 mg, 0.22 mmol) in DMF (1.5 mL) at 0 °C and let stir for 10 min. A solution of (R)-3-(1- (2-ammonio-4-methylpentanamido)cyclopropyl)-1-hydroxypyridin-1-ium 2,2,2- trifluoroacetate (0.21 g, 0.44 mmol) in DMF (1 mL) was then added and let stir for 16 h at rt. The reaction was diluted with H2O (3 mL) and directly chromatographed (C18, 5 - 100% MeCN in H2O) to give 3-(1-((R)-2-((R)-1-(tert-butoxycarbonyl)piperidine-2-carboxamido)-4- methylpentanamido)cyclopropyl)pyridine 1-oxide (99 mg, 95%) as a white solid. 1H NMR (400 MHz, CDCl3) 6 8.06 (s, 1H), 8.03 (d, J = 5.9 Hz, 1H), 7.29 (br s, 1H), 7.22 - 7.10 (m, 2H), 6.41 (d, 7 = 7.1 Hz, 1H), 4.71 - 4.58 (m, 1H), 4.42 - 4.31 (m, 1H), 4.01 - 3.86 (m, 1H), 2.90 - 2.77 (m, 1H), 2.19 (d, J = 13.6 Hz, 1H), 1.65 - 1.53 (m, 8H), 1.46 (s, 9H), 1.32 - 1.22 (m, 4H), 0.94 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H). LCMS Rf (min) = 3.370. MS m/z = 475.1 [M+H]+.
3-(1-((R)-4-methyl-2-((R)-1-(phenylsulfonyl)piperidine-2- carboxamido)pentanamido)cydopropyl)pyridine 1-oxide (MIPS-0052695)
Figure imgf000174_0001
A solution of benzenesulfonyl chloride (30 μL, 0.25 mmol) in anhydrous DCM (0.5 mL) was added into a mixture of 1-hydroxy-3-(1-((R)-4-methyl-2-((R)-piperidin-1-ium-2- carboxamido)pentanamido)cyclopropyl)pyridin-1-ium 2,2,2-trifluoroacetate (0.13 g, 0.21 mmol), DIPEA (0.18 mL, 1.05 mmol) in anhydrous DCM (1.5 mL) at 0 °C, then warm to rt over 2 h. After this time, the reaction was concentration, re-dissolved in DCM and added NEt3 (~0.1 mL). This mixture was then concentrated again, taken up in DCM and chromatographed (SiO2, 0 - 20% MeOH in DCM), to give 3-( 1-((R)-4-methyl-2-((R)-1- (phenylsulfonyl)piperidine-2-carboxamido)pentanamido)cyclopropyl)pyridine 1 -oxide (95.7 mg, 89%) as a white solid upon freeze-drying.1H NMR (400 MHz, CDCl3) 6 8.10 (s, 1H), 8.06 - 8.03 (m, 1H), 7.86 (dd, J = 8.5, 1.3 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.38 (s, 1H), 7.23 - 7.17 (m, 2H), 6.82 (d, J = 8.4 Hz, 1H), 4.50 - 4.40 (m, 1H), 4.37 (s, 1H), 3.71 (dt, 7 = 7.3, 3.4 Hz, 1H), 3.14 (ddd, 7 = 13.8, 10.9, 3.0 Hz, 1H), 2.23 - 2.13 (m, 1H), 1.90 - 1.47 (m, 4H), 1.40 - 1.20 (m, 8H), 0.97 (d, 7 = 6.3 Hz, 3H), 0.92 (d, 7 = 6.4 Hz, 3H); LCMS Rf (min) = 3.097. MS m/z = 515.3 [M+H]+. HRMS calcd. for C26H35N4O5S (M+H)+ = 515.2323, found 515.2344. Tert-butyl (S)-4,4-difluoro-2-(((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4- yl)methyl)amino)-1-oxopentan-2-yl)carbamoyl)piperidine-1-carboxylate
Figure imgf000175_0001
EDCI.HCl (44 mg, 0.23 mmol), HOBt (31 mg, 0.23 mmol) and DIPEA (0.09 mL, 0.53 mmol) were added to a solution of (S)-1-(tert-butoxycarbonyl)-4,4-difluoropiperidine-2- carboxylic acid (50 mg, 0.19 mmol) in DMF (1 mL) at 0 °C and let stir for 10 min. A solution of 4-(((S)-2-ammonio-4-methylpentanamido)methyl)-1-methyl-1H-pyrazol- 1 -ium 2,2,2- trifluoroacetate (0.13 g, 0.29 mmol), DIPEA (0.09 mL, 0.53 mmol) in DMF (1 mL) was then added and let stir for 16 h at rt. The reaction was diluted with H2O (~3 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine (5 mL), dried (MgSO4 ) and concentrated to give a crude residue, which was then taken up in DCM and chromatographed (0 - 20% MeOH in DCM) to give Tert-butyl (S)-4,4-difluoro-2-(((S)-4- methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1-oxopentan-2- yl)carbamoyl)piperidine-1-carboxylate (74.9 mg, 84%) as a clear solid. 1H NMR (400 MHz, CDCl3) 6 7.40 (s, 1H), 7.33 (s, 1H), 6.30 (d, J = 8.3 Hz, 1H), 6.26 - 6.20 (m, 1H), 4.96 - 4.87 (m, 1H), 4.45 - 4.36 (m, 1H), 4.33 - 4.14 (m, 3H), 3.88 (s, J = 5.2 Hz, 3H), 3.16 - 3.01(m, 1H), 2.95 - 2.82 (m, 1H), 2.11 - 1.80 (m, 4H), 1.70 - 1.55 (m, 2H, masked by H2O resonance) 1.48 (s, 9H), 0.93 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H); 19F NMR (376 MHz, CDCl3) 6 -75.77 (s), -90.83 (d, J = 240.1 Hz); Rf (min) = 3.452. MS m/z = 472.2 [M+H]+.
(2S)-4,4-difluoro-2-(((2S)-4-methyl-1-(((1-methyl-1H-pyrazol-1-ium-4-yl)methyl)amino)-
1-oxopentan-2-yl)carbamoyl)piperidin-1-ium 2,2,2-trifluoroacetate
Figure imgf000175_0002
A solution of tert-butyl (S)-4,4-difluoro-2-(((S)-4-methyl-1-(((l-methyl-l//-pyrazol-4- yl)methyl)amino)-1-oxopentan-2-yl)carbamoyl)piperidine-1-carboxylate (74.9 mg, 0.16 mmol) in TFA (3 mL) was let stir for 5 h. After this time, the reaction was concentrated under reduced pressure to give (2S)-4, 4-difluoro-2-(((2S)-4-mcthyl- 1 -((( 1 -methyl-1H-pyrazol- 1 - ium-4-yl)methyl)amino)-1-oxopentan-2-yl)carbamoyl)piperidin- 1-ium 2,2,2-trifluoroacetate (0.19 g, quant.) as a yellow gum, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO) δ 9.30 (br s, 1H), 9.02 (br s, 1H), 8.71 (d, J = 7.9 Hz, 1H), 8.32 (t, J = 5.5 Hz, 1H), 7.51 (s, 1H), 7.27 (s, 1H), 4.40 - 3.92 (m, 3H, masked by H2O resonance), 3.78 (s, 3H), 3.49 - 3.31 (m, 1H), 3.14 - 3.05 (m, 1H), 2.74 - 2.63 (m, 1H), 2.37 - 1.95 (m, 4H), 1.67 - 1.56 (m, 1H), 1.55 - 1.41 (m, 2H), 0.90 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H); 19F NMR (376 MHz, DMSO) δ -74.7, -91.7 (d, J = 239.0 Hz), -100.4 (d, J = 239.0 Hz); LCMS Rf (min) = 2.792. MS m/z = 372.2 [M]+.
(S)-4,4-difluoro-N-((S)-4-methyl-1-(((1-methyl-1H-pyrazol-4-yl)methyl)amino)-1- oxopentan-2-yl)-1-((2,4,6-trifluorobenzyl)sulfonyl)piperidine-2-carboxamide (MIPS- 0052755)
Figure imgf000176_0001
A solution of (2,4,6-trifluorophenyl)methanesulfonyl chloride (0.19 g, 0.77 mmol) in anhydrous DCM (1 mL) was added into a mixture of (25)-4,4-difluoro-2-(((25)-4-methyl-1- ((( 1 -methyl-1H-pyrazol- 1 -ium-4-yl)methyl)amino)- 1 -oxopentan-2-yl)carbamoyl)piperidin- 1 - ium 2,2,2-trifluoroacetate (0.12 g, 0.16 mmol, calc, from the synthesis of ), DIPEA (0.22 mL, 1.28 mmol) in anhydrous DCM (1.5 mL) at 0 °C, then warm to rt and let stir for 16 h. After this time, concentration, the crude residue was taken up in DMSO and chromatographed (Cl 8, 5 - 100% MeCN in H2O), to give the titled compound (34.7 mg, 37%) as a white solid upon freeze-drying. 1 H NMR (400 MHz, CDCl3) 8 7.40 (s, 1H), 7.32 (s, 1H), 6.82 (t, J = 8.0 Hz, 2H), 6.41 (d, J = 8.4 Hz, 1H), 6.25 (t, J = 5.3 Hz, 1H), 4.71 - 4.64 (m, 1H), 4.56 - 4.42 (m, 3H), 4.28 (d, J = 5.4 Hz, 2H), 3.99 - 3.83 (m, 4H), 3.58 - 3.45 (m, 1H), 2.91 - 2.79 (m, 1H), 2.27 - 1.97 (m, 3H), 1.80 - 1.71 (m, 1H), 1.68 - 1.62 (m, 2H), 0.98 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H); 19F NMR (376 MHz, CDCl3) 6 -91.5 (d, J = 244.5 Hz), -102.5 (d, J = 244.6 Hz), -104.0, -109.6; LCMS Rf (min) = 3.610. MS m/z = 580.1 [M+H]+. HRMS calcd. for C24H31F5N5O4S (M+H)+ = 580.2011, found 580.2035.
Example 5: Synthesis of the FP probe.
(S)-1-(Benzylsulfonyl)-N-((S)-3-(4-((l-(3-(3',6'-dihydroxy-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-5-carboxamido)propyl)-1H-l,2,3-triazol-4- yl)methoxy)phenyl)-1-oxo-1-((pyridin-3-ylmethyl)amino)propan-2-yl)piperidine-2- carboxamide (29)
According to a modified protocol by Donnelly et al. (2008) to a solution of 5, 5-2111 (37.6 mg, 65.4 μmol) in a mixture of acetonitrile, DCM and EtOH (10:4:1, 15 mL), was given sodium ascorbate (23.0 mg, 116 μmol), copper(II) sulfate pentahydrate (20.0 mg, 80.1 μmol), tris((1-benzyl-4-triazolyl)methyl)amine (TBTA, 11.0 mg, 20.7 μmol) and N-(3- Azidopropyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamide75 (30, 30.0 mg, 65.4 μmol) at rt. After stirring for 9 d at rt, the mixture was loaded directly onto silica gel and purified by flash chromatography (run 1: SiO2, A: CHCl3 + 1% FA, B: MeOH + 1% FA, gradient: 0→ 30% B; run 2: RP 18, A: H2O, B: MeOH, gradient: 5→ 100% B). 29 was obtained as an orange solid (52.0 mg, 50.3 μmol, 77%). Rf : 0.40 (DCSIL; DCM/MeOH = 20:3), mp: 160 °C. IR (ATR), [cm-1]: 3600 - 3000, 3058, 2930,
Figure imgf000177_0001
1749, 1648, 1604, 846, 818, 791, 698. 1H NMR (DMSO-d6, δ [ppm], J [Hz]): 8.94 (s, 1H), 8.61 (s, 1H), 8.49 (s, 1H), 8.32-8.18 (m, 2H), 8.08 (d, J = 7.7, 1H), 7.78-7.27 (m, 8H), 7.15 (d, J = 7.6, 2H), 6.89 (d, J = 7.6, 2H), 6.80-6.43 (m, 5H), 5.03 (s, 2H), 4.63-4.52 (m, 1H), 4.52-4.43 (m, 2H), 4.40-4.29 (m, 3H), 4.26 (d, J = 13.6, 1H), 4.13 (d, J = 13.6, 1H), 3.24- 3.20 (m, 1H), 3.02-2.96 (m, 1H), 2.87-2.76 (m, 1H), 2.21-2.07 (m, 2H), 1.99 (d, J = 12.4, 1H), 1.54-1.40 (m, 3H), 1.23 (s, 1H), 1.17-1.07 (m, 1H), 1.06-1.01 (m, 1H). The signals from linker-CH2 (4H), amide-NH (1H), and hydroxy-OH (2H) are masked by the signal of residual water (3.62-3.27). 13C NMR (DMSO-d6, δ [ppm], J [Hz]): 171.1, 170.5, 168.3, 165.0, 160.8 (2C), 157.1, 156.7, 152.3 (2C), 148.8, 147.9, 142.7, 137.7, 136.1, 134.8, 134.3, 130.9 (2C), 130.2 (4C), 129.83, 129.77, 129.2, 128.2 (2C), 128.0, 124.61, 124.58, 124.54, 123.8, 114.3 (2C), 113.4 (2C), 109.3 (2C), 102.4 (2C), 62.0, 61.1, 57.0, 54.7, 54.2, 47.4, 42.7, 36.7, 36.6, 29.7, 27.6, 25.5, 24.4, 19.1. LC/MS (m/z) 517.30 [M+2H]2+, 345.15 [M+3H]3+. Purity (HPLC): 98.5%, tR = 8.57 min. HRMS C55H52N8O11S calculated 1033.35490, found 1033.35225 [M+H]+, error: 2.6 ppm.
Compound correspondence table Table A: Compound correspondence table for the compounds and intermediates disclosed herein.
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Example 6: Initial Screening of BpMip Inhibitors by Fluorescence Polarization and Subsequent Investigation of Inhibition of PPIase Activity
The potent tracer molecule 29 was used for performing the FPA with BpMip. In active site titration with BpMip, 29 reached a KD of 63 nM, which is within the potency of the inhibitors investigated and thus allows precise determination of activities even with minimal structural differences. The framework of the fluorescein-labeled probe 29 corresponds to that of the inhibitors to be tested, thus guaranteeing good comparability and specificity.
Ki values for the Mip inhibitors with BpMip were determined using fluorescence polarization, according to similar procedures by Kozany et al. (2009). Briefly, the tracer 29 was diluted in DMSO to obtain a stock solution of 40 μM. All further dilution steps were performed with the assay buffer (20 mM HEPES, 0.002% TritonX-100, 13.4 mM KC1) to a final concentration of 10 nM, which is four times higher than the final concentration in the well. All compounds were also prepared as a stock DMSO solution, followed by three different dilution series on three different days. Subsequently, 15 pl each of the compound and tracer were mixed with 30 μL of BpMip (stock solution 0.5 μM) into black 384-well plates (Greiner Bio-One, Kremsmunster, Austria, #781900) to obtain final concentrations of 2.5 nM of the fluorescent ligand and 250 nM of BpMip in the well. To ensure a state of equilibrium, incubation was performed for 30 minutes in the dark at room temperature. Finally, fluorescence polarization was measured (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany), and the competition curves were analyzed using GraphPad Prism 8.0.1.
The results of the FPA, which was used as a first screening method, and the protease- coupled BpMip-PPIase assay are largely congruent (cf. Table 1) Substances with a high binding affinity in the FPA also show an increased inhibition of PPIase activity at an inhibitor concentration of 400 nM, with only few deviations (e.g., R/S,S-10b and R/S, S- 10c). The introduction of an additional side chain appears to have a drastic amplifying effect on the inhibition of BpMip PPIase activity and on the binding affinity measured in the FPA, which is evident, for example, when comparing 9 (Ki, FP ≈ 4000 nM) with S,S- 10a (Ki, FP = 122 nM). By adding a side chain, the Ki can decrease from a micromolar to a two-digit nanomolar range (cf. Ki, FP (S,S-21i) = 17 nM). Here, the correct configuration at both stereo centers is essential for the binding affinity and enzymatic inhibition of BpMip. A comparison of all four stereo isomers (cf. S,S-10a, S,R-10a, R,R-10a, and R,S-10a) demonstrates clearly that an S-con figuration at both stereocenters (S,S-10a) is a preferred configuration, whereas the other stereo isomers show significantly reduced binding in the FPA and also reduced inhibition in the enzyme assay. This observation is confirmed for all other comparisons of stereo isomers with varying degrees of manifestation (cf. S,S-10d, S,S-10e, and S,S-21e). Enzymatic activity of BpMip can be almost entirely prevented (up to 95.8%) with compounds such as S,S-21d and S,S-21h. Replacement of piperidine-2-carboxylate (X = O, Scheme 1) by an amide (X = NH) seems to be well tolerated (cf. S,S-10e and S,S-21e). The amides also demonstrated good chemical and/or metabolic stability, and an affinity loss-free exchange supports this approach. With the right configuration, both aromatic and alkyl residues in the side chain (R2) work as possible substituents. Para-fluorination of both benzene rings (as in S,S-21g) also shows slightly better inhibition than the non-fluorinated derivative (S,S-21a). In addition, the fluorine substituent in para position was intended to block enzymatic metabolization. Consequently, the influence of the substitution on the anti-PPIase activity and the activity in the cell-based assay was to be tested. For aromatic side chains, all lengths of connecting methylene units (0-2) used between the attachment point and the benzene ring appear to be well tolerated (cf. S,S-28i, S,S-21a, and S,S-21e). The importance of the pyridine moiety becomes evident when replacing it with an allyl group (S,S-25g), resulting in a drastic loss of affinity. Oxidation of the pyridine moiety to the N-oxide results in highly increased solubility could argue for such replacement in future development towards drug like compounds.
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Example 7: Mip Inhibitors Reduce B. pseudomallei-induced Cytotoxicity in RAW264.7 Murine Macrophages
B. pseudomallei is an intracellular pathogen that replicates within macrophages. Infection of macrophages with B. pseudomallei leads to a high level of cell death 24 hours after infection, which can be measured by the release of lactose dehydrogenase (LDH). Deletion of Mip from B. pseudomallei has been shown to result in less severe infection, especially a decrease in cytotoxicity in macrophages.
The cytotoxicity assays were performed as described by Begley et al. (2014). Wild- type B. pseudomallei cells were pre-treated with 50 μM compounds for 1 hour before the bacteria could infect RAW264.7 murine macrophages with a multiplicity of infection of 10 (i.e., 10 bacterial cells per 1 macrophage cell). Infection was continued for 24 hours in the presence of 50 p M of the Mip inhibitor compound before a cytotoxicity measurement was performed by the detection of LDH in the supernatant. After infection with B. pseudomallei, treatment with new inhibitors bearing a side chain, such as S,S/R-10f, S,R-21a, S/R,S-22a,
S,S-211, and S,S-21d resulted in a significant reduction in cytotoxicity of up to 70% compared to the DMSO-only control (Table 2). This reduction was significantly higher than the levels observed for previous generations of Mip inhibitors (cf. the reduction of up to 40% in J774A.1 macrophages, determined by Begley et al. (2014) or the reduction of up to 32% for 9 and 11, see Table 2). Therefore, the general trend observed in the FPA and PPIase assay of the side chain’s positive effect on BpMip inhibition is supported by the cell-based assay data by the enhanced reduction of B. pseudomallei-induced cytotoxicity (cf. 9 and S,S/R-10f). Replacement of the pyridine moiety with a pyridine -A-oxide is well tolerated (cf. S,S-22d vs.
S,S-21d), sometimes even superior (cf. S,S-22g vs. S,S- 21g, S/R,S-22a vs. S,S-21a). In the side chain, the benzyl group seems superior to an ethylbenzene group (cf. S,S-21a vs.
S,S-21e). Interestingly, the replacement of the benzene ring by a (second) pyridine in the side chain increases the effectiveness considerably (S,S/R-10f vs. S,S/R-lOe).
Replacement of the pipecolic ester by an amide seems to be well tolerated, if not superior sometimes (cf. S,S-21a vs. S,S-10a and S,R-21a vs. S,R-10a). A para-fluoro substitution at the aromatic of the sulfonamide moiety results in a more pronounced reduction in B. pseudomallei-induced cytotoxicity on macrophages (cf. S,S-21i vs. S,S-28i and S,S-21d vs. S,S-10d). To examine a possible influence of the compounds on cell viability of RAW264.7 cells, all compounds were incubated with non-infected cells as a control. All compounds were found to induce only low cytotoxicity by themselves. The improved activity of novel Mip inhibitors such as S,R-21a, S,S-21i, and S,S-21d against B. pseudomallei- induced cytotoxicity (reduction of up to 70%) highlights the benefit of an additional side chain and the potential of these compounds as novel antibacterial agents for the treatment of melioidosis.
Table 2. Percent reduction in B. pseudomallei (B.ps. (-induced cytotoxicity in RAW264.7 murine macrophages when treated with Mip inhibitors. Percentage of survival of N. meningitidis and N. gonorrhoeae in RAW264.7 murine macrophages treated with Mip inhibitors at 50 μM compared to untreated control. Mean of n repetitions (at least triplicate).
Figure imgf000195_0001
Figure imgf000196_0001
Example 8: Screening Enzymatic Anti-PPIase Activity Against Mips of Different Neisseria Species
The high conservation of the PPIase domain of Mip proteins results in Mip inhibitors exhibiting activity in a wide range of pathogens. Consequently, the compound library optimized against BpMip was subsequently tested against the two Mip proteins of N. meningitidis - NmMip and NEIS0004 (see Table 1) The use of NmMip was also expected to provide information on the effect of compounds against N. gonorrhoeae since the analogs NgMip and NmMip possess more than 99% similarity in the gene sequence of the PPIase region. As in the case of BpMip, for both Mip proteins of N. meningitidis, the introduction of a side chain has a drastic effect on the activity of the inhibitors in the protease-coupled enzyme assay (cf. 9 with a ~38% reduction against NEIS0004 and S,S-10a). Compounds such as S/R,S -10d, S,S-10d, S,S-21d, and S,S-10a result in up to 96% reduction in NEIS0004 enzyme activity.
In the case of NmMip, compounds with a side chain, especially an aromatic one, such as S,S- 10a, S,S-21 g, and S,S-21 h, show significantly increased anti-PPIase activity with only -10% enzymatic activity remaining compared to non-sidechain bearing inhibitors, such as 9 (52.2% remaining). Against NEIS0004, substances with an (S, S)-configuration show strong anti-PPIase activity (cf. S,S-10e and S,S-10a), and also substances partially containing the (S, S)-diastereomer (cf. S/R,S- 10d and S/R,S -10a) show a high inhibition of the enzymatic activity of the Mip protein-.
The introduction of para-fluoro substituents at the sulfonamide benzene ring as well as at the aromatic side chain seems to be beneficial for PPIase inhibition of NmMip (cf.
S,S-21a vs. S,S- 21g and S,S-21i vs. S,S-28i). Particularly in the case of NmMip, there is a preference for a benzyl moiety as a side chain (cf. S,S-10a and S,S-21g). Replacement of the ester by an amide seems to be well tolerated for both proteins, NmMip and NEIS0004 (cf.
S,S-21e vs. S,S-10e). All in all, the results of inhibitors such as S,S-10a or S,S-21h in the protease-coupled enzyme assay clearly support the introduction of an additional side chain with a preference for an (S, S)-configuration.
Example 9: Mip Inhibitors Reduce N. meningitidis and N. gonorrhoeae Survival in RAW264.7 Murine Macrophages
RAW264.7 murine macrophages were seeded into 96-well tissue culture plates and used at a density of 1x106 cells/mL in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and IX GlutaMAX (Gibco)). N. meningitidis serogroup B or N. gonorrhoeae FA19 were harvested from a 14 hour agar plate culture, resuspended in DMEM and normalized to an optical density of 0.4 (550 nm). For each experiment, the actual number of bacteria was confirmed by viable count of the inoculum. Bacteria were pre-incubated with 50 μM of either DMSO or relevant inhibitors for 1 hour prior to infection of RAW274.7 murine macrophages at a multiplicity of infection of 100 whereby they were incubated for six hours and incubated in 5% CO2 at 37 °C. Following six hours of incubation, extracellular bacteria were then removed by washing the monolayers once with PBS and then the addition of DMEM with 100 pg/mL gentamicin (Agri-Bio) for 1 hour. The monolayers were washed once again with PBS to remove gentamycin, and macrophage cells were lysed with 1% (w/v) saponin (Sigma) in DMEM to release the intracellular bacteria. The number of intracellular bacteria was enumerated by viable count. Three biological replicates containing two technical repeats were performed for each strain. All statistical analyses were carried out using the Student’s t-test using Graphpad Prism v.8.
N. meningitis survival
The cell-based assay shows the enormous effectiveness of the studied Mip inhibitors in sensitizing macrophages to killing N. meningitidis almost completely as bacterial survival drops to as low as 13% (cf. S,S-10d, and S,S-10e, see Table 2). The promising results of sensitizing macrophages to kill bacteria highlight the efficacy of Mip inhibitors such as S,S-10d, and S,S-10e against N. meningitidis and the potential for using Mip inhibitors to combat invasive meningococcal disease.
N. gonorrhoeae survival
Mip inhibitors containing a side chain were investigated to determine whether they sensitize infected RAW264.7 murine macrophages to eliminate N. gonorrhoeae (see Table 2) As a result, it was shown that the studied Mip inhibitors cause a significant reduction in the survival of N. gonorrhoeae in RAW264.7 murine macrophages. Substances such as S,S-10d, and S,S-10e (at a concentration of 50 μM) lead to a reduction in pathogen load by up to 80%. Overall, compounds such as the pipecolic ester derivatives S,S-10d, and S,S-10e demonstrate that the Mip inhibitors tested are potent enzyme inhibitors of NgMip and strongly sensitize infected macrophages to eliminate N. gonorrhoeae, thus showing promise as agents to combat gonorrhoea and emerging resistances.
Example 10: Solubility and cytotoxicity studies To determine thermodynamic solubility, the continuous shake flask protocol of Hiltensperger et al. (2016) was applied, using an extended stirring time of up to 48 h to ensure thermodynamic equilibrium. Solubility studies of a selection of substances were evaluated (see Table 3).
Possible cytotoxicity of the Mip inhibitors was investigated by a formazan (WST-1) assay following a protocol by Griesbeck et al (2016). NIH 3T3 and HEK 293T cells were seeded in triplets using 96-well plates (NIH 3T3 : 40 x 103 cells/mL, HEK 293T: 50 x 103 cells/mL, 100 μL per well) and were grown for 48 h (method A), respectively 24 h (method B) at 37 °C and 5% CO2. Dilution series of each Mip inhibitor from 2 μM to 200 μM (method A) or from 0.391 μM to 200 μM (method B) were prepared in growth medium and added (1:1 [V/V]) to the cells, leading to inhibitor concentrations of (method A) 1 μM to 100 μM or (method B) 0.195 μM to 100 μM. The final concentration of DMSO was 1% at the highest inhibitor concentration and lower at the dilutions, respectively. The cells were then incubated for 24 h at 37 °C and 5% CO2. Untreated cells were used as control. Cell viability was assessed using the WST-1 colorimetric assay following the manufacturer's instructions. 10 μL WST-1 reagent was added to each well, and cells were incubated at 37 °C and 5% CO2, according to manufacturer instructions. After 1 h, 2 h, and 3 h, the absorbance of the soluble formazan product at 450 nm as well as background noise at 630 nm was determined using a SPECTRAmax 250 microplate reader (Molecular Devices, Sunnyvale, USA). Essentially all Mip inhibitors exhibit an excellent cytotoxicity profile with IC50 values above 100 μM.
Table 3. Cytotoxicity and solubility data of a selection of Mip inhibitors.
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Example 11: Demonstration of compound efficacy of against recombinant Mip proteins
Protease-coupled PPIase assay
Mip proteins from B. pseudomallei (BpMip) was recombinantly expressed and purified. Compounds were screened for activity against purified BpMip protein using the protease-coupled PPIase assay developed by Fischer et al. (1984) and as described in Vivoli et al. (2017). NJS224 and NJS227 were screened using a Fluorescence Polarization Assay inspired by Kozany et al. (2009). The results are shown in Table 4. Table 4: Ki values of compounds against BpMip.
Figure imgf000201_0002
Figure imgf000202_0001
Example 12: Demonstration of compound efficacy of against recombinant Mip proteins - inhibition of Kobs
Mip proteins from different pathogenic bacterial species including B. pseudomallei (BpMip), C. burnetii (CbMip) and Neisseria meningitidis (NmMip1 and AmMip2) were recombinantly expressed and purified. Compounds were screened for activity against purified Mip proteins using the protease-coupled PPIase assay developed by Fischer et al. (1984) and as described in Vivoli et al. (2017). A decrease in the observed rate of reaction (Kobs) correlates to a decrease in the ability of Mip to catalyze the conversion of the Suc-Ala-Phe- Pro-Phe-pNa substrate from the cis to the trans conformation. Co-incubation of Mip with compounds that are inhibitors of Mip will result in a decrease in Kobs compared to Mip co- incubated with the DMSO vehicle control. The results are shown in Table 5.
Table 5: Percent remaining observed rates of reactions (Kobs) for recombinant Mip proteins compared to DMSO vehicle control.
Figure imgf000202_0002
Figure imgf000203_0001
Example 13: Demonstration of compound efficacy against bacterial Mip using in vitro cell infection models
Burkholderia pseudomallei
B. pseudomallei induced cytotoxicity is reduced by Mip compounds in three macrophage cell line models; J774 murine macrophage cell line, RAW264.7 murine macrophages and THP-1 human derived monocytes (Table 6):
Infection of macrophages with B. pseudomallei results in high levels of cell death at 24 hours post infection. Macrophage cell death is measured using the release of lactose dehydrogenase (LDH Cytotoxicity kit, Roche), thus a percentage cytotoxicity associated with B. pseudomallei infection can be determined. Deletion of mip from B. pseudomallei has been shown to lead to less severe infection, particularly a decrease in cytotoxicity in macrophages (Norville et al. (2011) and Begley et al. (2014)). Wild-type B. pseudomallei cells are pre- treated with 50 μM compounds for one hour before allowing bacteria to infect macrophages at a multiplicity of infection of 10 (i.e. 10 bacterial cells per 1 macrophage cell). The infection is allowed to continue for 24 hours in the presence of 50 μM compound before a cytotoxicity measurement is taken.
Table 6: Percent remaining of B. pseudomallei induced cytotoxicity in different macrophage cell lines relative to DMSO vehicle control.
Figure imgf000203_0002
Figure imgf000204_0001
Percentage of B. pseudomallei colony forming units are significantly reduced inside macrophage cells treated with Mip compound:
The colony forming units of B. pseudomallei cells inside the macrophages was enumerated (Table 7). After 24 hr of infection, murine macrophages are lysed and the bacterial burden is assessed by serial dilution followed by growth on nutrient agar (Luria Bertani agar) at 37 °C overnight.
Table 7: Percent remaining of intracellular B. pseudomallei numbers in macrophage cell lines relative to DMSO vehicle control.
Figure imgf000204_0002
Figure imgf000205_0001
Neisseria meningitidis
Mip compounds reduce N. meningitidis adhesion and invasion into Detroit 562 human nasopharyngeal epithelial cells: In order to colonise the body, N. meningitidis must adhere and subsequently invade the nasopharyngeal epithelial before migrating to the bloodstream where it can cause systemic infection. Inhibition of Mip in N. meningitidis has been previously shown to result in decreased capacity to adhere to and invade Detroit 562 human nasopharyngeal epithelial cells (Reimer et al. (2016)). N. meningitidis cells were pre-treated with 50 μM of Mip compound and allowed to adhere to and invade Detroit 562 human nasopharyngeal cells.
Following 6 hours of incubation, Detroit 562 cells are lysed and bacterial adhesion was enumerated by serial dilution followed by growth on nutrient agar. To determine the rate of invasion, extracellular bacteria were killed by incubation with the antibiotic Gentamicin. Following this, Detroit 562 cells were lysed and bacterial invasion was enumerated by serial dilution followed by growth on nutrient agar (Table 8).
Table 8: Percent remaining of N. meningitidis adherence and invasion in Detroit 562 human epithelial cell line relative to DMSO vehicle control.
Figure imgf000205_0002
Figure imgf000206_0001
Mip compounds reduce N. meningitidis survival in macrophage cells:
Following movement through the epithelial layer, N. meningitidis circulates around the body where it is phagocytosed by macrophages. Macrophages are able to kill bacteria through a number of intracellular killing mechanisms, which bacteria have evolved to be resistant to. To determine if treatment with compounds sensitises bacteria to intracellular killing, survival over 6 hours in RAW264.7 murine macrophages is assessed. N. meningitidis cells are pre-treated with 50μM compounds and then allowed to invade and be phagocytosed by RAW264.7 murine macrophages. Following 6 hours of incubation, RAW264.7 murine macrophages are lysed and bacterial invasion is enumerated by serial dilution followed by growth on nutrient agar (Table 9).
Table 9: Percentage remaining of N. meningitidis survival in RAW264.7 murine macrophages and THP-1 human derived monocyte cell line relative to DMSO vehicle control.
Figure imgf000206_0002
Figure imgf000207_0001
Neisseria gonorrhoeae
Survival in RAW264.7 murine macrophages:
N. gonorrhoea has adapted to become a resistant organism against many mechanisms of bacterial killing that the host has within its arsenal, in particular against intracellular killing as is commonly conducted by circulating macrophages. Previous work has shown that treatment of N. gonorrhoea with inhibitors against Mip results in decreased survival in neutrophils (Reimer et al. (2016)). To assess this with the compounds described here, the RAW264.7 murine macrophage model was adopted. N. gonorrhoea cells are pre-treated with compounds and then allowed to invade and be phagocytosed by RAW264.7 murine macrophages. Following 6 hours of incubation, RAW264.7 murine macrophages are lysed and bacterial invasion is enumerated by serial dilution followed by growth on nutrient agar (Table 10). Table 10: Percent remaining of N. gonorrhoeae survival in RAW264.7 murine macrophage cell line relative to DMSO vehicle control.
Figure imgf000207_0002
Figure imgf000208_0001
Coxiella burnetii
Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection.
Intracellular replication of C. burnetii in THP-1 human derived monocytes:
Infection of differentiated THP- 1 human derived monocytes with C. burnetii results in high levels of C. burnetii replication over seven days of infection. C. burnetii replication is measured by quantifying the number of genome equivalents (GE) of C. burnetii at indicated time points by quantitative PCR (qPCR), using ompA specific primers (Jaton et al. (2013); Kuba et al. (2020)) and is reported as the fold change in C. burnetii genome equivalent (GE) from day 0, relative to the DMSO control. Therefore, the percentage reduction in C. burnetii replication in the presence of compound can be determined.
Pre-treatment:
C. burnetii cells are pre-treated with 50 μM of compound for one hour before allowing bacteria to infect THP- 1 human derived monocytes at a multiplicity of infection of 5 (i.e. 5 bacterial cells per 1 macrophage cell). The infection is allowed to continue for 7 days in the presence of 50 μM compound with cells harvested for genomic extraction and C. burnetii GE quantification at Days 0, 1, 3, 5 and 7 of infection. Percentage decrease in replication of C. burnetii in THP-1 cells on Day 5 of infection:
Treatment with compounds AN296, AN263, AN258 and AN259 at 50 μM resulted in a decreased capacity of C. burnetii to replicate in THP-1 human derived monocytes. For Day 5 of infection, this results in only 13.7%, 2.2%, 24.6% and 14.9% replication compared to the DMSO vehicle control (100 %) (Figure 1A).
Treatment with compounds AN258 and NJS227 at 50 μM resulted in a decreased capacity of C. burnetii to replicate in THP-1 human derived monocytes. For Day 5 of infection, this results in only 21.7% and 8.2% replication compared to the DMSO (100 %) vehicle control (Figure IB).
Post-exposure treatment:
THP-1 human derived monocytes are infected with C. burnetii cells at a multiplicity of infection of 5. The infection is allowed to continue for 7 days. Infected THP-1 cells are dosed with 50 μM compound at Day 2 or Day 3 of infection. Infected THP-1 cells are harvested for genomic extraction and C. burnetii GE quantification at Days 0, 1, 3, 5 and 7 of infection.
THP-1 cells treated with compound on Day 2 of infection. Treatment with compound AN296 at 50 μM on Day 2 of infection resulted in a decreased capacity of C. burnetii to replicate in THP-1 human derived monocytes. For Day 5 and Day 7 of infection, this results in only 25.6% and 12.0% replication compared to the DMSO vehicle control (100 %) (Figure 2A).
Treatment with compound AN296 at 50 μM on Day 2 of infection resulted in a decreased capacity of C. burnetii to replicate in THP-1 human derived monocytes. For Day 5 and Day7 of infection, this results in only 55.1% and 21.9% replication compared to the DMSO vehicle control (100 %) (Figure 2B).
Mip compounds inhibit C. burnetii replication in axenic media:
C. burnetii in an obligate intracellular pathogen but can be grown in specialized axenic media such as acidified citrate cysteine medium-2 (ACCM-2) at 37 °C in 5% CO2 and 2.5% O2 (Omsland et al. (2011)). The growth of C. burnetii was evaluated in axenic media in the presence of different concentrations of compound. Inhibition of C. burnetii growth using luminescence assay:
Using C. burnetii-lux, a luciferase-expressing derivative of C burnetii NMII, light production can be measured and used as an indicator of replication. Stationary phase (6-7 day) ACCM-2 cultures of C. burnetii-lux were quantified using the PicoGreen assay (Thermo Fisher Scientific) and appropriately diluted to 1 x 106 genome equivalents (GE)/ml in white 96-well plates (Corning) in 0.1 ml of fresh ACCM-2 medium per well, in the presence of varying concentrations of compound. Light production is measured every 24 h over 5 days by a POLARStar plate reader (BMG Labtech). Data is presented as RLU (relative light units) relative to the DMSO control (Figures 3A, B and C).
Percent C. burnetii-lux replication in ACCM-2:
AN131 = 5.0% (100 μM), 22.0% (50 μM)
AN132 = 6.0% (100 μM), 31.3% (50 μM)
AN133 = 6.7% (100 μM), 26.8% (50 μM)
ANCH37 = 0.5% (100 μM), 17.2% (50 μM)
AN296 = 2.1% (100 μM), 36.4% (50 μM)
AN263 = 0.4% (100 μM), 46.2% (50 μM)
AN259 = 28.2% (100 μM), 60.0% (50 μM)
AN258 = 29.3% (100 μM), 57.3% (50 μM)
NJS227 = 25.5% (100 μM), 63.9% (50 μM)
NJS224 = 37.2% (100 μM), 56.6% (50 μM)
Treatment with 50 μM or 100 μM of all compounds reduced C. burnetii-lux replication in axenic media compared cells grown in the presence of the DMSO vehicle control. A dose response was seen in all compounds with 100 μM being the most effective concentration to reduce C. burnetii growth. inhibit C. burnetii when administered in axenic media:
Using C. burnetii-lux, a luciferase-expressing derivative of C burnetii NMII, light production can be measured and used as an indicator replication. Stationary phase (6-7 day) ACCM-2 cultures of C. burnetii-lux were quantified using the PicoGreen assay and the strain was inoculation at a concentration of 1 x 106 GE/ml into 0.1 ml of fresh ACCM-2 medium per well and growth was monitored over 5 days. Cultures were dosed with compound on Day 2 or Day 3 of the growth curve. Data is presented as RLU (relative light units) relative to the DMSO control (Figure 4A).
Percent C. burnetii-lux replication in ACCM-2 treated with AN296 from day of addition:
DAY 0 = 1.0% (100 μM), 43.1% (50 μM)
DAY 2 = 24.7% (100 μM), 59.4% (50 μM)
DAY 3 = 25.3% (100 μM), 52.4% (50 μM)
Treatment with 50 μM or 100 μM of compound AN296 at Day 2 or Day 3 of the assay reduced C. burnetii-lux replication in axenic media compared cells grown in the presence of the DMSO vehicle control.
Compounds inhibit C. burnetii growth when administered during log phase growth, measured by colony forming units:
Using wild type C. burnetii strain NMII and clear 96- well plates (Coming) the growth assay was prepared as described for the standard luminescence assay. Following 4 days of incubation, samples from test wells were taken and plated in serial dilutions on ACCM-2 agar plates which were then left for 7 days before viability counts were performed. Data is presented as fold increase in CFUs relative to Day 0 (Figure 4B).
Fold increase in CFU: 200 μM = 0.02 (n=l) 100 μM = 27.8 (n=4) 50 μM = 377.3 (n=4)
25 μM = 690 (n=3)
Compared to their respective DMSO controls = 533 (200 μM), 652 (100 μM), 750 (50 μM), 819 (25 μM). Treatment with 200 μM of compound AN296 reduced the number of C. burnetii CFUs compared to the starting inoculum indicating the compound AN296 has antimicrobial properties against C. burnetii.
Example 14: Mip inhibitors comprising additional sidechain demonstrate superior inhibition of intracellular replication of C.burnetii
Strain C. burnetii-NMII was pretreated with SF235 or AN296 for 1 h and used to infect differentiated THP-1 macrophage cells or HeLa epithelial cervical cancer cells. C. burnetii replication was then monitored by measuring genome equivalents (GE) over 7 days. In the presence of 50 μM AN296, intracellular replication of C. burnetii-NMII within THP-1 cells was significantly inhibited (Fig 7A), resulting in a 74%, 80% and 73% (p < 0.0001) reduction in C. burnetii-NMII replication compared to the control, 3, 5, and 7 days post infection, respectively. In contrast, 50 μM SF235 resulted in a 58%, 62% and 59% (p < 0.0001) reduction in C. burnetii-NMII replication 3, 5, and 7 days post infection, respectively, significantly lower than that achieved for AN296.
C. burnetii-NMII replication was also significantly reduced in HeLa cells when cultures were treated with higher concentrations AN296 compared to SF235. In the presence of 100 μM AN296, intracellular replication of C. burnetii-NMII was significantly inhibited in HeLa cells, resulting in a 92%, 90% and 92% (p < 0.0001) reduction in C. burnetii-NMII replication 3, 5, and 7 days post infection, respectively (Fig 7B). Furthermore, compared to the untreated control, treatment with 100 μM AN296 appeared to delay the replication kinetics of C. burnetii-NMII within HeLa cells by two days. The effect on C. burnetii-NMII replication in HeLa cells treated with 100 μM of SF235 was much less pronounced and resulted in a 62%, 70% (p < 0.05) and 76% (p < 0.01) reduction in C. burnetii-NMII replication at 3, 5, and 7 days post infection compared to control.
Example 15: Mip inhibitors comprising additional sidechain reduce C. burnetii-NMII growth in axenic culture in a dose dependant manner
A luciferase-expressing derivative of C. burnetii-NMII, C. burnetii-Iux, was evaluated in axenic media in the presence of different concentrations of SF235 and AN296. Luciferase activity of C. burnetii-Iux was inhibited in a dose-dependent manner compared to the control (Fig 8A and 85 Fig). Following normalization to DMSO, treatment with AN296 reduced bioluminescence by 87% and 55% (p < 0.0001) at 100 μM and 50 μM, respectively (Fig 4B). In contrast, SF235 only reduced C. burnetii-lux bioluminescence by 68% and 42% (p < 0.0001) at 100 μM and 50 μM, respectively. To confirm that the compounds were affecting C. burnetii growth and not simply preventing proper folding and function of the luciferase, the experiment was repeated using wild-type C. burnetii-NMII and colony forming units (CFU) were enumerated after 4 days of culture. The fold-increase in C. burnetii-NMII CFU/mL (logio) compared to day 0, in the presence of 100 μM or 50 μM of C6Mip inhibitor was 19% (p < 0.01) and 54% of the control, respectively, for SF235, and 2.1% (p < 0.0001) and 48% of the control, respectively, for AN296 (Fig 8C).
The potential of Mip inhibitors to act on exponentially growing C. burnetii cultures was also investigated using AN296 (Fig 9). Addition of AN296 to the culture on either day 2 (start of logarithmic growth) (Fig 9 A and 9B) or day 3 (mid logarithmic growth) (Fig 9C and 5D) (63) resulted in a significant decrease in bioluminescence. Following normalization to the control, addition of AN296 on day 2 reduced C. burnetii-lux bioluminescence by 75% and 41% (p < 0.0001) at 100 μM and 50 μM (Fig 9E), respectively. Similarly addition of AN296 on day 3 reduced bioluminescence by 75% and 48% (p < 0.0001) at 100 μM and 50 μM (Fig 9E), respectively. These data demonstrates that AN296 is effective at suppressing the growth of both starter cultures that are predominantly composed of C. burnetii SCVs and actively growing cultures which contain C. burnetii cells that are predominantly LCVs (64). Together these data suggest that Mip compounds having an additional side chain significantly perturb the ability of C. burnetii to replicate both intracellularly and in axenic media.
Example 16: Mip inhibitors comprising additional sidechain stops replication of virulent C. burnetii in axenic culture
For the C6Mip inhibitors to be therapeutically useful, they must be active against the virulent, phase I form of C. burnetii. Therefore, the activity of compounds AN296 and SF235 against the phase I parental strain C. burnetii-NMI was investigated. The growth of C. burnetii-NMI in axenic media while exposed to 100 μM of SF235 or AN296 versus a vehicle control was monitored over 7 days by enumerating CFU/mL for the first 4 days and then on day 7 (Fig 10A).
C. burnetii-NMI replication was significantly inhibited when grown in the presence of AN296 compared to the control culture at day 4 (p < 0.001) and 7 (p < 0.0001) of the growth curve (Fig 7A). In contrast, treatment with SF235 was less effective. The inhibitors were also tested against C. burnetii-NMI during later stages of growth (Fig 7B). Addition of 100 |aM of SF235 to the culture on day 3 had no impact on the growth of C. burnetii-NMI compared to the control. However, the addition of 100 μM of AN296 on day 3 significantly inhibited C. burnetii-NMI growth compared to the control on day 7 (p < 0.0001). Together these data indicate that AN296 directly impact on the ability of virulent C. burnetii-NMI to replicate in axenic media.
Example 17: Mip compounds AN296 and AN258 are efficacious in vivo using the Galleria mellonella infection model
To determine the efficacy of Mip compounds in vivo the greater wax moth, Galleria mellonella, insect model was used. G. mellonella is an alternative model of C. burnetii infection and has previously been shown to be susceptible to infection following subcutaneous inoculation (Norville et al.). Using this model it was observed that intracellular C. burnetii was present within haemocytes and that larval death occurred in a dose-dependent manner. Furthermore, the innate immune response of G. mellonella demonstrates notable similarities to the immune response in humans. Hence, this model can give us an accurate prediction of the efficacy of drugs and is a cost effective method to down select the most promising candidate, synthetically prepared molecules for future in vivo studies using small animal models (Tsai et al. (2016)). For the toxicity studies, candidate drugs were injected into the right proleg of G. mellonella larvae, which were then kept isolated at 37°C in the dark and survival was assessed for 10 days. Figure 5 demonstrates that even at the highest concentration of drug, there was minimal cytotoxicity to the G. mellonella larvae, indicating the drugs are safe to use for efficacy studies. To assess the effect of the candidate drugs on C. burnetii pathogenicity, C. burnetii was initially pre-incubated with each drug candidate for 1 hour prior to injection of larvae and survival was assessed for 10 days. Figure 6 shows that the compounds significantly increase the survival of the G. mellonella compared to the control. These results strongly suggest that the candidate drugs are both safe and efficacious in vivo.
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
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Claims

CLAIMS:
1. A compound of Formula (I), or a pharmaceutically acceptable salt, solvate or stereoisomer thereof,
Figure imgf000226_0001
wherein:
X is selected from O, S, and NR4;
A1, A2, A3 and A4 are each independently selected from the group consisting of CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1- 10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, each of which is optionally substituted;
L1 and L2 are each independently absent or selected from the group consisting of -C1- 10alk yl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O- ,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH-, -C(=O)NH( C1-10alkyl)-, - S(=O)NH-, -S(=O)NH( C1-10alkyl)-, -N( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH(CI- 10alk yl)-, and -OS(=O)2-, wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted and optionally substituted;
R1 and R3 are each independently selected from an optionally substituted carbocyclyl or an optionally substituted heterocyclyl;
R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl, each of which is optionally substituted; and R4 is selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl, each of which is optionally substituted.
2. The compound of claim 1, wherein:
X is selected from O, S, and NR4;
A1, A2, A3 and A4 are each independently selected from the group consisting of CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2-10alkynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1- 10alkyl-3-10-membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl; L1 and L2 are each independently absent or selected from the group consisting of - C1-10alkyl-, -O C1-10alkyl-, -C2-10alkenyl-, -OC2-10alkenyl-, -C(=O)-, -C(=O) C1-10alkyl-, -C(=O)O-,-C(=O)O( C1-10alkyl)-, -OC(=O)( C1-10alkyl)-, -C(=O)NH-, -C(=O)NH( C1-10alkyl)-, -S(=O)NH-, -S(=O)NH(C1- 10alk yl)-, -N( C1-10alkyl)-, -S(=O)2-, -S(=O)2NH-, -S(=O)2NH( C1-10alkyl)-, and -OS(=O)2-; wherein each C1-10alkyl or C2-10alkenyl is uninterrupted or interrupted with one or more groups selected from -O-, -C(=O)O-, -NH-, -C(=O)NH-, -S-, and -S(=O)2-, and is optionally substituted with one or more R7;
R1 and R3 are each independently selected from a carbocyclyl or a heterocyclyl;
R2 is selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, alkylcarbocyclyl, heteroalkyl, heterocyclyl, and alkylheterocyclyl; and
R4 is selected from the group consisting of H, C1-10alkyl, carbocyclyl, C1-10alkyl- carbocyclyl, heteroalkyl, heterocyclyl, and C1-10alkyl-heterocyclyl; wherein each of R , R1, R2, R3 and R4 is optionally substituted with one or more R5; each R5 is independently selected from the group consisting of H, halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, OC2-10alkenyl, OC2- 10alk ynyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-10alkyl-3-10- membered-carbocyclyl, C1-10alkyl-3-10-membered-heterocyclyl, -NO2, -CN, =O, -N(R6)2, - C(=O)N(R6)2, -S(=O)N(R6)2, -S(=O)2N(R6)2, -OR6, -SR6, -OC(=O)R6, -C(=O)R6, - C(=O)OR6, -S(=O)R6, -S(=O)2R6, -N(R6)C(=O)R6, -N(R6)S(=O)R6, -N(R6)C(=O)N(R6)2, and -N(R6)S(=O)2R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, C2-10alkynyl, 3-10 membered- carbocyclyl, and 3-10-membered-heterocyclyl is optionally substituted with one or more R7; each R6 is independently selected from the group consisting of H, C1-6alkyl, 3-10 membered carbocyclyl, 3-10-membered heterocyclyl, C1-6alkyl-3-10-membered-carbocyclyl, and C1-6alkyl-3- 10-membered-heterocyclyl; wherein each C1-6alkyl, 3-10-membered-carbocyclyl, and 3-10-membered heterocyclyl is optionally substituted with one or more R7; each R7 is independently selected from the group consisting of halogen, -NO2, - N(R8)2, -CN, =O, -C(=O)OR8, -N(R8)C(=O)R8, -OR8, -C1-6alkyl, and -OC1-6alkyl, or R7 together with the carbon it is attached forms a Cs-ecarbocyclic ring, and each R8 is independently selected from the group consisting of H and C1-6alkyl.
3. The compound of claim 1 or claim 2, wherein A1, A2 A3 and A4 are each independently selected from the group consisting CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1- 6haloalkyl, and OC1-6haloalkyl, each of which is optionally substituted.
4. The compound of any one of claims 1 to 3, wherein A1, A2 and A3 are each CH2, and A3 is selected from the group consisting CR'2, NR' , S and O, wherein each R is independently selected from the group consisting of H, halogen, C1-6alkyl, OC1-6alkyl, C1- 6haloalkyl, and OC1-6haloalkyl, each of which is optionally substituted.
5. The compound of any one of claims 1 to 4, wherein A1, A2 A3 and A4 are each CH2.
6. The compound of any one of claims 1 to 5, wherein R1 and R3 are each independently selected from an optionally substituted aryl or an optionally substituted heteroaryl.
7. The compound of any one of claims 1 to 6, wherein R1 and R3 are each independently selected from an optionally substituted 3-10-membered aryl or an optionally substituted 3-10- membered heteroaryl.
8. The compound of any one of claims 1 to 7, wherein R1 is an optionally substituted phenyl.
9. The compound of any one of claims 1 to 8, wherein R3 is an optionally substituted 5- 6-membered heteroaryl.
10. The compound of any one of claims 1 to 9, wherein R3 is an optionally substituted pyridyl, pyrazolyl or imidazolyl. ll. The compound of claim 10, wherein the pyridyl is pyridine-N-oxide.
12. The compound of claim 10 or claim 11, wherein R3 is independently selected from the group consisting of:
Figure imgf000229_0001
each of which is optionally substituted.
13. The compound of any one of claims 1 to 12, wherein each R1 and R3 are independently optionally substituted by one or more groups selected from H, halogen, C1- 10alk yl, O C1-10alkyl, C1-10haloalkyl, O C1-10haloalkyl, -NO2, -CN, -N(R6)2, -OR6,-S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl and C1-10haloalkyl is optionally substituted with one or more R7.
14. The compound of any one of claims 1 to 13, wherein each R1 and R3 are independently optionally substituted by one or more groups selected from H, halogen, C1- 6alkyl, OC1-6alkyl, C1-6haloalkyl, -NO2, -CN, -SO2H, -OH, -NH2, -N(H)C1-6alkyl, orC1- 6alkylNH2.
15. The compound of any one of claims 1 to 14, wherein R2 is selected from the group consisting of C3-10alkyl, cycloalkyl, C1-10alkylcycloalkyl, heteroalkyl, heterocyclyl, C1- 10alk ylheterocyclyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
16. The compound of any one of claims 1 to 15, wherein R2 is selected from the group consisting of C3-10alkyl, aryl, C1-10alkylaryl, heteroaryl, and C1-10alkylheteroaryl, each of which is optionally substituted.
17. The compound of any one of claims 1 to 16, wherein R2 is selected from the group consisting of C3-6alkyl, aryl, C1-6alkylaryl, heteroaryl, and C1-6alkylheteroaryl, each of which is optionally substituted.
18. The compound of any one of claims 1 to 17, wherein R2 is optionally substituted with one or more groups selected from halogen, C1-10alkyl, O C1-10alkyl, C1-10haloalkyl, OC1- whaloalkyl, C2-10alkenyl, OC2-10alkenyl, C2-10alkynyl, OC2-10alkynyl, -NO2, -CN -N(R6)2, - OR6, -S(=O)2R6, or -N(R6)C(=O)R6, wherein each C1-10alkyl, C1-10haloalkyl, C2-10alkenyl, or C2-10alkynyl is optionally substituted with one or more R7.
19. The compound of any one of claims 1 to 18, wherein R2 is optionally substituted with one or more groups selected from halogen, C1-6alkyl, OC1-6alkyl, C1-6haloalkyl, OC1- 6haloalkyl, C2-6alkenyl, OC2-6alkenyl, C2-6alkynyl, OC2-6alkynyl, -NO2, -CN, -SO2H, -OH, - NH2, -N(H)C1-6alkyl, or C1-6alkylNH2.
20. The compound of any one of claims 1 to 19, wherein L1 is absent or selected from - C1-10alkyl- or -N( C1-10alkyl)-.
121. The compound of any one of claims 1 to 20, wherein L2 is -C(=O)NH- or - C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted with one or more R7.
22. The compound of any one of claims 1 to 21, wherein L2 is -C(=O)NH( C1-10alkyl)-, wherein C1-10alkyl is optionally substituted with one or more R7.
23. The compound of any one of claims 1 to 22, wherein X is O or NH.
24. The compound of any one of claims 1 to 23, wherein the compound of Formula (I) is selected from the group consisting of:
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
or a pharmaceutically acceptable salt, solvate, stereoisomer or N-oxide thereof.
25. The compound of any one of claims 1 to 24, wherein the compound of Formula (I) is selected from the group consisting of:
Figure imgf000235_0002
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
or a pharmaceutically acceptable salt, solvate, or N-oxide thereof.
26. A pharmaceutical composition comprising a compound of any one of claims 1 to 25, and a pharmaceutically acceptable excipient.
27. A method of treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein, the method comprising administering to the subject an effective amount of compound of any one of claims 1 to 25, or a pharmaceutical composition of claim 26.
28. The method of claim 27, wherein the pathogen is a bacterial pathogen.
29. The method of claim 28, wherein the bacterial pathogen is a Gram-negative bacterium.
30. The method of any one of claims 27 to 29, wherein the Gram-negative bacterium is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
31. The method of any one of claims 27 to 30, wherein the pathogen is Coxiella burnetii, and the disease or condition is Q fever.
32. Use of a compound of any one of claims 1 to 25 or a pharmaceutical composition of claim 26 in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
33. A compound of any one of claims 1 to 25 or a pharmaceutical composition of claim 26, for use in treating and/or preventing a disease or condition mediated by a pathogen in a subject, wherein the pathogen is responsive to inhibition of macrophage infectivity potentiator (Mip) protein.
34. A method of treating and/or preventing a disease or condition mediated by a Gram- negative bacteria in a subject in which macrophage infectivity potentiator (Mip) protein is a virulence factor, comprising administering to the subject a compound of any one of claims 1 to 25, or a pharmaceutical composition of claim 26.
35. The method of claim 34, wherein the Gram-negative bacteria is selected from one or more of Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Legionella pneumophila and Coxiella burnetii.
36. The method of claim 34 or claim 35, wherein the Gram-negative bacteria is Coxiella burnetii, and the disease or condition is Q fever.
37. Use of a compound of any one of claims 1 to 25 or a pharmaceutical composition of claim 26, in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
38. A compound of any one of claims 1 to 25 or a pharmaceutical composition of claim 26, for use in treating and/or preventing a disease or condition mediated by a Gram-negative bacteria in which macrophage infectivity potentiator (Mip) protein is a virulence factor.
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