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WO2024200838A1 - New odilorhabdins analogues as antibiotics against multi-resistant bacteria - Google Patents

New odilorhabdins analogues as antibiotics against multi-resistant bacteria Download PDF

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Publication number
WO2024200838A1
WO2024200838A1 PCT/EP2024/058803 EP2024058803W WO2024200838A1 WO 2024200838 A1 WO2024200838 A1 WO 2024200838A1 EP 2024058803 W EP2024058803 W EP 2024058803W WO 2024200838 A1 WO2024200838 A1 WO 2024200838A1
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WO
WIPO (PCT)
Prior art keywords
alkyl
tert
mmol
butyl
carbamate
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Application number
PCT/EP2024/058803
Other languages
French (fr)
Inventor
Maxime Gualtieri
Emilie RACINE
Philippe Villain-Guillot
Frédéric JEANNOT
Eric Bacque
Michel Geslin
Pierre Despeyroux
Original Assignee
Nosopharm
Evotec International Gmbh
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Publication date
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Publication of WO2024200838A1 publication Critical patent/WO2024200838A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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

Definitions

  • the project leading to this application has received funding from the innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement n° 853979. This Joint Undertaking receives the support from the European Union’s Horizon 2020 research and innovation program and EFPIA. FIELD OF THE INVENTION
  • ODLs Odilorhabdins
  • the present invention relates to new Odilorhabdins (ODLs) analogues, and their use in medicine, in particular for treating or preventing bacterial infections, more specifically for treating or preventing multi-drug resistant bacterial infections, such as multi-drug resistant bacterial infections caused by P. aeruginosa.
  • Odilorhabdins are a new class of ribosome-targeting antibiotics [WO201304560 WO2016046409, Pantel et al., Mol Cell., 2018, 70(1):83-94.e7].
  • the first members of the class, NOSO-95 A-C were identified from cultures of Xenorhabdus nematophila, a bacterium symbiotically associated with entomopathogenic nematodes.
  • NOSO-502 a compound with potent pharmacological properties against multidrug-resistant Enterobacteriaceae including carbapenem-resistant, polymyxin-resistant, and ESBL (extended spectrum ⁇ -lactamase) isolates [Racine et al., Antimicrob. Agents Chemother., 2018, 62(9):e01016-18 ; Racine et al., Front. Microbiol., 2019, 10:2893].
  • This compound successfully completed the preclinical development stage recently.
  • the present invention also relates to a compound of formula (I) or a composition comprising thereof for use in medicine, more specifically for use in the treatment or prevention of bacterial infections. Further aspects of the invention are as disclosed herein and in the claims.
  • alkyl as used herein means a saturated, branched or straight monovalent hydrocarbon chain. As a matter of example, alkyl comprising from one to six carbon atoms are represented “-(C 1 -C 6 )-alkyl”.
  • hydroxyalkyl as used herein means an alkyl group as defined above substituted by one or more hydroxy radicals.
  • aminoalkyl as used herein means an alkyl group as defined above substituted by one or more amino groups (-NH 2 ).
  • haloalkyl as used herein means an alkyl group as defined above substituted by one or more halogen atoms (F, Cl, Br, I).
  • hydroxyaminoalkyl as used herein means an alkyl group as defined above substituted by one or more hydroxy radicals and one or more amino groups (-NH 2 ).
  • Me as used herein designates a methyl group.
  • alkoxy as used herein designates a group of formula -O-alkyl wherein alkyl is as defined herein.
  • alkanediyl as used herein means a saturated, branched or straight divalent hydrocarbon chain.
  • alkanediyl comprising from one to six carbon atoms are represented “C 1 -C 6 -alkanediyl”.
  • alkanediyl include, but are not limited to, methanediyl (-CH 2 -).
  • alkenediyl as used herein means an unsaturated, branched or straight divalent hydrocarbon chain comprising one or more double bonds.
  • alkenyl designates a univalent branched or straight hydrocarbon chain containing one or more double bonds, including di-enes, tri-enes, such as vinyl, propen-1-yl, propen-2-yl, but-1-en-1-yl, but-1-en-2-yl, but-2-en-1-yl.
  • alkynyl designates a univalent branched or straight hydrocarbon chain containing one or more triple bonds.
  • cycloalkyl as used herein means a monovalent monocyclic or bicyclic (fused) hydrocarbon chain.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • aryl refers to aromatic carbocyclic groups having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings (e.g., naphthyl,).
  • heteroaryl refers to aryl groups as defined herein comprising at least one heteroatom as a ring atom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like.
  • multidrug-resistant means a lack of susceptibility to at least one agent in three or more chemical classes of antibiotic, as defined in Clin Microbiol Infect.2012;18:268-81. doi: 10.1111/j.1469-0691.2011.03570.x. DETAILED DESCRIPTION
  • ODLs analogs compounds of formula (I), see below
  • the ODLs analogs have been found to exhibit improved in vitro and in vivo pharmacological properties against P.
  • the present invention relates to compounds of formula (I): R a -Xaa 1 -Xaa 2 -Xaa 3 - Xaa 4 -Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -(Xaa1 0 ) y -R b (I) wherein: R a is H or -(C 1 -C 3 )-alkyl; y is 0 or 1; Xaa 1 i wherein one or more carbon atoms of the chain bearing the NR 11 R’ 11 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl, wherein
  • physiologically acceptable salt refers to a relatively non-toxic, inorganic or organic acid addition salt of the compound of formula (I).
  • a suitable pharmaceutically acceptable salt of the compound of formula (I) may be, for example, an acid-addition salt of a compound of formula (I), such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3 -hydroxy-2 -naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3- phenylpropionic, picric, pival
  • the salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid with any of the compounds of the invention.
  • Solvates in the context of the invention are described as those forms of the compounds which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water.
  • the present invention includes all possible stereoisomers of the compounds of formula (I) as single stereoisomer, or as any mixture of said stereoisomers, in any ratio. Isolation of a single stereoisomer, e.g.
  • a single enantiomer or a single diastereomer, of a compound of formula (I) can be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
  • R a is H or methyl.
  • R a is H.
  • R a is methyl.
  • Xaa 1 is wherein R1, R 11 , R’ 11 are as disclosed herein above and n1 is an integer from 1-3, in particular n1 is 1, 2 or 3.
  • one or more carbon atoms of the chain bearing the NR 11 R’ 11 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl. In some embodiments, Xaa 1 is 1 .
  • Xaa 1 is wherein R 11 and R’ 11 are a hydrogen atom and n1 is 1, 2 or 3, preferably 3.
  • one or more carbon atoms of the chain bearing the NR 11 R’ 11 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl.
  • the carbon atom in alpha of the NR 11 R’ 11 group is substituted by a –(C 1 - C 3 )-alkyl, preferably a methyl group.
  • the configuration of the asymetric carbon atom bearing the radical R1 or –(CH 2 ) n1 -NR 11 R’ 11 is preferably S.
  • Xaa 1 is , more specifically .
  • Xaa 1 is more specifically .
  • one or more carbon atoms of the chain bearing the NR 11 R’ 11 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl.
  • Xaa 2 is in particular Xaa2 is or , wherein X 2 is NH or N(Me), preferably NH and n 2 , R 2 , R’ 2 and R 22 are as disclosed herein above or below .
  • Xaa2 is preferably , wherein X 2 is NH or N(Me), preferably NH; R 2 is H, methyl, ethyl, -C(O)-(C 1 -C 2 )-alkyl, or -C(O)-(C 1 -C 2 )-haloalkyl, preferably H, methyl, acetyl or trifluoroacetyl, and R’ 2 is H, methyl, ethyl, preferably H.
  • n2 is an integer from 1- 3, in particular 1, 2 or 3, advantageously n2 is 1.
  • R 22 is as disclosed herein above.
  • Xaa2 is , preferably , wherein R 22 is OH, halogen, -(C 1 -C 3 )-alkyl, -(C 1 -C 3 )-alkoxy, -O-C(O)-(C 1 - C 6 )-alkyl or –O-C(O)-(C 1 -C 6 )- haloalkyl, preferably OH, fluorine, methyl, methoxy, -O- C(O)-CH 3 , -O-C(O)-CF 3 , more preferably OH and R 2 and R’ 2 are independently H, -(C 1 - C 3 )-alkyl, -C(O)-(C 1 -C 3 )-alkyl or -C(O)-(C 1 -C 3 )-haloalkyl, preferably R 2 is H or methyl, and R’ 2 is H, more preferably
  • Xaa2 is , preferably , wherein R 22 is OH, fluorine, methyl, methoxy, -O-C(O)-CH 3 or -O-C(O)-CF 3 , in particular OH, -O-C(O)-CH 3 or -O-C(O)-CF 3 , more specifically OH.
  • R 2 and R’ 2 are as defined above.
  • R 2 is H, methyl, acetyl or trifluoroacetyl, advantageously H, and R’ 2 is H.
  • X 2 is preferably NH or N(Me), more preferably NH.
  • Xaa3 is , , X 3 is NH or N(Me), preferably NH. In some other embodiments, X 3 is O.
  • R3, R 33 , R’ 33 , R’’ 33 , R 333, R’ 333 , R’’ 333, R’’’ 333, n 3 and n 4 are as disclosed herein.
  • the configuration of asymetric carbon atom between –X 3 and –C(O) is preferably S.
  • R 3 is halogen, -(C 1 -C 4 )-alkyl, -(C 1 -C 4 )- haloalkyl, -(C 2 -C 4 )-alkenyl, -(C 2 -C 4 )-alkynyl, -(C 1 -C 4 )-alkyl-OH, -(C 1 -C 4 )-alkyl-NHR 33 , - (C 1 -C 4 )-alkyl-C(O)NHR 33 , -(C 1 -C 4 )-alkyl-C(O)OH, -(C 1 -C 4 )-alkyl-heteroaryl wherein said heteroaryl is selected from the group consisting of imidazole, pyrazole, oxazole, isoxazole, thiazole, pyrole, furane, thiophene, pyra
  • R 33 , R’ 33 and R’’ 333 are advantageously independently H or -(C 1 -C 3 )-alkyl, more advantageously H.
  • R 33 3, R’ 333 and R’’ 333 3 are advantageously independently H, OH, halogen, -(C 1 -C 3 )-alkyl, or -(C 1 -C 3 )-alkoxy, advantageously H.
  • Xaa3 is , more specifically , wherein X 3 is N(R 33 ); R3 is -(C 1 -C 4 )-alkyl-NR 33 R’ 33 , preferably -CH 2 -CH 2 - NR 33 R’ 33 and R 33 and R’ 33 being as disclosed herein, preferably are H.
  • Xaa3 is , more specifically , wherein X 3 is NH or N(Me), preferably NH.
  • R3 is as disclosed herein, more specifically -(C 1 -C 4 )-alkyl-NHR 33 with R 33 as disclosed herein.
  • Xaa 5 is , preferably , wherein R 5 and R’ 5 are independently H or -(C 1 -C 3 )-alkyl and n 5 as disclosed herein. In some embodiments, R 5 and R’ 5 are H. In some embodiments, n 5 is 2. In each of these embodiments, X 5 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa 5 is , preferably , wherein R 5 and R’ 5 are independently H or methyl and n 5 as disclosed herein. In some embodiments, R 5 and R’ 5 are H. In some embodiments, n 5 is 2.
  • X 5 is preferably NH or N(Me), more preferably NH.
  • Xaa 5 is and R5 is H or methyl, preferably H.
  • Xaa 5 is and R 5 is H or methyl, preferably H.
  • X 5 is preferably NH or N(Me), more preferably NH.
  • Xaa 6 is , wherein X6 is NH or N(Me), preferably NH. In some other embodiments, X6 is O.
  • X6 is preferably NH or N(Me), more preferably NH.
  • X 6 is preferably NH or N(Me), more preferably NH.
  • n6 is 0, 1 or 2.
  • Xaa 6 is , R 66 is H, CH 3 or halogen
  • R’ 66 is H, halogen, methyl, OH, -O-(C 1 -C 3 )-alkyl, -(C 1 -C 3 )-haloalkyl, -CH 2 -COOH or NH 2 and n 6 is an integer from 0-3.
  • n 6 is 0, 1 or 2.
  • the configuration of the carbon atom bearing R 66, R’ 66 or R 66 and R’ 66 can be S or R, advantageously R.
  • the configuration of the carbon atom linked to –X 6 and to – C(O)- is advantageously S.
  • Xaa 6 is , In some embodiments, in the above formula (I), Xaa 6 is , , , , , , o In some embodiments, in the above formula (I), Xaa 6 is , more specifically In some embodiments, in the above formula (I), Xaa 7 is , preferably , preferably , wherein X 7 is NH or N(Me), preferably NH. In some other embodiments, X 7 is O. R 7 , R 77 , R’ 77 and n 7 are disclosed herein.
  • R 7 is H, -(C 1 -C 6 )-alkyl, -(C 1 -C 6 )-haloalkyl, -(C 2 -C 6 )-alkenyl, -(C 2 -C 6 )-alkynyl, -(C 1 -C 6 )-alkyl-OR 77 , (C 1 -C 6 )-alkyl-SR 77 , -(C 1 -C 6 )-alkyl- S(O)R 77 , -(C 1 -C 6 )-alkyl-S(O) 2 R 77 , -(C 1 -C 6 )-alkyl-NR 77 R’ 77 , -(C 1 -C 6 )-alkyl-C(O)OR 77 , -(C 1 - C 6 )-alkyl-C(O)NR 77 R’ 77 ,
  • the heteroaryle is advantageously pyridine, indole, isoindole, benzothiophene, benzofurane, imidazole, oxazole, thiazole, furane, pyrrole, thiophene or triazole, more advantageously pyridine, isoindole, imidazole, thiophene.
  • the aryl or heteroaryle is advantageously mono-, di- or tri- substituted with OH, NH 2 , -COOH, -CONH 2 , -CN, -CF 3 , -(C 1 -C 3 )-alkyl, -(C 1 -C 3 )-alkoxy, -CH 2 -COOH, -O-CH 2 -COOH, -CH 2 -NH-CO-NH 2 , -CHCH 3 OH, -CH 2 -NH 2 or halogen.
  • X 7 is preferably NH or N(Me), more preferably NH.
  • Xaa 7 is , preferably , wherein X 7 is N(R 77 ); R 7 is -(C 1 -C 6 )-alkyl-heteroaryl or -(C 1 -C 6 )-alkyl-aryl, wherein said aryl or heteroaryl is optionally monosubstituted with –OH, -NH 2 , -COOH, - CONH 2 , -CN, -CF 3 , -(C 1 -C 6 )-alkyl or halogen, preferably -COOH; preferably the aryl is phenyl; preferably R 7 is -CH 2 -heteroaryl or -CH 2 -phenyl, wherein said phenyl is optionally monosubstituted with –OH, -NH 2 , -COOH, -CONH 2 , -CN, -CF 3 , -(C 1 -
  • R, R’ and R being independently OH, NH 2 , -COOH, -CONH 2 , -CN, -CF 3 , -(C 1 -C 3 )-alkyl, -(C 1 -C 3 )- alkoxy, -CH 2 -COOH, -O-CH 2 -COOH, -CH 2 -NH-CO-NH 2 , -CHCH 3 OH, -CH 2 -NH 2 or halogen.
  • X 7 is preferably NH or N(Me), more preferably NH.
  • the configuration of the carbon atom linked to –X 7 and to – (CO)- is advantageously S.
  • Xaa 7 is , with R, R’ and R” being independently OH, NH 2 , -COOH, -CONH 2 , -CN, -CF 3 , -(C 1 -C 3 )-alkyl, -(C 1 -C 3 )- alkoxy, CH 2 -COOH, O-CH 2 -COOH, CH 2 -NH-CO-NH 2 , CHCH 3 OH, CH 2 -NH 2 or halogen.
  • X 7 is preferably NH or N(Me), more preferably NH.
  • Xaa 7 is with R, R’ and R” being independently OH, NH 2 , - COOH, -CONH 2 , -CN, -CF 3 , -(C 1 -C 3 )-alkyl, -(C 1 -C 3 )-alkoxy, -CH 2 -COOH, -O-CH 2 -COOH, -CH 2 -NH-CO-NH 2 , -CHCH 3 OH, -CH 2 -NH 2 or halogen.
  • X 7 is preferably NH or N(Me), more preferably NH.
  • Xaa 8 wherein X 8 is NH or N(Me), preferably NH. In some other embodiments, X 8 is O. R 8 , R’ 8 , R 88 , R’ 88 , R’’ 88 , R’’’ 88 and n 8 are as disclosed herein. In some embodiment, Xaa 8 is In some embodiments, in the above formula (I), Xaa 8 is or . X 8 is preferably NH or N(Me), more preferably NH. R 8 , R’’ 88 and n 8 are as disclosed herein.
  • Xaa 8 is , O , In each of these embodiments, X 8 is preferably NH or N(Me), more preferably NH. In some embodiments, Xaa 8 is . In each of these embodiments, X 8 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa 8 is , preferably , with X 8 being preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa 9 is , , wherein X 9 is NH or N(Me), preferably NH. In some other embodiments, X 9 is O.
  • R 9 , R’ 9 , R 99 and n 9 are as disclosed herein.
  • R’9 is a hydrogen atom
  • R 99 is H or -(C 1 -C 3 )- alkyl
  • n 9 is an integer from 1-4.
  • Xaa 9 is ;
  • R 99 is H or -(C 1 -C 3 )-alkyl; and
  • n 9 is an integer from 1-4.
  • X 9 is preferably NH or N(Me), more preferably NH.
  • X 9 is preferably NH or N(Me), more preferably NH.
  • Xaa 9 is , , , , , , , , or In each of these embodiments, X 9 is preferably NH or N(Me), more preferably NH.
  • X 10 is NH; R 10 and R’ 10 are independently H and n 10 is an integer from 0-5, preferably 2 or 3.
  • the compounds of formula (I) may comprise any combinations of the Xaa 1 to Xaa 10 groups disclosed herein.
  • the present invention relates to compounds of formula (I): R a -Xaa 1 -Xaa 2 -Xaa 3 - Xaa4-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -(Xaa 10 )y-R b (I) wherein: R a is H or -(C 1 -C 3 )-alkyl; y is 0 or 1; Xaa 1 is wherein one or more carbon atoms of the chain bearing the NR 11 R’ 11 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl, preferably when the chain bearing the NR 11 R’ 11 group is substituted, the carbon atom in alpha of the NR 11 R’ 11 group is substituted by - (C 1 -C 3 )-alkyl, preferably a methyl group; wherein:
  • the compounds of the invention are compounds of formula (I) as disclosed herein above wherein: R a is as disclosed herein; y is 0 or 1; Xaa 1 is , more specifically , wherein one or more carbon atoms of the chain bearing the NH 2 group may be substituted by one or more substituents selected from the group consisting of –(C 1 -C 3 )-alkyl and carboxyl, preferably wherein the carbon atom in alpha of the NH 2 group is substituted by–(C 1 -C 3 )-alkyl, preferably a methyl group; Xaa 2 is , more specifically wherein: X 2 is NH or N(Me), preferably NH; R 22 is OH, fluorine, methyl, methoxy or -O-C(O)-CH 3 , -O-C(O)-CF 3 , in particular OH,
  • the compounds of the invention are compounds of formula (I) as disclosed herein above wherein: when R 5a is: -A 5 -NHR 6 A 5 is an unsubstituted C 2 -C 6 -alkenediyl group, an unsubstituted C 1 -C 6 -alkanediyl or a C 1 - C 6 -alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C 1 -C 3 )-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl, an amine and a –(C 1 -C 6 )-aminoalkyl; and R e is a hydrogen atom, a -(C 1 -C 3 )-alkyl or a -(C 1 -
  • the compounds of the invention are compounds of formula (Ia): wherein: R 1a and R 2a are, independently of each other, a hydrogen atom or a -(C 1 -C 3 )-alkyl; R 3a is H or NH 2 ; R 4a is y is equal to 0 or 1; R 5a and R 6a are as disclosed herein in relation to formula (I); or hydrates, solvates, or salts thereof.
  • R 5a is: -A 1 -NR a -A 2 -NHR b , -A 3 -CO-NR c -A 4 -NHR d , or -A 5 -NHR e wherein: A 1 is an unsubstituted (C 2 -C 3 )-alkanediyl or a (C 2 -C 3 )-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl and a –(C 1 -C 3 )-hydroxyalkyl; A 2 is an unsubstituted (C 2 -C 4 )-alkanediyl or a (C 2 -C 4 )-alkanediyl substituted by one or more hydroxyl; R a is a hydrogen atom, a –(C 1 -C 3 )-hydroxyalkyl, a –(C
  • R1a and R 2 a are a hydrogen atom.
  • R 4a is In some embodiments, in the above formula (Ia), R 5a is -A 1 -NR a -A 2 -NHR b withA 1 ,A 2 , R a and R b as disclosed herein above. More specifically, R 5a may be one the following groups: Even more specifically, R 5a may be one the following groups:
  • R 6a is preferably H.
  • R 5a in the above formula (Ia), R 5a is -A 3 -CO-NR c -A 4 -NHR d with A 3 , A 4 , R c and R d as disclosed herein above. More specifically, R 5a may be one the following groups: In these embodiments, R 6a is preferably H. In some embodiments, in the above formula (Ia), R 5a is -A 5 -NHR e with A 5 and R e as disclosed herein above. More specifically, R 5a may be one the following groups: In these embodiments, R 6a is preferably H.
  • -NR 5a R 6a is one of the following groups:
  • the compound according to the invention may be selected from the group consisting of compounds 1 to 80, represented below, and the salts, e.g. pharmaceutically acceptable salts, hydrates and/or solvates thereof.
  • the compound of formula (I) may be selected from the group consisting of compounds 1 to 7 as disclosed hereinabove and salts, e.g. pharmaceutically acceptable salts, hydrates and/or solvates thereof.
  • the compounds of formula (I) may be prepared by methods known to the skilled person. Exemplary methods are shown in the Examples.
  • the expression “a compound of formula (I)” refers to a compound of formula (I) as described herein, including any compounds of formula (I) disclosed in the “Examples” section, as well as any salts, hydrates, solvates, isomers, mixtures of isomers in any ratio and any combinations thereof.
  • a compound of formula (I) may refer to a single compound of formula (I) or a combination of two or more compounds of formula (I) or salts, hydrates, solvates, isomers or mixtures of isomers thereof.
  • Therapeutic indications The compounds of formula (I) have been found to be effective against bacteria, in particular against multi-drug resistant bacteria. Resultantly, the compounds of formula (I) may be useful in medicine.
  • the present invention relates to a compound of formula (I) for use in medicine, in particular for the treatment or prevention of a bacterial infection.
  • the bacterial infection is multi-drug resistant.
  • the bacterial strain is hospital-acquired.
  • the bacterial strain is nosocomial. In some embodiments, the bacterial infection comprises infection from Gram-negative bacteria. In some embodiments, the bacterial infection comprises infection from Gram-positive bacteria. In some embodiments, the bacterial infection comprises infection by more than one bacterial strain.
  • Multiresistant bacterial pathogens that cause infection comprise methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended spectrum ß-lactamase formers (ESBL), carbapenem-resistant Enterobacteriaceae, multiresistant Pseudomonas and Acinetobacter species.
  • MRSA methicillin-resistant Staphylococcus aureus
  • VRE vancomycin-resistant enterococci
  • ESBL extended spectrum ß-lactamase formers
  • carbapenem-resistant Enterobacteriaceae multiresistant Pseudomonas and Acinetobacter species.
  • the bacterial or microbial infection is an infection caused or suspected to be caused in whole or in part by bacteria of the Achromobacter, Actinobacillus, Actinomyces, Acinetobacter, Aeromonas, Anaplasma, Bacillus, Bacteroides, Bartonella, Bdellovibrio, Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Hemobartonella, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria, Mannheimi
  • the bacterial infection is an infection caused or suspected to be caused in whole or in part by bacteria of Acinetobacter sp., Bacillus sp., Burkholderia sp., Enterobacter sp., Enterococcus sp., Escherichia sp., Klebsiella sp., Staphylococcus sp., Stenotrophomonas sp., Serratia sp.and Pseudomonas sp..
  • the bacteria is selected from the group consisting of Staphylococus sp., Escherichia sp., Klebsiella sp., Pseudomonas sp., or Acinetobacter sp.
  • the bacterial infection is an infection caused or suspected to be caused in whole or in part by Acinetobacter baumannii, Bacillus subtilis, Burkholderia cepacia, Enterobacter clocae, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Serratia marcescens or Pseudomonas aeruginosa.
  • the bacterial infection is an infection caused or suspected to be caused in whole or in part by A. baumannii, E. cloacae, E.
  • the bacterial infection is an infection caused or suspected to be caused in whole or in part by Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by multidrug-resistant P. aeruginosa.
  • the present invention relates a method of treatment or prevention of a bacterial infection using a therapeutically effective amount of a compound of formula (I).
  • the present invention relates to a method of treating or preventing a bacterial infection in a subject in need thereof comprising administering to the subject, that may be human or animal, a therapeutically effective amount of a compound of formula (I).
  • the present invention provides a method for treating a subject suffering from a multi-drug resistant bacterial infection comprising administering to said subject a therapeutically amount of a compound of formula (I).
  • a “therapeutically effective amount” as used herein refers to an amount that (i) treats or prevents the infection, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the infection, or (iii) prevents or delays the onset of one or more symptoms of the infection described herein.
  • the amount of a compound which constitutes a therapeutically effective amount will vary depending on many factors, such as for instance the compound and its biological activity, the composition used for administration, the route of administration, the type of disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds, and the age, body weight, general health, sex, and diet of the patient. Such an effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge.
  • the present invention relates to a compound of formula (I) for use in a method of treating or preventing a bacterial infection, in particular a multi-drug resistant bacterial infection.
  • the present invention relates to the use of a compound of formula (I) for the preparation of a pharmaceutical composition, preferably a medicament, for the treatment or prevention of a bacterial infection, in particular a multi- drug resistant bacterial infection.
  • compositions The present invention also relates to pharmaceutical or veterinary compositions, in particular a medicament, comprising a compound of formula (I) and one or more excipients, in particular one or more pharmaceutically or veterinary acceptable excipient(s) and to their uses for the above-mentioned purpose.
  • the present invention relates to a pharmaceutical or veterinary composition, in particular a medicament, comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically or veterinary acceptable excipient.
  • the pharmaceutical or veterinary composition is particularly useful in the treatment of bacterial infections.
  • Pharmaceutically or veterinary acceptable excipients include fillers and carriers, ointment bases, bases for suppositories, solvents, surfactants, emulsifiers, dispersants or wetting agents, buffers, acids and bases, isotonicity agents, adsorbent, viscosity- increasing agents, gel formers, thickeners and/or binders, disintegrants, coating materials and film formers for films or diffusion membranes, capsule materials, natural or synthetic polymers, plasticizers, penetration enhancers, stabilizers, preservatives, colourants, flavourings, sweeteners, flavour- and/or odour-masking agents.
  • the compounds of formula (I) can be administered in therapeutically effective amounts in a combinational therapy with one or more other pharmaceutically active agents (pharmaceutical combinations). Therefore, the present invention also relates to such pharmaceutical combinations.
  • the compounds of the present invention can be combined with an antibiotic compound.
  • the compounds can be administered simultaneously (as a single preparation or separate preparation), sequentially or separately.
  • a compound of formula (I) is administered prior to the administration of one or more other pharmaceutically active agents.
  • a compound of formula (I) is administered concomitantly with the administration of one or more other pharmaceutically active agents.
  • a compound of formula (I) is administered immediately after administration of the other pharmaceutically active agent(s).
  • the pharmaceutically active agents may be packaged in a kit or separately.
  • the present invention relates to a kit comprising: - a composition comprising a compound of formula (I) as described herein; - a second composition comprising one or more other pharmaceutically active agents, and - preferably instructions for using said kit.
  • the other pharmaceutically active agent is preferably an antibiotic compound.
  • the antibiotic compound is preferably selected from the group consisting of beta- lactams, aminoglycosides, tetracyclines, glycylcyclines, macrolides, azalides, ketolides, synergistins, lincosamides, fluoroquinolones, phenicols, rifamycins, sulfamides, trimethoprim, glycopeptides, oxazolidinones, nitromidazoles, fosfomycins, polymyxins and lipopeptides.
  • antibiotics are amoxicillin, amoxicillin-clavulanic acid, ampicillin, ampicillin-subactam, benzylpenicillin, cloxacillin, phenoxymethylpenicillin, piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin- clavulanic acid, cefalexin, cefazolin, cefuroxime, cefixime, cefotaxime, cefepime, cefoxitin, ceftazidime, ceftazidime-avibactam, ceftriaxone, cefiderocol, ceftolozane-tazobactam, imipenem, imipenem-relebactam, meropenem, meropenem-vaborbactam, ertapenem, aztreonam, spectinomycin, amikacin, gentamicin, tobramycin, plazomicin, doxycycline, minocycline
  • the compounds of formula (I) and the compositions comprising thereof can have systemic and/or local activity.
  • they can be administered in a suitable manner, such as, for example, via the oral, dermal, transdermal or parenteral route.
  • suitable administration forms for oral administration include for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
  • Suitable administration forms for parenteral administration are preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.
  • Suitable administration forms for the dermal or transdermal administration routes are, for example, pharmaceutical forms for aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (for example patches), milk, pastes, foams or dusting powders.
  • Run time 12.5 minutes HPLC-MS analytical method for final purity check (AM3): Flow: 0.8 mL/min Mobile phase A: milliQ water, HFBA 0.2% Mobile phase B: acetonitrile, 0.05 % formic acid Gradient: 20% to 50% of B in 8 min then, 50 to 95 % of B in 2 min. Run time: 16 minutes Solvents and amino-acid building blocks 1.5-diaminopentane trityl resin, 1.3-diaminopropane trityl resin, Fmoc-rink amide resin and N1,N4-bis-Boc-norspermidine [122248-82-2] were purchased from Chem Impex International Inc (Wood dale, IL, USA).
  • Fmoc-(4-CO 2 tBu)Phe-OH [183070-44-2], Fmoc-Asp-(Alloc)-OH [146982-24-3] and t- butyl (3-amino-2-hydroxypropyl)carbamate [14412-84-5] were purchased from Fluorochem (Hadfield, United Kingdom).
  • tert-Butyl N-(3-amino-2,2-difluoropropyl)carbamate [1044675-84-4] was purchased from abcr Gmbh (Karlsruhe, Germany).
  • Fmoc- ⁇ -(D)-Ser(Trt)-OH [1820583-73-0] Fmoc-(S)-3-NH 2 -2-OH propionic acid [172721- 23-2], tert-butyl (4-aminobut-2-en-1-yl)carbamate [146394-99-2], tert-butyl N-[(2S)-3- amino-2-hydroxypropyl]carbamate [853944-08-8], tert-butyl N-[(2R)-3-amino-2- hydroxypropyl]carbamate [1042665-83-5] and tert-butyl-N-(2-amino-3- ⁇ [(tert- butoxy)carbonyl]amino ⁇ propyl)carbamate [149876-86-8] were purchased from Enamine (Kiev, Ukraine).
  • Step 3 ((2S,3S)-2-amino-4-(tert-butoxycarbonylamino)-3-hydroxy-butanoic acid) 2 Cu (1- 2) (17.1 g, 32.0 mmol) was suspended in water (300 mL). A solution of Na 2 EDTA (1.5 eq., 48.0 mmol, 15.9 g) and NaOH (3.0 eq., 96.0 mmol, 3.84 g) in water (300 mL) was added. The mixture was stirred at room temperature for 4 hours until full dissolution of the suspension.
  • Step 4 (2S,3S)-4-(tert-butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-3-hydroxy-butanoic acid (1-3) (29.1 g, 63.6 mmol) was suspended in a mixture of acetone and 2,2-dimethoxypropane (1:1, 480 mL). The suspension was cooled down with an ice bath then BF 3 .OEt2 (catalytic, 900 ⁇ L) was added drop wise. The reaction was stirred in the melting ice bath until obtaining a limpid orange/brown solution (about 2.5 hours, completion of the reaction was checked by LC- MS).
  • Step 1 A solution of benzyl carbamate (90.0 g, 0.59 mol, 1.0 eq.) and monohydrate dihydroxyacetic acid 1 (60.3 g, 1.1 mol, 1.1 eq.) in toluene (840 mL) was introduced in a 2 L flask and the solution was heated at 40°C for 1.5 hours. Half of the solvent was concentrated down under reduce pressure. Toluene (540 mL) was added and half of the solvent was concentrated down under reduce pressure. Toluene (540 mL) was added and the reaction was stirred at 40°C for 2 hours and then cooled down to 20°C. The white solid was filtered, rinsed with toluene and dried under vacuum.
  • Step 2 Step 2-1: 2-(((benzyloxy)carbonyl)amino)-2-hydroxyacetic acid 2 (133.0 g, 0.59 mol, 1.0 eq.) was diluted in methanol (480 mL). Trimethyl orthoformate (TMOF, 130.2 mL, 1.11 mol, 2.0 eq.) and hydrochloric acid in methanol (1.25 M, 24 mL, 0.03 mol, 0.05 eq.) were added successively. The mixture was stirred at 56 °C for 40 min. Solvent was concentrated down under reduce pressure and 600 mL of Et2O were added. If necessary starting material was filtered off. The filtrate was concentrated down and dried under reduce pressure.
  • TMOF Trimethyl orthoformate
  • Et2O Et2O
  • the previous intermediate (149.5 g, 0.59 mol, 1.0 eq.) was introduced under argon followed by anhydrous toluene (720 mL). Three drops of concentrated sulfuric acid were added. Phosphorus trichloride (80 mL, 0.69 mol, 1.2 eq.) was introduced in the dropping funnel. The mixture was heated at 75°C and PCl 3 was added over 1 h at this temperature. At the end of the addition, the mixture was stirred at 75°C for 13 h. After cooling down to room temperature, the solid was filtered off and the filtrate was concentrated down under vacuum to remove excess of PCl 3 . The crude mixture was diluted in 720 mL of anhydrous toluene under argon.
  • Triethyl phosphite (100 mL, 0.65 mol, 1.1 eq.) was then added and the mixture was stirred at 75°C for 2 h and then at 90°C for 30 min. The reaction mixture was cooled down to room temperature; solvent and excess of triethyl phosphite were removed under vacuum. The crude mixture was dissolved in EtOAc. Organic phase was washed twice with saturated Na 2 CO 3 , dried over MgSO 4 , filtered and concentrated down under vacuum. The crude mixture was precipitated in Et 2 O (1 h at 5 °C).
  • Step 3 Methyl 2-(((benzyloxy)carbonyl)amino)-2-(diethoxyphosphoryl)acetate 3 (20.1 g, 55.7 mmol, 1.0 eq.) was dissolved in 600 mL of EtOH.10% palladium on charcoal (2.0 g, cat.) was added and the reaction mixture was stirred under H 2 atmosphere for 8 to 14 h. Deprotection was monitored by 31 P NMR. After completion the mixture was filtered through celite. The filtrate was concentrated down under vacuum. The crude was dissolved in DCM and concentrated down under reduce pressure. The operation was repeated three times in order to remove traces of EtOH. The free amine was used directly in the next step. The crude product was dissolved in 60 mL of DCM.
  • Step 4 1.3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (50.0 g, 172.1 mmol, 1.0 eq.) was dissolved in 400 mL of DMF. Aminopropan-3-ol (52.5 mL, 688.0 mmol, 4.0 eq.) was added drop wise followed by dimethylaminopyridine (2.1 g, 17.2 mmol, 0.1 eq.). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was dissolved in 4 L of Et2O.
  • Step 5 and 5’ 1.2 eq. of the aldehyde was prepared for the Horner-Wadsworth-Emmons (1.0 eq. of compound 4 of Scheme 1).
  • Step 5 In a 2 L flask, compound 5 of Scheme 1 (20.5 g, 64.6 mmol, 1.2 eq.) was dissolved in DCM (stabilized over amylene, 410 mL). Pyridine (30.9 mL, 465.1 mmol, 7.2 eq.) was added followed by Dess-Martin periodinane (29.7 g, 70 mmol, 1.3 eq.). The reaction was stirred at room temperature for 3 h.
  • the Z/E ratio was determined as 86/14.
  • the crude product was purified by column chromatography on silica (40-63 ⁇ m, pore 60 ⁇ , 1.5 kg, 3/2 petroleum ether/EtOAc ⁇ 2 L, then 1/1 petroleum ether/EtOAc ⁇ 3 L then 2/3 petroleum ether/EtOAc).3 fractions were obtained, the first one containing the alkene E, the second a mixture of Z and E alkene and the third the alkene Z with a good purity (> 95% by HPLC). The second fraction was purified again by column chromatography.
  • Example 3 Synthesis of non-commercial building-blocks for C-terminal derivatization
  • Example 3.1 Under inert atmosphere and magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (1.19 g, 6.65 mmol, 1.0 eq.) was dissolved in 12 mL of anhydrous 2-propanol.
  • Step 2 To a solution of tert-butyl N-[3-[[3-(benzyloxycarbonylamino)-2-hydroxy- propyl]amino]propyl]carbamate (3.20 g, 6.29 mmol, 1.0 eq.) in 63 mL of methanol, at room temperature, was added tert-butoxycarbonyl tert-butyl carbonate (2.06 g, 9.44 mmol, 1.5 eq.) and N-ethyl-N-(propan-2-yl)propan-2-amine (2.2 mL, 12.6 mmol, 2.0 eq.). The reaction mixture was stirred 1 h at 65 °C then concentrated under vacuum.
  • Step 3 At room temperature, under inert atmosphere and magnetic stirring, tert-butyl N- [3-(benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)propyl] carbamate (1.98 g, 3.70 mmol, 1.0 eq.) was dissolved in 75 mL of methanol. Palladium (787 mg, 0.740 mmol, 2.0 eq.) and cyclohexene (10 mL, 92.5 mmol, 25 eq.) were added. The mixture was stirred for 2 h at 70 °C then filtered through a Millipore filter.
  • Step 3 To a solution of allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate (744 mg, 4.73 mmol, 1.0 eq.) in 12 mL of 2-propanol, at 70 °C, was added dropwise over 15 min tert-butyl N- [(2S)-3-amino-2-hydroxypropyl]carbamate (900 mg, 4.73 mmol, 1.0 eq.). The mixture was stirred for 1 h at 70 °C then concentrated under vacuum.
  • tert-Butyl N-[(2S)-3-[[(2R)- 3-(allyloxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained (1.6 g).
  • Step 4 At 5 °C and under magnetic stirring, tert-butyl N-[(2S)-3-[[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]amino]-2-hydroxy-propyl]carbamate (1.60 g, 4.61 mmol, 1.0 eq.) was dissolved in 30 mL of CH 2 Cl 2 .
  • Step 5 To allyl-N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.48 g, 3.43 mmol, 1.0 eq.) in CH 2 Cl 2 (60 mL) at 0 °C, was added dropwise 2,2,2-trifluoroacetic acid (5.5 mL, 71.9 mmol, 21 eq.). The mixture was risen to room temperature, stirred 3 h then concentrated under vacuum. The crude product was dried overnight under vacuum.
  • Step 4 To a solution of tert-butyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]-2-hydroxy-propyl]carbamate (1.47 g, 4.15 mmol, 1.0 eq.) in 43 mL of ethanol, was added by portion tert-butoxycarbonyl tert-butyl carbonate (1.02 g, 4.51 mmol, 1.01 eq.). The mixture was stirred for 1.5 h at room temperature then concentrated under vacuum.
  • tert-butyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2S)-3- (tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (1.73 g).
  • Step 5 Under inert atmosphere and magnetic stirring, tert-butyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2S)-3-(tert-butoxycarbonylamino)-2- hydroxy-propyl]carbamate (1.73 g, 3.83 mmol, 1.0 eq.) was dissolved in 32 mL of anhydrous CH 2 Cl 2 .
  • Phenylsilane (2.4 mL, 19.1 mmol, 5.0 eq.) was added. The mixture was stirred for 15 min under argon bubbling, then palladium-tetrakis(triphenylphosphine) (221 mg, 0.191 mmol, 0.05 eq.) was added. The mixture was stirred for 1.5 h at room temperature then concentrated under vacuum. The product was purified by silica gel flash column chromatography (CH 2 Cl 2 /(CH 2 Cl 2 -MeOH-7N NH 3 in MeOH 90-9-1) 100/0 to 0/100 then CH 2 Cl 2 -MeOH-7N NH 3 in MeOH 80-19-1).
  • Triethylamine (151 mL, 1.08 mol, 4.0 eq.) was added, followed by a dropwise addition of prop-2-en-1-yl carbonochloridate (58 mL, 540 mmol, 2.0 eq.).
  • the reaction mixture was stirred for 1 h at room temperature. Water was added to the reaction mixture. The phases were separated and the organic layer was washed with brine. The phases were separated and the organic phase was dried over MgSO 4 , filtered and concentrated under vacuum to give a crude oil.
  • the crude product was purified by silica gel flash column chromatography (CH 2 Cl 2 /MeOH, 0 to 10 % of MeOH in 30 min).
  • the organic phase was washed with 250 mL of saturated NH 4 Cl then 150 mL of brine, dried over MgSO 4 , filtered and concentrated under vacuum to give a crude oil.
  • the crude product was purified by silica gel flash column chromatography (CH 2 Cl 2 /MeOH, 0 to 10 % of MeOH in 30 min) to give two batches of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate.
  • Step 3 To a solution of tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]carbamate (2.5 g, 12.5 mmol, 1.0 eq.) in 2-propanol (34 mL), stirred at 70 °C, was added dropwise over 25 min a solution of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate (2.07 g, 12.5 mmol, 1.0 eq.) in 2-propanol (6 mL). The mixture was stirred for 1 h at 70 °C.
  • Step 4 To a solution of tert-butyl N-[(2S)-3-[[(2S)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]-2-hydroxy-propyl]carbamate (2.7 g, 7.84 mmol, 1.0 eq.) in ethanol (80 mL), stirred at room temperature, was added, by portion, tert-butoxycarbonyl tert-butyl carbonate (1.9 g, 8.44 mmol, 1.1 eq.). The mixture was stirred for 1h30 at room temperature then concentrated under vacuum.
  • Phenylsilane (4.9 mL, 38.2 mmol, 5.0 eq.) was added and the mixture was stirred 15 min under argon bubbling. Then, palladium- tetrakis(triphenylphosphine) (442 mg, 0.382 mmol, 0.05 eq.) was added. The reaction mixture was stirred for 6 h. Additional phenylsilane (2.4 mL) and palladium- tetrakis(triphenylphosphine) (220 mg) were added to the reaction. The mixture was stirred overnight then concentrated under vacuum.
  • Step 6 Under inert atmosphere and magnetic stirring, tert-butyl N-[(2R)-3-amino-2- hydroxy-propyl]-N-[(2R)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.82 g, 4.91 mmol, 1.0 eq.) was dissolved in 7 mL of anhydrous pyridine and 21 mL of anhydrous CH 2 Cl 2 . Fmoc-Lys(Boc)-OH (2.37 g, 4.91 mmol, 1.0 eq.) was added.
  • Step 7 To a solution of tert-butyl N-[(2R)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxypropyl]-N-[(2R)-3- (tert-butoxycarbonylamino)-2-hydroxypropyl]carbamate (2.9 g, 3.49 mmol, 1.0 eq.) in THF (66 mL) was added N-ethylethanamine (9.2 mL, 88.8 mmol, 25 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum.
  • Step 4 To a solution of allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]amino]-2-hydroxy- propyl]carbamate (738 mg, 1.39 mmol, 1.0 eq.) in CH 2 Cl 2 (14 mL) was added 2,2,2- trifluoroacetic acid (0.53 mL, 6.95 mmol, 5.0 eq.). The mixture was stirred overnight at room temperature.
  • Step 5 Under inert atmosphere, allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-[(2R)-3-amino-2-hydroxy-propyl]amino]-2-hydroxy-propyl]carbamate dihydrochloride (720 mg, 1.36 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (8 mL). Anhydrous pyridine (6 mL) was added followed by Fmoc-Lys(Boc)-OH (670 mg, 1.39 mmol, 1.02 eq.).
  • N-ethyl-N-isopropyl-propan-2-amine (1.2 mL, 6.79 mmol, 5.0 eq.), Fmoc-Lys(Boc)-OH (670 mg, 1.39 mmol, 1.02 eq.) and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine;hydrochloride (286 mg, 1.49 mmol, 1.1 eq.) were added. The mixture was stirred overnight at room temperature then concentrated (co-evaporation with toluene). The residue was taken up in a small volume of MeOH/EtOAc and washed with water, 50 mL citric acid, saturated NaHCO 3 then brine.
  • Step 6 To a solution of allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-[(2R)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]amino]-2-hydroxy- propyl]carbamate (1200 mg, 0.70 mmol, 1.0 eq.) in THF (25 mL) was added N- ethylethanamine (2.5 mL, 24.2 mmol, 34 eq.).
  • Example 3.6 Step 1 To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (20 g, 0.14 mol, 1.0 eq.) in CH 2 Cl 2 (100 mL) at -5 °C, were added N,N-diethylethanamine (40 mL, 0.29 mol, 2.1 eq.) and dropwise prop-2-en-1-yl carbonochloridate (15 mL, 0.14 mol, 1.05 eq.). The reaction was stirred for 2 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water and 1 M HCl. The organic phase was dried over Na2SO 4 , filtered then concentrated under vacuum.
  • Step 3 To allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate (1.0 g, 6.36 mmol, 1.0 eq.) in 2- propanol (15 mL) at 70 °C, was added dropwise (15 min) a solution of tert-butyl 3- (aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.33 g, 6.36 mmol, 1.0 eq.) in 2- propanol (2 mL).
  • Step 4 To tert-butyl 3-[[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (2.2 g, 6.12 mmol, 1.0 eq) in ethanol (60 mL) was added tert-butoxycarbonyl tert-butyl carbonate (1.47 g, 6.73 mmol, 1.1 eq.). The reaction was stirred 2 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash chromatography (CH 2 Cl 2 /MeOH 94/6).
  • Step 6 To tert-butyl 3-[[[(2S)-3-amino-2-hydroxy-propyl]-tert- butoxycarbonylamino]methyl]-3-hydroxy-azetidine-1-carboxylate (0.94 g, 2.50 mmol, 1.0 eq.) in CH 2 Cl 2 (15 mL) and pyridine (8 mL) at 0 °C, were added Fmoc-Lys(Boc)-OH (1.21 g, 2.50 mmol, 1.0 eq.) then N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (388 mg, 2.50 mmol, 1.0 eq.).
  • Step 7 To tert-butyl 3-[[tert-butoxycarbonyl-[(2S)-3-[[(2S)-6-(tert-butoxycarbonylamino)- 2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy- propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.12 g, 1.36 mmol, 1.0 eq.) in THF (30 mL) was added N-ethylethanamine (5.0 mL, 47.6 mmol, 35 eq.). The reaction mixture was stirred 4 h at room temperature then concentrated under vacuum.
  • Step 2 Under inert atmosphere, at 0 °C and under magnetic stirring, tert-butyl N-[3- (allyloxycarbonylamino)propyl]carbamate (13.1 g, 50.7 mmol, 1.0 eq.) was dissolved in 128 mL of anhydrous 1,4-dioxane. A solution of 4 M HCl (127 mL, 0.51 mol, 10 eq.) was added dropwise in 30 min. The mixture was risen to room temperature, stirred overnight then concentrated under vacuum. Allyl N-(3-aminopropyl)carbamate hydrochloride was obtained (9.5 g).
  • Step 3 Under inert atmosphere and magnetic stirring, (2R)-3-aminopropane-1,2-diol (24.08 g, 0.256 mol, 1.0 eq.) and N,N-diethylethanamine (52.6 mL, 384.6 mmol, 1.5 eq.) were dissolved in 723 mL of anhydrous MeOH. The mixture was cooled down to 0 °C then tert-butoxycarbonyl tert-butyl carbonate (83.94 g, 0.385 mol, 1.5 eq.) was added by portion. The resulting mixture was risen to room temperature, stirred 2 h at room temperature then concentrated under vacuum.
  • Step 6 Under inert atmosphere, tert-butyl N-[3-(allyloxycarbonylamino)propyl]-N-[(2R)- 3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.25 g, 2.90 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (25 mL). Phenylsilane (0.74 mL, 5.79 mmol, 2.0 eq.) then palladium-tetrakis(triphenylphosphine) (335 mg, 0.290 mmol, 0.1 eq.) were added under argon bubbling. The mixture was stirred for 3 h then concentrated under vacuum.
  • Step 8 Under inert atmosphere, tert-butyl N-[3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]propyl]-N-[(2R)-3-(tert- butoxycarbonyl amino)-2-hydroxy-propyl]carbamate (1.35 g, 1.69 mmol, 1.0 eq.) was dissolved in anhydrous THF (15 mL). N-ethylethanamine (3.5 mL, 33.8 mmol, 20 eq.) was added. The mixture was stirred for 12 h at room temperature then concentrated under vacuum.
  • Example 3.8 Step 1 Under inert atmosphere and magnetic stirring, tert-butyl N-(3-amino-2-hydroxy- propyl)carbamate (0.39 g, 2.05 mmol, 1.0 eq.) was dissolved in 4.5 mL of isopropanol. The mixture was heated under reflux (90 °C) then benzyl N-(oxiran-2- ylmethyl)carbamate (0.50 g, 2.05 mmol, 1.0 eq.) was added dropwise. The reaction mixture was stirred for 1 h under reflux then cooled down to room temperature and concentrated under vacuum.
  • Step 4 Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (498 mg, 1.06 mmol, 1.2 eq.) was dissolved in 10 mL of CH 2 Cl 2 . 3-(ethyliminomethyleneamino)-N,N- dimethyl-propan-1-amine hydrochloride (204 mg, 1.06 mmol, 1.2 eq.) and pyridine (5.1 mL, 0.063 mol, 71 eq.) were added.
  • Step 5 Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2- hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (245 mg, 0.301 mmol, 1.0 eq.) was dissolved in anhydrous THF (4 mL). N-ethylethanamine (22 mg, 0.301 mmol, 1.0 eq.) was added. The mixture was stirred for 3 h at room temperature then concentrated under vacuum.
  • Example 3.9 Step 1: To tert-butyl N-(3-amino-2-hydroxypropyl)carbamate (5.0 g, 25.0 mmol, 1.0 eq.) solubilized in anhydrous CH 2 Cl 2 (100 mL) under inert atmosphere, was added N,N- diethylethanamine (3.5 mL, 25.0 mmol, 1.0 eq.). The mixture was cooled down at 0°C and tert-butyl(chloro)dimethylsilane (4.52 g, 30.0 mmol, 1.2 eq.) was added. The mixture was risen up to room temperature, stirred for 4 h then concentrated under vacuum.
  • Step 2 To a solution of 9H-fluoren-9-ylmethyl (3-hydroxypropyl)carbamate (5.0 g, 16.0 mmol, 1.0 eq.) in CH 2 Cl 2 (50 mL) at 0 °C, was added 1,1,1-tris(acetyloxy)-1,1-dihydro- 1,2-benziodoxol-3-(1H)-one (7.13 g, 16.0 mmol, 1.0 eq.). The mixture was stirred for 10 min at 0 °C then risen to room temperature and stirred for 1 h. The mixture was concentrated, and the residue was taken up in EtOAc (100 mL) and washed with saturated NaHCO 3 (30 mL).
  • Step 3 Under inert atmosphere, tert-butyl N-[3-amino-2-[tert-butyl(dimethyl)silyl]oxy- propyl]carbamate (3.0 g, 9.85 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane (125 mL).9H-fluoren-9-ylmethyl N-(3-oxopropyl)carbamate (2.91 g, 9.85 mmol, 1.0 eq.) and acetic acid (0.96 mL, 16.7 mmol, 1.7 eq.) were added.
  • Step 4 To a solution of 9H-fluoren-9-ylmethyl N-[3-[[3-(tert-butoxycarbonylamino)-2- [tert-butyl(dimethyl)silyl]oxy-propyl]amino]propyl]carbamate (1.50 g, 2.57 mmol, 1.0 eq.) in CH 2 Cl 2 (37.5 mL) were added N,N-diethylethanamine (0.54 mL, 3.85 mmol, 1.5 eq.), DMAP (31 mg, 0.257 mmol, 0.1 eq.) and di-tert-butyl dicarbonate (0.84 g, 3.85 mmol, 1.5 eq.).
  • Step 5 Under inert atmosphere, tert-butyl N-[3-(tert-butoxycarbonylamino)-2-[tert- butyl(dimethyl)silyl]oxy-propyl]-N-[3-(9H-fluoren-9- ylmethoxycarbonylamino)propyl]carbamate (1.1 g, 1.61 mmol, 1.0 eq.) was dissolved in anhydrous THF (20 mL). N-ethylethanamine (5.0 mL, 48.3 mmol, 30 eq.) was added. The mixture was stirred for 12 h at room temperature then concentrated under vacuum.
  • Example 3.10 Step 1: Tert-butyl N-[(2R)-2,3-dihydroxypropyl]carbamate was obtained as a white solid (33.36 g) following the step 3 described in example 3.7. Step 2: Tert-butyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained as a white solid (17.42 g) following the step 4 described in example 3.7.
  • Step 4 Tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-hydroxybutyl]carbamate (1.2 g, 3.55 mmol, 1.0 eq.) was dissolved in CH 2 Cl 2 (20 mL). A solution of 4 M hydrogen chloride in dioxane (9.0 mL, 36.0 mmol, 10 eq.) was added. The mixture was stirred overnight at room temperature then concentrated under vacuum. The crude product was triturated with diisopropyl ether and the precipitate was filtered and dried under vacuum. Benzyl N-[(1S)-3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride was obtained as a white solid (900 mg).
  • Step 5 To a solution of benzyl N-[(1S)-3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride (900 mg, 3.28 mmol, 1.0 eq.) in CH 2 Cl 2 (30 mL) were added N,N- diethylethanamine (1.6 mL, 11.5 mmol, 3.5 eq.) and a solution of tert-butyl-chloro- dimethylsilane (dropwise , 987 mg, 6.55 mmol, 2.0 eq.) in CH 2 Cl 2 (10 mL). The reaction mixture turned pink. It was stirred for 3 h then concentrated under vacuum.
  • Step 6 Benzyl N-[(1S)-3-amino-1-[[tertbutyl(dimethyl)silyl]oxymethyl]propyl] carbamate (600 mg, 1.7 mmol, 1.0 eq.) was suspended in isopropanol (3.4 mL).
  • Step 7 To a solution of tert-butyl N-[(2S)-3-[[(3S)-3-(benzyloxycarbonylamino)-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]-2-hydroxypropyl]carbamate (590 mg, 1.12 mmol, 1.0 eq.) in CH 2 Cl 2 (50 mL) were added tert-butoxycarbonyl tert-butyl carbonate (0.49 g, 2.24 mmol, 2.0 eq.), N,N-diethylethanamine (0.39 mL, 2.81 mmol 2.5 eq.) and N,N-dimethylpyridin-4-amine (27 mg, 0.22 mmol, 0.2 eq.).
  • Step 8 Tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy- butyl]-N-[(2R)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate and [(1R)-1- [[[(3S)-3-(benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-tert- butoxycarbonyl-amino]methyl]-2-(tert-butoxycarbonylamino)ethyl] tert-butyl carbonate (640 mg, 0.88 mmol, 1.0 eq.) were dissolved in MeOH (50 mL) and hydrogenated with a H-cube ® hydrogenation reactor (1 mL/min, room temperature and full H 2 ) on a Pd/C (0.88 mmol, 1.0
  • Step 9 Under inert atmosphere, Fmoc-Lys(Boc)-OH (375 mg, 0.78 mmol, 1.0 eq.) and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (164 mg, 0.85 mmol, 1.1 eq.) was dissolved in dry pyridine (9.9 mL, 0.12 mol).
  • Step 10 Tert-butyl N-[(3S)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N-[(2R)- 3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate and [(1R)-1-[(tert- butoxycarbonylamino)methyl]-2-[tertbutoxycarbonyl-[(3S)-3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]ethyl] tert
  • Step 1 Allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a colourless liquid (11 g) following the step 1 described in example 3.2.
  • Step 2 Allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a colourless liquid (4.8 g) following the step 2 described in example 3.2.
  • Step 3 To tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]carbamate (1.50 g, 7.88 mmol, 1.0 eq.) in isopropanol (20 mL) at 70 °C was added dropwise over 15 min allyl N-[[(2S)- oxiran-2-yl]methyl]carbamate (1.24 g, 7.88 mmol, 1.0 eq.). The reaction was stirred for 40 min at 70 °C then concentrated under vacuum.
  • Step 5 Under inert atmosphere, to tert-butyl N-[(2S)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.6 g, 3.58 mmol, 1.0 eq.) in anhydrous CH 2 Cl 2 (40 mL) was added phenylsilane (1.4 mL, 10.7 mmol, 3.0 eq.) and tetrakis(triphenylphosphine)-palladium (289 mg, 0.25 mmol, 0.07 eq.). The reaction was stirred 3 h at room temperature then concentrated under vacuum.
  • Step 6 To a solution of Fmoc-Lys(Boc)-OH (1.69 g, 3.50 mmol, 1.0 eq.) in CH 2 Cl 2 (16 mL) and pyridine (8 mL) at 5 °C were added N-[3-(dimethylamino)propyl]-N'- ethylcarbodiimide (652 mg, 4.20 mmol, 1.2 eq.) then after 5 min tert-butyl N-[(2S)-3- amino-2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy- propyl]carbamate (1.27 g, 3.50 mmol, 1.0 eq.).
  • Example 3.12 Step 1 To a solution of tert-butyl N-(3-aminopropyl)carbamate (2.0 g, 11.5 mmol, 1.0 eq.) in MeOH (50 mL) was added prop-2-enenitrile (0.76 mL, 11.5 mmol, 1.0 eq.). The reaction mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water. The organic layer was dried over Na2SO 4 , filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH 2 Cl 2 /MeOH 97/3).
  • Step 3 To a solution of ethyl 2-[3-(tert-butoxycarbonylamino)propyl-(2- cyanoethyl)amino]acetate (3.75 g, 12 mmol, 1.0 eq.) in ethanol (50 mL) was added by portion sodium borohydride (760 mg, 20 mmol, 1.7 eq.). The reaction mixture was heated to 60 °C and stirred for 2 h. After 2 h, the reaction was not complete: sodium borohydride (300 mg, 7.9 mmol, 0.7 eq.) was added and the mixture was stirred at 70 °C for 1.5 h.
  • sodium borohydride 300 mg, 7.9 mmol, 0.7 eq.
  • Step 4 To a solution of tert-butyl N-[3-[2-cyanoethyl(2- hydroxyethyl)amino]propyl]carbamate (1.20 g, 4.42 mmol, 1.0 eq.) in CH 2 Cl 2 (56 mL) was added tert-butyl-chloro-dimethyl-silane (1.67 g, 11.1 mmol , 2.5 eq.), triethylamine (1.6 mL, 11.5 mmol, 2.6 eq.) and N,N-dimethylpyridin-4-amine (0.11 g, 0.884 mmol, 0.2 eq.). The mixture was stirred for 18 h at room temperature.
  • Step 5 Tert-butyl N-[3-[2-[tert-butyl(dimethyl)silyl]oxyethyl-(2- cyanoethyl)amino]propyl]carbamate (0.93 g, 2.41 mmol, 1.0 eq.) was dissolved in MeOH (15 mL). The mixture was cooled down to 0 °C. Nickel(II) chloride hexahydrate (250 mg, 1.05 mmol, 0.4 eq.) was added followed by an addition by portion, over 40 min, of sodium borohydride (630 mg, 16.7 mmol, 6.9 eq.).
  • Step 6 To a solution of tert-butyl N-[3-[3-aminopropyl-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]propyl]carbamate (770 mg, 1.98 mmol, 1.0 eq.) in CH 2 Cl 2 (8 mL) and pyridine (4 mL), were added Fmoc-Lys(Boc)-OH (1.05 g, 2.17 mmol, 1.1 eq.) and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (337 mg, 2.17 mmol, 1.1 eq.).
  • Step 7 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1- [3-[3-(tert-butoxycarbonylamino)propyl-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]propylcarbamoyl]pentyl]carbamate (450 mg, 0.536 mmol, 1.0 eq.) in THF (15 mL) was added N-ethylethanamine (3.0 mL, 28.7 mmol, 54 eq.). The reaction was stirred overnight at room temperature then concentrated under vacuum.
  • Step 1 To a solution at 0-5 °C of tert-butyl N-(4-amino-3-hydroxybutyl)carbamate (1000 mg, 4.90 mmol, 1.0 eq.) in CH 2 Cl 2 (20 mL), were added triethylamine (0.8 mL, 5.88 mmol, 1.2 eq.) and chloro(triethyl)silane (0.9 mL, 5.39 mmol, 1.1 eq.). The mixture was risen to room temperature, stirred overnight then concentrated under reduced pressure. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over MgSO 4 , filtered then concentrated under vacuum.
  • tert-Butyl N-(4-amino-3- triethylsilyloxy-butyl)carbamate was obtained (1.36 g).
  • Step 2 tert-Butyl N-(4-amino-3-triethylsilyloxy-butyl)carbamate (860 mg, 2.7 mmol, 1.0 eq.) was dissolved in 5 mL of isopropanol. The mixture was heated to reflux.
  • Step 3 Under inert atmosphere, tert-butyl N-[4-[[3-(benzyloxycarbonylamino)-2-hydroxy- propyl]amino]-3-triethylsilyloxy-butyl]carbamate (1.25 g, 2.38 mmol, 1.0 eq.) was dissolved in 9.5 mL of anhydrous CH 2 Cl 2 . The mixture was cooled down at 0-5 °C and triethylamine (1.17 mL, 8.56 mmol, 3.6 eq.) then chloro(triethyl)silane (1.2 mL, 7.13 mmol, 3.0 eq.) were added.
  • Step 4 tert-Butyl N-[4-[[3-(benzyloxycarbonylamino)-2-triethylsilyloxypropyl] amino]-3-triethylsilyloxy-butyl]carbamate (1.7 g, 2.66 mmol, 1.0 eq.) and tert- butoxycarbonyl tert-butyl carbonate (1159 mg, 5.31 mmol, 2.0 eq.) were dissolved in CH 2 Cl 2 (13 mL). The mixture was cooled down at 0-5 °C and N,N-diethylethanamine (0.80 mL, 5.84 mmol, 2.2 eq.) was added.
  • Step 5 To a solution of tert-butyl N-[3-(benzyloxycarbonylamino)-2- triethylsilyloxypropyl]-N-[4-(tert-butoxycarbonylamino)-2-triethylsilyloxybutyl]carbamate (1.4 g, 1.89 mmol, 1.0 eq.) in 50 mL of MeOH, were added cyclohexene (4.8 mL, 47.3 mmol, 25 eq.) then palladium (10%, 403 mg, 0.378 mmol, 0.2 eq.). The mixture was heated to reflux, stirred for 1 h then cooled down to room temperature and stirred for 2 h.
  • Step 6 Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (399 mg, 0.825 mmol, 1.0 eq.) and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (174 mg, 0.908 mmol, 1.1 eq.) were dissolved in anhydrous pyridine (10 mL).
  • Step 7 Under inert atmosphere, tert-butyl N-[4-(tert-butoxycarbonylamino)-2- triethylsilyloxybutyl]-N-[3-[[rac-(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-triethylsilyloxy-propyl]carbamate (660 mg, 0.62 mmol, 1.0 eq.) was dissolved in 10 mL of a solution of diethylamine at 20% in anhydrous THF. The mixture was stirred overnight then concentrated under vacuum.
  • Step 2 Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]amino]propyl]carbamate (4.70 g, 12.3 mmol, 1.0 eq.) and N, N-diisopropylethylamine (4.7 mL, 27.1 mmol, 2.2 eq.) were dissolved in anhydrous CH 2 Cl 2 (47 mL).
  • Step 3 Under inert atmosphere and magnetic stirring, tert-butyl N-[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (3.15 g, 6.54 mmol, 1.0 eq.) was dissolved in MeOH (194 mL) then cyclohexene (17 mL, 0.164 mol, 25 eq.) was added. The mixture was stirred for 5 min then palladium (139 mg, 1.31 mmol, 0.2 eq.) was added. The mixture was heated at 70 °C, stirred for 4 h then concentrated under vacuum.
  • Step 4 Under inert atmosphere and magnetic stirring, tert-butyl N-(3-amino-2-hydroxy- propyl)-N-[3-(tert-butoxycarbonylamino)propyl]carbamate (1.00 g, 2.88 mmol, 1.0 eq.) was dissolved in anhydrous dichloroethane (31 mL). Ethyl oxoacetate (0.67 mL, 3.17 mmol, 1.1 eq.) and acetic acid (0.28 mL, 4.89 mmol, 1.7 eq.) were added and the reaction mixture was stirred 30 min.
  • Step 5 Under inert atmosphere and magnetic stirring, tert-butyl N-[3-(tert- butoxycarbonylamino)propyl]-N-[2-hydroxy-3-(2-hydroxyethylamino)propyl]carbamate (1.24 g, 3.17 mmol, 1.0 eq.) and N-ethyl-N-isopropyl-propan-2-amine (5.5 mL, 31.7 mmol, 10 eq.) were dissolved in anhydrous CH 2 Cl 2 (15 mL).
  • Step 6 Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (301 mg, 0.629 mmol, 1.2 eq.), tert-butyl N-[3-(tert-butoxycarbonylamino)propyl]-N-[2-[tert- butyl(dimethyl)silyl]oxy-3-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]propyl]carbamate (365 mg, 0.524 mmol, 1.0 eq.) and anhydrous pyridine (5 mL) were dissolved in anhydrous CH 2 Cl 2 (10 mL).
  • Step 7 Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]-2-[tert-butyl(dimethyl)silyl]oxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (424 mg, 0.392 mmol, 1.0 eq.) and N- ethylethanamine (2.0 mL, 19.3 mmol, 49 eq.) were dissolved in anhydrous THF (8 mL).
  • Example 3.15 Step 1 To a solution of Cbz-Dab(Boc)-OH (5.0 g, 14.2 mmol, 1.0 eq.) in THF (81.2 mL) at -20 °C, were added N,N-diethylethanamine (2.2 mL, 15.6 mmol, 1.1 eq.) and isobutyl carbonochloridate (2.2 mL, 17 mmol, 1.2 eq.). The reaction was stirred for 20 min at -20 °C then 2.4 M lithium aluminum hydride (10 mL, 24 mmol, 1.7 eq.) was added (temperature -12 °C to -3 °C). The reaction mixture was raised to room temperature then stirred for 2 h.
  • N,N-diethylethanamine 2.2 mL, 15.6 mmol, 1.1 eq.
  • isobutyl carbonochloridate 2.2 mL, 17 mmol, 1.2 eq.
  • Step 2 To a solution of tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-hydroxy- butyl]carbamate (1.9 g, 5.61 mmol, 1.0 eq.) in CH 2 Cl 2 (60 mL) was added 4 M HCl (16 mL, 64.0 mmol, 11 eq.). The reaction was stirred for 18 h at room temperature then concentrated under vacuum. The residue was triturated with diisopropylether then the resulting mixture was filtered. The solid residue was dried under vacuum.
  • Step 3 To benzyl N-[(1S)- 3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride (920 mg, 3.35 mmol, 1.0 eq.) in CH 2 Cl 2 (30 mL) were added N,N-diethylethanamine (1.6 mL, 11.7 mmol, 3.5 eq.), a solution of tert-butyl-chloro-dimethyl-silane (dropwise , 1.01 g, 6.70 mmol, 2.0 eq.) in CH 2 Cl 2 (4 mL) and N,N-dimethylpyridin-4-amine (82 mg, 0.670 mmol, 0.2 eq.).
  • Step 4 To benzyl N-[(1S)-3-amino-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl]carbamate (814 mg, 2.31 mmol, 1.0 eq.) in isopropanol (10 mL) at 70 °C, was added dropwise over 15 min a solution of tert-butyl N-[[(2S)-oxiran-2-ylmethyl]carbamate (400 mg, 2.31 mmol, 1.0 eq.) in isopropanol (2 mL). The reaction mixture was stirred 1 h at 70 °C then concentrated under vacuum.
  • Step 5 To a solution of tert-butyl N-[(2R)-3-[[(3S)-3-(benzyloxycarbonylamino)-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]-2-hydroxypropyl]carbamate (1.21 g, 2.30 mmol, 1.0 eq.) in CH 2 Cl 2 (30 mL) were added tert-butoxycarbonyl tert-butyl carbonate (1.00 g, 4.60 mmol, 2.0 eq.), N,N-diethylethanamine (0.80 mL, 5.75 mmol, 2.5 eq.) and N,N- dimethylpyridin-4-amine (56 mg, 0.460 mmol, 0.2 eq).The mixture was stirred overnight at room temperature then concentrated under vacuum.
  • Step 7 To a solution of tert-butyl N-[(3S)-3-amino-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N- [(2S)-3-(tertbutoxycarbonylamino)-2-hydroxypropyl]carbamate (480 mg, 0.976 mmol, 1.0 eq.) in CH 2 Cl 2 (8 mL) and pyridine (3 mL), were added Fmoc-Lys(Boc)-OH (471 mg, 0.976 mmol, 1.0 eq.) and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (167 mg, 1.07 mmol, 1.1 eq.).
  • Step 8 To a solution of tert-butyl N-[(3S)-3-[[(2S)-6-(tertbutoxycarbonylamino)- 2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4-[tert- butyl(dimethyl)silyl]oxy-butyl]-N-[(2S)-3-(tert-butoxycarbonylamino)-2- hydroxypropyl]carbamate (700 mg, 0.743 mmol, 1.0 eq.) in THF (30 mL) was added N- ethylethanamine (8.0 mL, 76.6 mmol, 103 eq.).
  • Step 3 To tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.00 g, 4.94 mmol, 1.0 eq.) in 2-propanol (12 mL) at 70 °C was added dropwise over 15 min allyl N- [[rac-(2S)-oxiran-2-yl]methyl]carbamate (777 mg, 4.94 mmol, 1.0 eq.). The reaction was stirred 50 min at 70 °C then concentrated under vacuum. The crude was then purified by silica gel flash column chromatography (CH 2 Cl 2 /MeOH 94/6 to 90/10).
  • Step 7 To allyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate (1.12 g, 1.41 mmol, 1.0 eq.) in THF (35 mL) was added N-ethylethanamine (6.0 mL, 57.7 mmol, 41 eq.). The reaction mixture was stirred for 4 h at room temperature then concentrated under vacuum.
  • Step 3 Tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (2.0 g, 9.39 mmol, 1.0 eq.) was dissolved in isopropanol (15 mL).
  • Step 4 To a solution of tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (2.0 g, 5.56 mmol, 1.0 eq.) in ethanol (60 mL) was added tert-butoxycarbonyl tert-butyl carbonate (1.34 g, 6.12 mmol, 1.1 eq.). The reaction mixture was stirred for 3 h at room temperature then concentrated under vacuum.
  • Phenylsilane (1.91 mL, 15.01 mmol, 3.0 eq.) was added then the mixture was stirred for 20 min.
  • Palladium - triphenylphosphine (1:4) (405 mg, 0.350 mmol, 0.07 eq.) was added.
  • the reaction mixture was stirred for 1 h at room temperature then concentrated under vacuum.
  • the crude product was purified by silica gel flash chromatography to give tert-butyl 3-[[[(2R)- 3-amino-2-hydroxy-propyl]-tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1- carboxylate (1.4 g).
  • Step 7 Under inert atmosphere, tert-butyl 3-[[tert-butoxycarbonyl-[(2R)-3-[[(2S)-6- (tertbutoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]- 2-hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.5 g, 1.82 mmol, 1.0 eq.) was dissolved in a solution of N-ethylethanamine in anhydrous THF (solution at 20% in THF). The mixture was stirred overnight then concentrated under vacuum. The crude product was purified on silica gel flash column chromatography.
  • Step 4 Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]amino]propyl]carbamate (3.16 g, 9.54 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (50 mL) then triethylamine (2.9 mL, 21.0 mmol, 2.2 eq.) was added. The reaction mixture was a yellow solution. Tert- butoxycarbonyl tert-butyl carbonate (4166 mg, 19.1 mmol, 2.0 eq.) was added by portion. The mixture was stirred at room temperature for 2 h then washed with water.
  • Step 5 Under N 2 atmosphere and magnetic stirring, tert-butyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (2.20 g, 5.09 mmol, 1.0 eq.) and phenylsilane (3.1 mL, 25.5 mmol, 5.0 eq.) were dissolved in anhydrous THF (25 mL). This solution was stirred under argon bubbling for 15 min then palladium-tetrakis(triphenylphosphine) (1.18 g, 1.02 mmol, 0.2 eq.) was added.
  • Example 3.19 Step 1 Under inert atmosphere and magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (5.36 g, 29.8 mmol, 1.0 eq.) and N-ethyl-N-(propan-2-yl)propan- 2-amine (11 mL, 65.6 mmol, 2.2 eq.) were dissolved in anhydrous CH 2 Cl 2 (54 mL). The solution was cooled down to 0 °C then prop-2-en-1-yl carbonochloridate (3.8 mL, 35.8 mmol, 1.2 eq.) was added dropwise. The reaction mixture was risen to room temperature, stirred for 1 h then washed with water.
  • tert-butyl N-(3- aminopropyl)carbamate 5.36 g, 29.8 mmol, 1.0 eq.
  • N-ethyl-N-(propan-2-yl)propan- 2-amine 11
  • Step 2 Under inert atmosphere and magnetic stirring, tert-butyl N-[3- (allyloxycarbonylamino)propyl]carbamate (7.35 g, 28.5 mmol, 1.0 eq.) was dissolved in anhydrous 1,4-dioxane (72 mL) then a solution of 4 M HCl in 1,4-dioxane (41 mL, 0.285 mol, 10 eq.) in 1,4-dioxane was added dropwise. The mixture was stirred overnight at room temperature then concentrated under vacuum to give allyl N-(3- aminopropyl)carbamate hydrochloride as a white solid (5.5 g).
  • Step 3 To a solution of allyl N-(3-aminopropyl)carbamate hydrochloride (3.37 g, 17.3 mmol, 1.0 eq.) in isopropanol (30 mL) was added N-ethyl-N-(propan-2-yl)propan-2- amine (6.0 mL, 34.6 mmol, 2.0 eq.). The mixture was heated to 70 °C and a solution of tert-butyl [(2S)-oxiran-2-ylmethyl]carbamate (3.0 g, 17.3 mmol, 1.0 eq.) in isopropanol (9 mL) was added dropwise.
  • Phenylsilane (1.7 mL, 13.3 mmol, 2.0 eq.) was added then the mixture was stirred under argon bubbling for 10 min.
  • Palladium-tetrakis(triphenylphosphine) (0.77 g, 0.667 mmol, 0.1 eq.) was added and the mixture was stirred 4 h at room temperature. The mixture was concentrated under vacuum.
  • Example 3.20 Step 1 To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (50.0 g, 0.342 mol, 1.0 eq.) in CH 2 Cl 2 (500 mL) at -5 °C, were added N,N-diethylethanamine (143 mL, 1.03 mol, 3.0 eq.) and prop-2-en-1-yl carbonochloridate (dropwise, 44 mL, 0.411 mol, 1.2 eq.). The reaction was risen to room temperature, stirred for 2 h then concentrated under vacuum. The residue was taken up in EtOAc and washed with water (2 x 100 mL) then 1 M HCl.
  • Step 2 To a solution of allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (36.45 g, 0.188 mol, 1.0 eq.) in MeOH (290 mL) was added dropwise sodium methanolate (25%, 86 mL, 0.376 mol, 2.0 eq.) 25% w/w. The reaction was stirred for 2 h at room temperature then partially concentrated.
  • Step 3 A solution of tert-butyl N-(3-aminopropyl)carbamate (1.0 g, 5.74 mmol, 1.0 eq.) in isopropanol (8 mL) was heated to 70 °C, then a solution of allyl N-[[(2S)-oxiran-2- yl]methyl]carbamate (0.90 g, 5.74 mmol, 1.0 eq.) in isopropanol (2 mL) was added dropwise over 15 min. The reaction mixture was stirred 1 h at 70 °C then concentrated under vacuum.
  • Step 5 Under inert atmosphere, to a solution of tert-butyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (1.6 g, 3.71 mmol, 1.0 eq.) in anhydrous CH 2 Cl 2 (74 mL) was added phenylsilane (2.4 mL, 18.5 mmol, 5.0 eq.). The mixture was stirred under bubbling of N 2 for 20 min then palladium-tetrakis(triphenylphosphine) (300 mg, 0.260 mmol, 0.07 eq.) was added.
  • phenylsilane 2.4 mL, 18.5 mmol, 5.0 eq.
  • Step 2 Under inert atmosphere, Fmoc-Lys(Boc)-OH (582 mg, 1.2 mmol, 1.0 eq.) and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine;hydrochloride (254 mg, 1.33 mmol, 1.33 eq.) were dissolved in anhydrous pyridine (10 mL). Tert-butyl N-[3-[2-[tert- butyl(dimethyl)silyl]oxyethylamino]-2-hydroxy-propyl]carbamate (420 mg, 1.20 mmol, 1.2 eq.) was added. The mixture was stirred overnight at room temperature then concentrated to dryness.
  • Step 3 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1-[[3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]-[2- [tertbutyl(dimethyl)silyl]oxyethyl]carbamoyl]pentyl]carbamate (580 mg, 0.73 mmol, 1.0 eq.) was suspended in 15 mL of a solution of N-ethylethanamine at 20% in THF. The mixture was stirred overnight then concentrated to dryness to give 498 mg of a crude product.
  • Step 4 Under inert atmosphere, the mixture of tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-[2-[tertbutyl(dimethyl)silyl]oxyethyl]amino]-2- hydroxy-propyl]carbamate and tert-butyl N-[3-[[(2S)-2-amino-6-(tert- butoxycarbonylamino)hexanoyl]-(2-hydroxyethyl)amino]-2-hydroxy-propyl]carbamate (300 mg, 0.65 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (2.6 mL).
  • Step 4 To a solution at 0-5 °C of tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.0 g, 2.78 mmol, 1.0 eq.) in CH 2 Cl 2 (20 mL) were added prop-2-en-1-yl carbonochloridate (0.32 mL, 3.00 mmol, 1.1 eq.) then N,N-diethylethanamine (0.97 mL, 6.96 mmol, 2.5 eq.). The mixture was stirred for 20 min at 0-5 °C then risen to room temperature for 1 h.
  • Step 5 To a solution of tert-butyl 3-[[allyloxycarbonyl-[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (880 mg, 1.98 mmol, 1.0 eq.) in dioxane (4 mL) was added a solution of 4 N HCl in dioxane (10 mL, 39.7 mmol, 20 eq.). The mixture was stirred for 3 h at room temperature, concentrated under vacuum then triturated with diisopropylether.
  • Step 7 Allyl N-[(2R)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate (626 mg, 0.79 mmol, 1.0 eq.) was dissolved in 10 mL of a 20% solution of N-ethylethanamine in THF (2 mL, 19.3 mmol, 25 eq.). The mixture was stirred for 2 h then concentrated under vacuum.
  • Example 3.23 Step 1 To a solution of tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.35 g, 6.47 mmol, 1.0 eq.) in CH 2 Cl 2 (35 mL) at 5 °C, were added N-ethyl-N-isopropyl- propan-2-amine (1.6 mL, 9.06 mmol, 1.4 eq.) and allyl chloroformate (0.76 mL, 7.12 mmol, 1.1 eq.). The reaction mixture was stirred for 3 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water then with saturated NaHCO 3 .
  • Step 3 To a solution of Fmoc-Lys(Boc)-OH (2.73 g, 5.66 mmol, 1.0 eq.) in DMF (15 mL) at 5 °C, were added allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1.26 g, 5.66 mmol, 1.0 eq.), N-ethyl-N-isopropyl-propan-2-amine (2.5 mL, 14.1 mmol, 2.5 eq.) and benzotriazol-1-yloxy(tripyrrolidin-1-yl)phosphonium;hexafluorophosphate (3.24 g, 6.22 mmol, 1.1 eq.).
  • Step 1 Under inert atmosphere and magnetic stirring, Fmoc-(D)-Dab(Boc)-OH (10 g, 22.0 mmol, 1.0 eq.) and N,N-diethylethanamine (3.7 mL, 26.4 mmol, 1.2 eq.) were dissolved in anhydrous THF (188 mL). This solution was cooled down at -5 °C and isobutyl carbonochloridate (3.1 mL, 24.2 mmol, 1.1 eq.) was added dropwise. The mixture turned white heterogeneous. The mixture was stirred for 30 min.
  • Step 2 Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1R)-3- (tert-butoxycarbonylamino)-1-(hydroxymethyl)propyl]carbamate (6.46 g, 15.1 mmol, 1.0 eq.), N-ethyl-N-isopropyl-propan-2-amine (16 mL, 90.9 mmol, 6.0 eq.) and N,N- dimethylpyridin-4-amine (185 mg, 1.51 mmol, 0.1 eq.) were dissolved in anhydrous CH 2 Cl 2 (70 mL).
  • Step 3 Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1R)-3- (tert-butoxycarbonylamino)-1-[[tert-butyl(dimethyl)silyl]oxymethyl]propyl]carbamate (5.55 g, 10.3 mmol, 1.0 eq.) and N-ethylethanamine (12 mL, 10.3 mmol, 1 eq.) were dissolved in anhydrous THF (48 mL). The reaction mixture was stirred overnight at room temperature then concentrated under vacuum to give a crude oil.
  • Step 4 Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (0.92 g, 1.92 mmol, 1.2 eq.) and tert-butyl N-[(3R)-3-amino-4-[tert- butyl(dimethyl)silyl]oxybutyl]carbamate (510 mg, 1.60 mmol, 1.0 eq.) were dissolved in anhydrous CH 2 Cl 2 (10 mL) then pyridine (5 mL) was added. A yellow solution was obtained.
  • reaction mixture was cooled down to 0 °C then a solution of propylphosphonic anhydride at 50 % in DMF (1.9 mL, 3.20 mmol, 2.0 eq.) was added dropwise.
  • the reaction mixture was risen to room temperature and stirred overnight.
  • the mixture was diluted in 50 mL of CH 2 Cl 2 and washed with 10 mL of water.
  • the organic layer was dried over MgSO 4 , filtered and concentrated under vacuum to give a yellow oil.
  • Step 5 Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1S)-5- (tert-butoxycarbonylamino)-1-[[(1R)-3-(tert-butoxycarbonylamino)-1-[[tert- butyl(dimethyl)silyl]oxymethyl]propyl]carbamoyl]pentyl]carbamate (1.07 g, 1.39 mmol, 1.0 eq.) and N-ethylethanamine (4.0 mL, 1.39 mmol, 1.0 eq.) were dissolved in anhydrous THF (16 mL). The reaction mixture was stirred for 4 h then concentrated under vacuum.
  • Step 1 To a solution of (2R)-3-aminopropane-1,2-diol (12.70 g, 0.139 mol, 1.0 eq.) in ethanol (400 mL) was added slowly tert-butoxycarbonyl tert-butyl carbonate (33.46 g, 0.153 mol, 1.1 eq.). The mixture was stirred at room temperature for 1h40 then concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (CH 2 Cl 2 /MeOH 100/0 to 90/10). Tert-butyl N-[rac-(2R)-2,3- dihydroxypropyl]carbamate was obtained as a white solid (30.3 g).
  • Step 2 To a solution of tert-butyl N-[rac-(2R)-2,3-dihydroxypropyl]carbamate (30 g, 0.149 mol, 1.0 eq.) in pyridine (200 mL) at 0-5 °C, was added dropwise (30 min) methanesulfonyl chloride (13 mL, 0.164 mol, 1.1 eq.). The mixture was stirred for 10 min. This solution was added dropwise (30 min) to a solution of sodium hydroxide (17.89 g, 0.447 mol, 3.0 eq.) in water (200 mL) and DMSO (132 mL), stirred at 0-5 °C.
  • Step 4 To a solution of tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine- 1-carboxylate (6.96 g, 23.8 mmol, 1.0 eq.) in CH 2 Cl 2 (48 mL) was added 4 M hydrogen chloride (15 mL, 60.0 mmol, 2.5 eq.). The mixture was stirred for 5 h then 24 mL of MeOH were added to homogenize the mixture. The mixture was stirred for 1 h then concentrated under vacuum.
  • Step 5 To a suspension of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1000 mg, 3.59 mmol, 1.0 eq.) in 2-propanol (20 mL) at 70 °C was added dropwise, during 15 min, a solution of tert-butyl[(2R)-oxiran-2-ylmethyl]carbamate (635 mg, 3.59 mmol, 1.0 eq.) and N,N-diethylethanamine (0.76 mL, 5.39 mmol, 1.5 eq.) in 2- propanol (5 mL).
  • Step 6 Under inert atmosphere, at room temperature, tert-butyl N-[(2R)-3-[3- [(allyloxycarbonylamino)methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (1100 mg, 2.97 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (40 mL). Phenylsilane (1.8 mL, 14.8 mmol, 5.0 eq.) was added and the resulting mixture was stirred under argon bubbling for 15 min, then palladium-tetrakis(triphenylphosphine) (172 mg, 0.148 mmol, 0.05 eq.) was added.
  • Step 7 To a solution of tert-butyl N-[(2R)-3-[3-(aminomethyl)-3-hydroxy-azetidin-1-yl]-2- hydroxy-propyl]carbamate (370 mg, 1.26 mmol, 1.0 eq.) in CH 2 Cl 2 (13 mL) and pyridine (6.5 mL) was added Fmoc-Lys(Boc)-OH (610 mg, 1.26 mmol, 1.0 eq.). The mixture was cooled down to 0-5 °C and stirred at this temperature.
  • Step 8 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1- [[1-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]-3-hydroxy-azetidin-3- yl]methylcarbamoyl]pentyl]carbamate (315 mg, 0.434 mmol, 1.0 eq.) in THF (15 mL) was added N-ethylethanamine (1.5 mL, 14.1 mmol, 32 eq.). The mixture was stirred at room temperature for 4 h then concentrated under vacuum.
  • Step 4 To a solution of tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine- 1-carboxylate (4.3 g, 14.3 mmol, 1 eq.) in CH 2 Cl 2 (30 mL) at room temperature was added 4M hydrogen chloride (9.0 mL, 36.0 mmol, 2.5 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum. Allyl N-[(3- hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained as a light brown gum (3.2 g).
  • Step 5 To a solution of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1.5 g, 6.60 mmol, 1.0 eq.) in 2-propanol (39 mL) in MeOH (13 mL) at 70 °C, was added dropwise (during 30 min) a solution of tert-butyl [(2R)-oxiran-2-ylmethyl]carbamate (1.174 mg, 6.64 mmol, 1.1 eq.) and N,N-diethylethanamine (1.4 mL, 9.90 mmol, 1.5 eq.) in 2-propanol (13 mL).
  • Step 6 To a solution of tert-butyl N-[(2R)-3-[3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (1.0 g, 2.78 mmol, 1.0 eq.) in CH 2 Cl 2 (30 mL) and MeOH (10 mL) stirred at room temperature, was added 4 M hydrogen chloride (4.2 mL, 16.7 mmol, 6.0 eq.). The mixture was stirred overnight then concentrated under vacuum.
  • Step 8 To a solution of tert-butyl N-[(5S)-6-[[(2R)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5-(9H-fluoren-9- ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate (815 mg, 1.10 mmol, 1.0 eq.) in THF (37 mL) was added N-ethylethanamine (3.7 mL, 35.8 mmol, 32.5 eq.). The mixture was stirred at room temperature for 4 h then concentrated under vacuum.
  • Step 4 Allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained as a light brown gum (5.36 g) following step 4 described in example 3.25.
  • Step 5 To a solution of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (3.37 g, 12.1 mmol, 1.0 eq.) in 2-propanol (70 mL) and MeOH (20 mL) at 70 °C, was added dropwise (during 15 min) a solution of tert-butyl [(2S)-oxiran-2- ylmethyl]carbamate (2.1 g, 12.1 mmol, 1.0 eq.) and N,N-diethylethanamine (2.6 mL, 18.2 mmol, 1.5 eq.) in isopropanol (20 mL).
  • Step 6 To a solution of tert-butyl N-[(2S)-3-[3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (500 mg, 1.39 mmol, 1.0 eq.) in CH 2 Cl 2 (14 mL) was added 4 M hydrogen chloride (1.7 mL, 6.96 mmol, 5.0 eq.). The mixture was stirred at room temperature. After 5 min, a precipitate appeared.1,4-dioxane (10 mL) was added and the mixture was stirred for 30 min. MeOH (5 mL) was added to homogenize the solution.
  • Step 7 Under inert atmosphere, allyl N-[[1-[(2S)-3-amino-2-hydroxy-propyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate dihydrochloride (500 mg, 1.46 mmol, 1.0 eq.) was dissolved in anhydrous CH 2 Cl 2 (15 mL). N,N-diethylethanamine (0.62 mL, 4.38 mmol, 3.0 eq.), anhydrous pyridine (7.5 mL) and Fmoc-Lys(Boc)-OH (705 mg, 1.46 mmol, 1.0 eq.) were added.
  • Step 8 To a solution of tert-butyl N-[(5S)-6-[[(2S)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5-(9H-fluoren-9- ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate (365 mg, 0.51 mmol, 1.0 eq.) in THF was added N-ethylethanamine (2 mL, 19.3 mmol, 37.6 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum.
  • Example 4 Loading measurement Before starting the synthesis of peptides, the loading of the first amino acid (or dipeptide) on resin was measured following the steps 1, 2 or 3 (depending on the resin used) and step 4 described below.
  • Step 1 Resin swelling. The resin (5.10 -4 mol, 1.0 eq.) was put in the reaction vessel. CH 2 Cl 2 (3.0 mL) was added to immerse all the resin, the mixture was shaken for 30 min. The solvent was removed by filtration under vacuum.
  • Step 2 Esterification on the 2-chlorotrityl resin.
  • the first amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was dissolved in 2.5 mL of CH 2 Cl 2 .
  • DIPEA 2.0 mmol, 4.0 eq.
  • This solution was immediately added to the swollen 2-chlorotrityl resin (5.10 -4 mol, 1.0 eq.).
  • the reaction vessel was closed with a cap and the reaction mixture was shaken for 4 h then filtered.
  • DMF 2.5 mL was added and the mixture was shaken for 20 seconds then filtered. This washing was repeated 3 times.
  • a solution of CH 2 Cl 2 /MeOH/DIPEA 80/15/5 (2 mL) was added to the resin.
  • Step 3 For the 1,3-diaminopropane trityl resin, 1,5-diaminopentane trityl resin and Rink amide resin, the anchoring of the first amino acid or dipeptide was performed with a standard coupling procedure. DMF (2.5 mL) was added to the swollen resin (5.10 -4 mol, 1.0 eq.). The mixture was shaken for 20 seconds, and the solvent was removed under vacuum.
  • the protected amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was added followed by 4.0 mL of DMF. The mixture was shaken for 20 seconds then DIPEA (2.0 mmol, 4.0 eq.) was added and the mixture was shaken until dissolution of the amino-acid or dipeptide.
  • HBTU (1.45 mmol, 2.9 eq.) was added then the reaction vessel was closed with a cap and shaken for 2 h. The mixture was filtered under vacuum. DMF (2.5 mL) was added to the resin and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times. This coupling step was repeated once.
  • Step 4 Loading measurement. Preparation of the sample solution.
  • the resin obtained in step 2 or 3 was dried for 2 h under vacuum then air-dried overnight. About 10 mg of the dry resin was introduced in an Eppendorf tube. 200 ⁇ L of a solution of DMF/piperidine 80/20 were added. The mixture was shaken for 10 min then the supernatant was collected and introduce in a 25 mL volumetric flask.
  • Step 1 The 2-chlorotrityl resin was swollen following step 1 of example 4.
  • Step 2 Esterification on the 2-chlorotrityl resin.
  • the first amino-acid or dipeptide was esterified on the 2-chlorotrityl resin following Step 2 from example 1.
  • a solution of DMF/piperidine 80:20 (4.0 mL) was added to the resin.
  • the mixture was shaken for 20 minutes then filtered under vacuum to remove the solvent. This step was repeated once.
  • DMF was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times.
  • MeOH was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered.
  • CH 2 Cl 2 was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered.
  • Step 3 Standard coupling procedure and Fmoc removal.
  • DMF 2.5 mL was added to the swollen resin (5.10 -4 mol, 1.0 eq.). The mixture was shaken for 20 seconds, and the solvent was removed under vacuum. The protected amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was added followed by 4.0 mL of DMF. The mixture was shaken for 20 seconds then DIPEA (2.0 mmol, 4.0 eq.) was added and the mixture was shaken until dissolution of the amino-acid or dipeptide. HBTU (1.45 mmol, 2.9 eq.) was added then the reaction vessel was closed with a cap and shaken for 2 h.
  • Step 5 Coupling in solution of the C-terminal building-block.
  • the fully-protected peptide (2.25.10 -4 mol, 1.0 eq.) obtained at the end of step 4 was dissolved in CH 2 Cl 2 (4 mL).
  • DIPEA (1.8 mmol, 8.0 eq.)
  • HOBt (1.3 mmol, 5.8 eq.)
  • EDC.HCl (1.3 mmol, 5.8 eq.) were added, followed by a solution of the amine for C-terminal derivatization (1.35 mmol, 6.0 eq.) in a minimum volume of CH 2 Cl 2 .
  • the reaction mixture was stirred for 18 h at room temperature. The mixture was quenched with water and the phases were separated.
  • Step 6 Cleavage of alloc then acid sensitive protecting groups.
  • the peptide from Step 5 (0.25 mmol) was dissolved under argon in anhydrous CH 2 Cl 2 (20 mL). Phenylsilane (1.25 mmol or 2.5 mmol, 5.0 eq. and 10 eq.) was added.
  • Step 7 Cleavage of acid sensitive protecting groups (Boc and silylated groups): the peptide from Step 5 (2.25.10 -4 mol), was dissolved in 4 mL of a solution TFA/triisopropylsilane/water (85/7.5/7.5).
  • the crude product was dissolved in milliQ water ( ⁇ 250 mg/mL) and was purified by preparative HPLC using TFA as additive. Tubes containing pure product were combined and the solution was frozen to -80 °C and freeze-dried to TFA salt of the peptide. This product was analyzed by HPLC-MS applying the HPLC-MS analytical method for final purity check described above. When the peptide obtained was not pure enough, the product was dissolved in milliQ water ( ⁇ 150 mg/mL) and was purified by preparative HPLC using HFBA as additive. Tubes containing pure product were combined and the solution was frozen to -80 °C and freeze-dried to give the HFBA salt of the peptide.
  • Step 9 Dowex 1,4 chloride resin (938 mg, 5.0 eq., resin loading at 1.2 meq/g) was introduced in a reaction vessel. A solution of purified peptide from step 8 (2.25.10 -4 mol, 1.0 eq.) in water (15 mL) was added. The mixture was shaken for 2 h then filtered. Water (15 mL) was added to the resin and the mixture was shaken then filtered. This washing was repeated twice. All the filtrates were combined, frozen at -80 °C then freeze-dried.
  • Example 6 Synthesis of peptide with CO 2 H at the C-terminal position Peptides were synthesized following step 1 of example 4 then steps 2 and 3 of example 5.
  • Step 4 The cleavage of the peptide from the resin and the deprotection of all the lateral chains were performed in one step as follows. A solution of TFA/triisopropylsilane/water 85/7.5/7.5 (4 mL) was added to the resin (2.5.10 -4 mol). The mixture was shaken for 2 h then filtered. The filtrate was poured dropwise to 30 mL of cold (0 °C) TBME.
  • Step 6 The salt exchange was performed following the step 9 described in example 5.
  • Example 7 Synthesis of peptide with NH(CH 2 ) 3 NH 2 or NH(CH 2 )5NH 2 at the C-terminal position Peptides were synthesized from 1,3-diaminopropane or 1,5-diaminopentane trityl resin. The resin was swollen following the step 1 described in example 4. The coupling of amino acid or dipeptide was achieved following the step 3 described in example 5 and the peptide was cleaved from the resin following the step 4 described in example 6. Final purification and salt exchange were performed following the steps 8 and 9 described in example 5.
  • Example 8 Synthesis of peptides with CONH 2 at the C-terminal position Peptides with CONH 2 at the C-terminal position were synthesized from Rink amide resin. The resin was swollen following step 1 described in example 4. Then the Fmoc protecting group was removed from the resin following the procedure described in step 3, example 5. The peptide chain was grown by introduction of amino acids or dipeptide following the procedure described in step 3 in example 5. Then the peptide was cleaved from the resin following the procedure described in step 4 in example 6. The final purification and salt exchange were performed following the procedures described in steps 8 and 9 in example 5.
  • Example 9 Synthesis of compound 33 The peptide was synthesized by using a Rink amide resin.
  • Step 4 Alloc cleavage. Under inert atmosphere, phenylsilane (370 ⁇ L, 3 mmol, 10 eq.) and palladium tetrakis-(triphenylphosphine) (35 mg, 0.03 mmol, 0.1 eq.) were dissolved in anhydrous CH 2 Cl 2 (10 mL). This solution was added to the dried resin (0.3 mmol).
  • Step 7 The final purification was performed following the step 8 described in example 5.
  • Step 8 The salt exchange was made following the step 9 described in example 5.
  • the following compounds of formula (I) were prepared in accordance with the methods described herein (table 1). Structures of compounds 1 to 80 are as shown herein before.
  • Table 1 Compounds of formula (I) II – In vitro pharmacological properties Minimum inhibitory concentration (MIC): MIC values were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methodology, colony direct suspension, as described in CLSI document M07-A10 (Clinical and Laboratory Standards Institute. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—10th ed.
  • CLSI Clinical and Laboratory Standards Institute
  • BMD broth microdilution
  • RNA Integrity Number (RIN) were determined, and reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen ref. 18064-022) and random hexamer from Applied Biosystems ref. N8080127.
  • RT-qPCR to follow mexY gene expression was carried out using a LightCycler 480 (Roche) with Sensi-Fast SYBR no rox commercialized by Bioline (BIO-98050) and with primers MexY1a (5′-CTA CAA CAT CCC CTA TGA CAC CTC-3′) and MexY1b (5′- ATGGTCAGCACGTTGATCGAGAA-3′).
  • Results were expressed in fold change compared to mexY expression in Pa PAO1 culture (arbitrarily equal to 1). (-) not tested because the strains are MexXY-OprM deficient.
  • c RT-qPCR comparative analysis of mexY gene (as a measure of mexXY) differentially expressed in P. aeruginosa culture treated with NOSO-502 (A), NOSO-95C (B) or compound 1 (128 ⁇ g/mL, 45 min) relative to untreated culture.
  • R e sults were expressed in fold change compared to mexY expression in P. aeruginosa untreated culture.
  • NOSO-502, NOSO-95C, and compound 1 exhibit very potent and close antibacterial activity against strains with deficient MexXY-OprM efflux pump (11B, PAO1T).
  • NOSO-502 is inactive against P. aeruginosa strains expressing the MexXY-OprM efflux pump. Indeed, NOSO-502 strongly enhances the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates and is susceptible to the efflux activity of this pump, resulting in a high NOSO-502 resistance level of these strains.
  • NOSO-95C does not induce the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates. However, its antibacterial activity against P.
  • aeruginosa strains varies widely depending on their constitutive MexXY expression level, clearly showing that this compound remains susceptible to the MexXY-OprM efflux activity.
  • Compound 1 according to the invention does not induce the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates. Its antibacterial activity against P. aeruginosa strains remains constant, independently from the MexXY expression level, clearly showing that this compound is not susceptible to the MexXY-OprM efflux activity.
  • mice Male CD-1 mice (18-22g) were allowed to acclimatize for 7 days, then rendered neutropenic by IP injection of cyclophosphamide (150 mg/kg on day 4 and 100 mg/kg on day 1 before infection). Mice were infected by intranasal route (5 x 10 5 c.f.u./mouse) under parenteral anaesthesia.
  • test compounds were formulated in phosphate-buffered saline (PBS, pH 7.4). Treatment was initiated 2 h post infection and 6 doses of each compound (from 0.5 mg/kg to 64 mg/kg depending on the compounds) were administered once by subcutaneous injection in a single dose volume of 4 mL/kg (2 mice per dose). A non-treated group (vehicle-only) was included to serve as a negative treatment control. At 2 h post infection, one infected group was humanely euthanized, and lungs processed for pre-treatment quantitative culture to determine P. aeruginosa burdens. At 8 h post infection, all remaining mice were humanely euthanized, and lungs were aseptically removed.
  • PBS phosphate-buffered saline
  • mice The number of viable bacteria in lung was determined by plating serial tenfold dilutions of homogenates onto LB agar for 24 h at 37 °C. Results are presented in table 5.
  • Table 5 Efficacy of compounds of the present invention in an immunocompromised mice respiratory tract infection model with P. aeruginosa ATCC 27853. Mice (2 mice/group) were infected by intranasal instillation at t0. Antibacterial administration was performed by subcutaneous route at t0+2h. Bacterial counts in lungs were done at t0+2h for the control group (stasis) and at t0+8h for the treated and vehicle groups.
  • Results were expressed as the doses (in mg/kg) required to achieve bacteriostatic effect and 1-log10 CFU in lungs reduction compared to stasis.
  • Compound 1 was also challenged in P. aeruginosa ATCC 27853 and A. baumannii ATCC BAA-1710 respiratory tract infection models in neutropenic mice, with minor changes compared to the protocol previously described.
  • mice were infected by intranasal route with 8 x 10 5 CFU/mouse of P. aeruginosa ATCC 27853 or with 3 x 10 6 CFU/mouse of A. baumannii ATCC BAA-1710. Treatment was initiated 2 h post infection and Compound 1 (1, 2, 4, 8, 16, and 32 mg/kg) was administered every 6 h in different groups (2 mice per dose) by subcutaneous injection (0.1 mL).26 h post infection, mice were humanely killed, and bacterial load in lungs was determined.

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Abstract

The present disclosure relates to new Odilorhabdins (ODLs) analogues, and their use in medicine, in particular for treating or preventing bacterial infections, more specifically for treating or preventing multi-drug resistant bacterial infections, such as multi-drug resistant bacterial infections caused or suspected to be caused by P. aeruginosa.

Description

NEW ODILORHABDINS ANALOGUES AS ANTIBIOTICS AGAINST MULTI- RESISTANT BACTERIA The project leading to this application has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement n° 853979. This Joint Undertaking receives the support from the European Union’s Horizon 2020 research and innovation program and EFPIA. FIELD OF THE INVENTION The present invention relates to new Odilorhabdins (ODLs) analogues, and their use in medicine, in particular for treating or preventing bacterial infections, more specifically for treating or preventing multi-drug resistant bacterial infections, such as multi-drug resistant bacterial infections caused by P. aeruginosa. BACKGROUND OF THE INVENTION The emergence and spread of bacteria resistant to most or all known antibiotics is a major issue for human health and should be our next big concern to prevent a new pandemic like COVID-19 from occurring. If the scientific community fails to renew our antibiotic arsenal to treat drug-resistant infections, nearly 10 million extra deaths are predicted by 2050 [Kraker et al., PLoS Med., 2016, 13(11): e1002184]. In a post antibiotic era, treatment of immunocompromised patients, transplantation of solid organ, or implantation in orthopedic surgery will be compromised (DeNegre AA, Ndeffo Mbah ML, Myers K, Fefferman NH. Emergence of antibiotic resistance in immunocompromised host populations: a case study of emerging antibiotic resistant tuberculosis in AIDS patients. PLoS One. 2019;14:e0212969. doi:10.1371/journal.pone.0212969 ; Bartoletti M, Giannella M, Tedeschi S, Viale P. Multidrug-resistant bacterial infections in solid organ transplant candidates and recipients. Infect Dis Clin North Am.2018;32:551- 580. doi:10.1016/j.idc.2018.04.004 ; Ravi S, Zhu M, Luey C, Young SW. Antibiotic resistance in early periprosthetic joint infection. ANZ J Surg.2016;86:1014-1018. doi:10.1111/ans.13720). The care of patients with cancer are already profoundly impacted by the lack of new antibiotics able to fight multidrug resistant bacteria (Antibiotic resistance in the patient with cancer: Escalating challenges and paths forward , Amila K. Nanayakkara PhD,Helen W. Boucher MD,Vance G. Fowler Jr MD, MHS,Amanda Jezek,Kevin Outterson JD, LLM,David E. Greenberg MD). Presently, over 700,000 deaths occur annually as a consequence of infections caused by antibiotic-resistant bacteria. Gram-negative pathogens are particularly alarming because they are becoming resistant to almost all available antibiotic creating therapeutic impasses, especially in intensive care units. The most serious Gram- negative infections are commonly caused by carbapenem-resistant Enterobacteriaceae (mostly Klebsiella pneumoniae and Escherichia coli), multidrug-resistant, including carbapenem-resistant, Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannnii with high morbidity and mortality rates [Guidelines for the prevention and control of carbapenem-resistant Enterobacteriaceae, A. baumannii and P. aeruginosa in health care facilities, World Health Organization, 2017]. This situation highlights the need for innovation and urgent development of new antimicrobial compounds. Odilorhabdins (ODLs) are a new class of ribosome-targeting antibiotics [WO201304560 WO2016046409, Pantel et al., Mol Cell., 2018, 70(1):83-94.e7]. The first members of the class, NOSO-95 A-C, were identified from cultures of Xenorhabdus nematophila, a bacterium symbiotically associated with entomopathogenic nematodes. First medicinal chemistry efforts have led to the selection of NOSO-502, a compound with potent pharmacological properties against multidrug-resistant Enterobacteriaceae including carbapenem-resistant, polymyxin-resistant, and ESBL (extended spectrum β-lactamase) isolates [Racine et al., Antimicrob. Agents Chemother., 2018, 62(9):e01016-18 ; Racine et al., Front. Microbiol., 2019, 10:2893]. This compound successfully completed the preclinical development stage recently.
Figure imgf000003_0001
Figure imgf000004_0001
Unfortunately, P. aeruginosa isolates have demonstrated variable resistance level to NOSO-502 and NOSO-95C mediated by the MexXY-OprM efflux pump. Efflux pumps are transport proteins involved in the extrusion of substrates such as antibiotics. NOSO- 502 favors a strong overexpression of this efflux pump resulting in high resistance degree of P. aeruginosa isolates while the antibacterial activity of NOSO-95C against P. aeruginosa strains correlates with their constitutive MexXY-OprM expression. Regrettably, the MexXY-OprM pump is frequently reported to be overexpressed among P. aeruginosa clinical isolates obtained from cystic fibrosis or non-cystic fibrosis patients (Antimicrob Agents Chemother. 2004 May; 48(5): 1676–1680.). These observations preclude the use of the previously reported ODLs as drugs to treat infections caused or suspected to be caused by Pseudomonas strains. Therefore, a strong need remains for the provision of new antibacterial compounds, in particular effective at treating multi-drug resistant bacterial infections, such as infections caused by Pseudomonas strains. SUMMARY OF THE INVENTION The present invention relates to a compound of formula (I) as recited in claim 1. The present invention also relates to a compound of formula (I) or a composition comprising thereof for use in medicine, more specifically for use in the treatment or prevention of bacterial infections. Further aspects of the invention are as disclosed herein and in the claims. DEFINITIONS The term “alkyl” as used herein means a saturated, branched or straight monovalent hydrocarbon chain. As a matter of example, alkyl comprising from one to six carbon atoms are represented “-(C1-C6)-alkyl”. The term “hydroxyalkyl” as used herein means an alkyl group as defined above substituted by one or more hydroxy radicals. The term “aminoalkyl” as used herein means an alkyl group as defined above substituted by one or more amino groups (-NH2). The term “haloalkyl” as used herein means an alkyl group as defined above substituted by one or more halogen atoms (F, Cl, Br, I). The term “hydroxyaminoalkyl” as used herein means an alkyl group as defined above substituted by one or more hydroxy radicals and one or more amino groups (-NH2). The term “Me” as used herein designates a methyl group. The term “alkoxy” as used herein designates a group of formula -O-alkyl wherein alkyl is as defined herein. The term “alkanediyl” as used herein means a saturated, branched or straight divalent hydrocarbon chain. As a matter of example, alkanediyl comprising from one to six carbon atoms are represented “C1-C6-alkanediyl”. Examples of alkanediyl include, but are not limited to, methanediyl (-CH2-). The term “alkenediyl” as used herein means an unsaturated, branched or straight divalent hydrocarbon chain comprising one or more double bonds. The term “alkenyl” as used herein designates a univalent branched or straight hydrocarbon chain containing one or more double bonds, including di-enes, tri-enes, such as vinyl, propen-1-yl, propen-2-yl, but-1-en-1-yl, but-1-en-2-yl, but-2-en-1-yl. The term “alkynyl” as used herein designates a univalent branched or straight hydrocarbon chain containing one or more triple bonds. The term “cycloalkyl” as used herein means a monovalent monocyclic or bicyclic (fused) hydrocarbon chain. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like. The term “aryl” as used herein refers to aromatic carbocyclic groups having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings (e.g., naphthyl,). The terms “heteroaryl” as used herein refers to aryl groups as defined herein comprising at least one heteroatom as a ring atom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, selenium and the like. In some cases, a heteroaryl is a cyclic aromatic radical having from five to ten ring atoms of which at least one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. The term “carbamoyl” as used herein means the group (-(CO)-NH2). The term “hydroxy” or “hydroxyl” as used herein means the group -OH. The term “carboxyl” as used herein means the group -COOH. The term “multidrug-resistant” as used herein means a lack of susceptibility to at least one agent in three or more chemical classes of antibiotic, as defined in Clin Microbiol Infect.2012;18:268-81. doi: 10.1111/j.1469-0691.2011.03570.x. DETAILED DESCRIPTION After extensive research, the inventors have succeeded in developing ODLs analogs (compounds of formula (I), see below) useful for treating or preventing bacterial infections, more specifically for treating or preventing multi-drug resistant bacterial infections, such as multi-drug resistant bacterial infections caused or suspected to be caused by P. aeruginosa. The ODLs analogs have been found to exhibit improved in vitro and in vivo pharmacological properties against P. aeruginosa. The proposed ODLs analogs retain a strong antibacterial activity against multidrug-resistant P. aeruginosa isolates independently of the MexXY-OprM efflux pump expression. Compounds of formula (I) The present invention relates to compounds of formula (I): Ra-Xaa1-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-(Xaa10)y-Rb (I) wherein: Ra is H or -(C1-C3)-alkyl; y is 0 or 1; Xaa1 i
Figure imgf000006_0001
wherein one or more carbon atoms of the chain bearing the NR11 R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl, wherein: R1, R11 and R’11 are independently H or -(C1-C3)-alkyl; n1 is an integer from 1-4; Xaa2 is
Figure imgf000007_0001
wherein: X2 is NH, N(Me) or O; R2 and R’2 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl or -C(O)-(C1- C3)-haloalkyl; R22 is OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -O-C(O) -(C1-C6)-alkyl or –O- C(O)-(C1-C6)- haloalkyl; n2 is an integer from 1-3;
Figure imgf000007_0002
wherein: X3 is N(R33) or O; R3 is halogen, NH2, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C3-C8)-cycloalkyl, -(C2- C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)-alkyl-OR33, -(C1-C6)-alkyl-N R33R’33, -(C1-C6)-alkyl- C(O)NR33R’33, -(C1-C6)-alkyl-C(O)OR33 or -(C1-C6)-alkyl-heteroaryl wherein said alkyl is optionally substituted by one or more substituents selected from the group consisting of –OH, –O-C(O)-(C1-C6)-alkyl and –O-C(O)-(C1-C6)-haloalkyl, wherein said heteroaryl is optionally substituted by one or more substituents selected from the group consisting of -(C1-C3)-alkyl and (C1-C6)-alkyl-aryl wherein said aryl is optionally substituted by one or more substituents selected from the group consisting of –OH, -(C1-C3)-alkyl, -(C1-C3)- alkoxy and halogen; R33, R’33 and R’’333 are independently H, -C(=NH)-NH2, -(C1-C3)-alkyl, -C(O)-(C1- C3)-alkyl, -C(O)-(C1-C3)-haloalkyl or -(C1-C6)-alkyl-phenyl, wherein said phenyl may be substituted by one or more substituents selected from the group consisting of –OH, - NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl, -(C1-C6)-alkyl-COOH, -(C1-C6) hydroxyalkyl, -(C1-C6) aminoalkyl, -(C1-C6) alkyl-NHCONH2, -(C1-C3)-alkoxy, -(C1-C3)- alkoxy-COOH and halogen; R333, R’333 , R’’333 and R’’’333 are independently H, OH, halogen, -(C1-C3)-alkyl, - (C1-C3)-alkoxy, –O-CO-(C1-C3)-alkyl, –O-CO-(C1-C3)-haloalkyl or -NR’R” wherein R’ and R’’ are independently hydrogen or C1-C6-alkyl; n3 is an integer from 1-3; n4 is an integer from 0-3; Xaa4 is
Figure imgf000008_0001
wherein: X4 is NH, N(Me) or O; R4 and R’4 are independently H, -(C1-C3)-alkyl or -(C1-C3)-haloalkyl; Xaa5 is
Figure imgf000008_0002
wherein: X5 is NH, N(Me) or O; R5, R’5 and R’’5 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, or -C(O)- (C1-C3)-haloalkyl; R555, R’555, R’’555 and R’’’555 are independently H, OH, halogen, -(C1-C3)-alkyl, or - (C1-C3)-alkoxy; n5 is an integer from 1-3;
Figure imgf000009_0001
wherein: X6 is N(R66) or O; R6 is H, halogen, NH2, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C3-C8)-cycloalkyl, -(C2- C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)-alkyl-OR66, -(C1-C6)-alkyl-SR66, -(C1-C6)-alkyl- NR66R’66, -(C1-C6)-alkyl-C(O)NR66R’66, -(C1-C6)-alkyl-C(O)OR66 or -(C1-C6)-alkyl- heteroaryl wherein the alkyl in the -(C1-C6)-alkyl-heteroaryl is optionally substituted by one or more substituents selected from the group consisting of –OH, –O-C(O)-(C1-C6)- alkyl and –O-C(O)-(C1-C6)-haloalkyl and the heteroaryl is optionally substituted by one or more substituents selected from the group consisting of -(C1-C3)-alkyl, and (C1-C6)- alkyl-aryl wherein said aryl is optionally substituted by one or more –OH, -(C1-C3)-alkyl, -(C1-C3)-alkoxy or halogen; R66 and R’66 are independently H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -O- (C1-C3)-alkyl, -(C1-C3)-haloalkyl, -(C1-C6)-alkyl-COOH, -C(O)-(C1-C3)-alkyl, -C(O)-(C1- C3)-haloalkyl, -NH2, -NH(C1-C3)-alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl]; n6 is an integer from 0-3; Xaa7 is wherein
Figure imgf000009_0002
: X7 is N(R77) or O; R7 is H, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C2-C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)- alkyl-OR77, -(C1-C6)-alkyl-SR77, -(C1-C6)-alkyl-S(O)-R77, -(C1-C6)-alkyl-S(O)2-R77, -(C1-C6)- alkyl-NR77 R’77, -(C1-C6)-alkyl-C(O)OR77, -(C1-C6)-alkyl-C(O)NR77R’77, -(C1-C6)-alkyl- heteroaryl, -(C1-C6)-alkyl-aryl or -(C1-C6)-alkyl-aryl-heteroaryl, wherein said aryl or heteroaryl in the -(C1-C6)-alkyl-heteroaryl, -(C1-C6)-alkyl-aryl or -(C1-C6)-alkyl-aryl- heteroaryl groups is optionally mono- or poly- substituted with –OH, -NH2, -COOH, - CONH2, -CN, -CF3, -(C1-C6)-alkyl, -(C1-C6)-alkyl-COOH, -(C1-C6)-hydroxyalkyl, -(C1-C6)- aminoalkyl, -(C1-C6)-alkyl-NHCONH2, -(C1-C3)-alkoxy, -(C1-C3)-alkoxy-COOH, or halogen; R77 and R’77 are independently H, OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-haloalkyl, - C(O)-NH2, -C(=NH)-NH2, -C(O)-(C1-C3)-alkyl, -C(O)-(C1-C3)-haloalkyl, -NH2, NH(C1-C3)- alkyl, or N[(C1-C3)-alkyl][(C1-C3)-alkyl]; n7 is an integer from 0-3; Xaa8 is
Figure imgf000010_0001
wherein: X8 is NH, N(Me) or O; R8 and R’8 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, -C(=NH)-NH2, or -C(O)-(C1-C3)-haloalkyl; R88, R’88 , R’’88 and R’’’88 are independently H, OH, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, or halogen; n8 is an integer from 1-4; Xaa9 is
Figure imgf000011_0001
or
Figure imgf000011_0002
wherein: X9 is NH, N(Me) or O; R9 and R’9 are independently H, -C(=NH)-NH2, -C(O)NH2, -(C1-C6)-alkyl, -(C1-C6)- haloalkyl, -C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R99 is H or -(C1-C3)-alkyl; n9 is an integer from 1-4; Xaa10 is
Figure imgf000011_0003
or
Figure imgf000011_0004
wherein: X10 is NH or O; R10 and R’10 are independently H, -C(=NH)-NH2, -(C1-C3)-alkyl, -(C1-C6)-alkyl- NH2,-C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R100 is H, OH, -NH2, -(C1-C3)-alkyl, -(C1-C3)-alkoxy or halogen; n10 is independently an integer from 0-5; Rb is -NR5aR6a, wherein R5a is: -A1-NRa-A2-NHRb, -A3-CO-NRc-A4-NHRd, or -A5-NHRe wherein: A1 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; A2 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; Ra is a hydrogen atom, a –(C1-C5)-hydroxyalkyl, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyaminoalkyl or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A1 or A2 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Ra; Rb is a hydrogen atom or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A2 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rb; A3 is an unsubstituted C1-C4-alkanediyl or a C1-C4-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5-hydroxyalkyl, a carboxyl and a carbamoyl; A4 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; Rc is a hydrogen atom, a –(C1-C5)-hydroxyalkyl, a –(C1-C5)-aminoalkyl, a –(C1-C6)-hydroxyaminoalkyl or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A4 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rc; Rd is a hydrogen atom or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A4 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rd; A5 is an unsubstituted C2-C6-alkenediyl group, an unsubstituted C1-C6- alkanediyl or a C1-C6-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl, an amine and a –(C1-C6)- aminoalkyl; Re is a hydrogen atom, a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl, provided that Re is a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl when A5 is an unsubstituted C1-C6-alkanediyl; R6a is a hydrogen atom, a –(C1-C5)-hydroxyalkyl or a C1-C6-alkanediyl group that forms a cycle with a carbon atom of A1, A3 or A5 of R5a in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing R6a; or hydrates, solvates, or salts thereof. It shall be clear from the above representation that Ra is linked to the nitrogen atom of Xaa1 and that Xaa1 and Xaa2 are linked via a covalent bond between the carbon atom of the carbonyl group of Xaa1 and the X2 group of Xaa2 and so on. In other words, bonds between the different Xaa groups are amide bonds (-(C=O)-NH-) or ester bonds (-(C=O)- O-) (depending on whether Xn is NH, N(Me) or O). Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds of formula (I). However, the invention also encompasses salts which themselves are unsuitable for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention. The term “physiologically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of the compound of formula (I). A suitable pharmaceutically acceptable salt of the compound of formula (I) may be, for example, an acid-addition salt of a compound of formula (I), such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3 -hydroxy-2 -naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3- phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethane sulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluene sulfonic, methansulfonic, 2- naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric, or thiocyanic acid, for example. The salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid with any of the compounds of the invention. Solvates in the context of the invention are described as those forms of the compounds which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water. The present invention includes all possible stereoisomers of the compounds of formula (I) as single stereoisomer, or as any mixture of said stereoisomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of formula (I) can be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example. In some embodiments, in the above formula (I), Ra is H or methyl. In some embodiments, Ra is H. In some embodiments, Ra is methyl. In some embodiments, in the above formula (I), Xaa1 is
Figure imgf000014_0001
wherein R1, R11, R’11 are as disclosed herein above and n1 is an integer from 1-3, in particular n1 is 1, 2 or 3. In each of these embodiments wherein Xaa1 is
Figure imgf000014_0002
O , one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl. In some embodiments, Xaa1 is 1
Figure imgf000014_0004
. In some embodiments, Xaa is
Figure imgf000014_0003
In some embodiments, in the above formula (I), Xaa1 is
Figure imgf000015_0001
wherein one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl, preferably when the chain bearing the NR11R’11 group is substituted, the carbon atom in alpha of the NR11R’11 group is substituted by -(C1-C3)-alkyl, preferably a methyl group; R11 and R’11 are independently H or -(C1-C3)-alkyl, preferably H; and n1 is an integer from 1-4, preferably 3. In some embodiments, in the above formula (I), Xaa1 is
Figure imgf000015_0002
wherein R11 and R’11 are a hydrogen atom and n1 is 1, 2 or 3, preferably 3. In these embodiments, one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl. In some embodiments, the carbon atom in alpha of the NR11R’11 group is substituted by a –(C1- C3)-alkyl, preferably a methyl group. In the above formula (I), the configuration of the asymetric carbon atom bearing the radical R1 or –(CH2)n1-NR11R’11 is preferably S. In some embodiments, in the above formula (I), Xaa1 is
Figure imgf000015_0003
, more specifically . In particular, Xaa1 is
Figure imgf000015_0006
Figure imgf000015_0004
Figure imgf000015_0005
more specifically
Figure imgf000016_0002
. In each
Figure imgf000016_0003
of these embodiments, one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl. In some embodiments, the carbon atom in alpha of the NR11R’11 group is substituted by a –(C1-C3)-alkyl, preferably a methyl group. In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000016_0001
or
Figure imgf000016_0004
wherein X2 is NH or N(Me), preferably NH. In some other embodiments, X2 is O. In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000016_0005
Figure imgf000016_0006
wherein X2 is NH or N(Me), preferably NH. In some other embodiments, X2 is O. In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000017_0001
in particular Xaa2 is
Figure imgf000017_0002
or , wherein X2 is NH or N(Me), preferably NH and n2, R2, R’2 and R22 are as disclosed herein above or below . In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000017_0003
preferably
Figure imgf000017_0004
, wherein X2 is NH or N(Me), preferably NH; R2 is H, methyl, ethyl, -C(O)-(C1-C2)-alkyl, or -C(O)-(C1-C2)-haloalkyl, preferably H, methyl, acetyl or trifluoroacetyl, and R’2 is H, methyl, ethyl, preferably H. n2 is an integer from 1- 3, in particular 1, 2 or 3, advantageously n2 is 1. R22 is as disclosed herein above. In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000017_0005
, preferably
Figure imgf000017_0006
, wherein R22 is OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -O-C(O)-(C1- C6)-alkyl or –O-C(O)-(C1-C6)- haloalkyl, preferably OH, fluorine, methyl, methoxy, -O- C(O)-CH3, -O-C(O)-CF3, more preferably OH and R2 and R’2 are independently H, -(C1- C3)-alkyl, -C(O)-(C1-C3)-alkyl or -C(O)-(C1-C3)-haloalkyl, preferably R2 is H or methyl, and R’2 is H, more preferably R2 and R’2 are H. In some embodiments, in the above formula (I), Xaa2 is
Figure imgf000018_0001
, preferably , wherein R22 is OH, fluorine, methyl, methoxy, -O-C(O)-CH3 or -O-C(O)-CF3,
Figure imgf000018_0002
in particular OH, -O-C(O)-CH3 or -O-C(O)-CF3, more specifically OH. In these embodiments, R2 and R’2 are as defined above. In particular, in some embodiments, R2 is H, methyl, acetyl or trifluoroacetyl, advantageously H, and R’2 is H. In these embodiments, X2 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa3 is
Figure imgf000018_0003
, ,
Figure imgf000018_0004
X3 is NH or N(Me), preferably NH. In some other embodiments, X3 is O. R3, R33, R’33, R’’33, R333, R’333, R’’333, R’’’333, n3 and n4 are as disclosed herein. The configuration of asymetric carbon atom between –X3 and –C(O) is preferably S. In some embodiments, in the above formula (I), R3 is halogen, -(C1-C4)-alkyl, -(C1-C4)- haloalkyl, -(C2-C4)-alkenyl, -(C2-C4)-alkynyl, -(C1-C4)-alkyl-OH, -(C1-C4)-alkyl-NHR33, - (C1-C4)-alkyl-C(O)NHR33, -(C1-C4)-alkyl-C(O)OH, -(C1-C4)-alkyl-heteroaryl wherein said heteroaryl is selected from the group consisting of imidazole, pyrazole, oxazole, isoxazole, thiazole, pyrole, furane, thiophene, pyrazine, pyridazine, pyridine, and pyrimidine, preferably from the group consisting of imidazole, pyrole, thiazole, furane and thiophene, more preferably from the group consisting of imidazole, oxazole and thiazole, even more preferably is imidazole, or -(C1-C4)-alkyl-aryl wherein said aryl is phenyl. The heteroaryl and aryl may be substituted as disclosed herein. In some embodiments, in the above formula (I), R33, R’33 and R’’333 are advantageously independently H or -(C1-C3)-alkyl, more advantageously H. In some embodiments, in the above formula (I), R333, R’333 and R’’3333 are advantageously independently H, OH, halogen, -(C1-C3)-alkyl, or -(C1-C3)-alkoxy, advantageously H.
Figure imgf000019_0001
In some embodiments, in the above formula (I), Xaa3 is , more specifically
Figure imgf000019_0002
, wherein X3 is N(R33); R3 is -(C1-C4)-alkyl-NR33R’33, preferably -CH2-CH2- NR33R’33 and R33 and R’33 being as disclosed herein, preferably are H. In some embodiments, in the above formula (I), Xaa3 is
Figure imgf000019_0003
, more specifically
Figure imgf000019_0004
, wherein X3 is NH or N(Me), preferably NH. In these embodiments, R3 is as disclosed herein, more specifically -(C1-C4)-alkyl-NHR33 with R33 as disclosed herein. In some embodiments, R33 is H. In some embodiments, in the above formula (I), Xaa4 is
Figure imgf000019_0005
, wherein X4 is NH or N(Me), preferably NH. In some other embodiments, X4 is O. R4 and R’4 are disclosed herein. In some embodiments, in the above formula (I), R4 and R’4 are H.
Figure imgf000019_0006
In some embodiments, in the above formula (I), Xaa4 is or , wherein X4 is NH or N(Me), preferably NH. In some embodiments, in the above formula (I), Xaa5 is
Figure imgf000020_0001
, preferably
Figure imgf000020_0002
, wherein X5 is NH or N(Me), preferably NH. In some other embodiments, X5 is O.
Figure imgf000020_0003
In some embodiments, in the above formula (I), Xaa5 is
Figure imgf000020_0006
, preferably , wherein R5 and R’5 are independently H or -(C1-C3)-alkyl and n5 as disclosed herein. In some embodiments, R5 and R’5 are H. In some embodiments, n5 is 2. In each of these embodiments, X5 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa5 is
Figure imgf000020_0005
, preferably
Figure imgf000020_0004
, wherein R5 and R’5 are independently H or methyl and n5 as disclosed herein. In some embodiments, R5 and R’5 are H. In some embodiments, n5 is 2. In each of these embodiments, X5 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa5 is
Figure imgf000020_0007
and R5 is H or methyl, preferably H. In some embodiments, Xaa5 is
Figure imgf000020_0008
and R5 is H or methyl, preferably H. In each of these embodiments, X5 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000021_0001
Figure imgf000021_0002
, wherein X6 is NH or N(Me), preferably NH. In some other embodiments, X6 is O. In these embodiments, R6 is preferably H, -(C1-C3)-alkyl, or -(C1- C3)-haloalkyl, more preferably H or CH3; R66 is preferably H, CH3 or halogen and R’66 is preferably H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -O-(C1-C3)-alkyl, -(C1-C3)- haloalkyl, -CH2-COOH, -C(O)-(C1-C3)-alkyl, -C(O)-(C1-C3)-haloalkyl, -NH2, -NH(C1-C3)- alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl] and n6 is an integer from 0-3. X6 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000021_0003
Figure imgf000021_0004
wherein R6 is preferably H, -(C1-C3)-alkyl, or -(C1-C3)- haloalkyl, more preferably H or CH3; R66 is preferably H, CH3 or halogen and R’66 is preferably H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -O-(C1-C3)-alkyl, -(C1-C3)- haloalkyl, -CH2-COOH, -C(O)-(C1-C3)-alkyl, -C(O)-(C1-C3)-haloalkyl, -NH2, -NH(C1-C3)- alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl] and n6 is an integer from 0-3. X6 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000021_0005
, wherein R66 and R’66 are independently H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -(C1-C3)-haloalkyl, or -NH2, preferably H and -NH2; and n6 is an integer from 0-3, preferably 1. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000022_0001
wherein R66 is H, CH3 or halogen and R’66 is preferably H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -O- (C1-C3)-alkyl, -(C1-C3)-haloalkyl, -CH2-COOH, -C(O)-(C1-C3)-alkyl, -C(O)-(C1-C3)- haloalkyl, -NH2, -NH(C1-C3)-alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl]. Advantagously, n6 is 0, 1 or 2. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000022_0002
, R66 is H, CH3 or halogen, R’66 is H, halogen, methyl, OH, -O-(C1-C3)-alkyl, -(C1-C3)-haloalkyl, -CH2-COOH or NH2 and n6 is an integer from 0-3. Advantagously, n6 is 0, 1 or 2. The configuration of the carbon atom bearing R66, R’66 or R66 and R’66 can be S or R, advantageously R. In all these embodiments, the configuration of the carbon atom linked to –X6 and to – C(O)- is advantageously S. In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000022_0003
,
Figure imgf000022_0004
In some embodiments, in the above formula (I), Xaa6 is
Figure imgf000023_0001
, ,
Figure imgf000023_0007
, , , , o In some embodiments, in the above formula (I), Xaa6 is , more
Figure imgf000023_0002
specifically
Figure imgf000023_0003
In some embodiments, in the above formula (I), Xaa7 is
Figure imgf000023_0004
, preferably
Figure imgf000023_0005
, preferably
Figure imgf000023_0006
, wherein X7 is NH or N(Me), preferably NH. In some other embodiments, X7 is O. R7, R77, R’77 and n7 are disclosed herein. In some embodiments, in the above formula (I), R7 is H, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C2-C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)-alkyl-OR77, (C1-C6)-alkyl-SR77, -(C1-C6)-alkyl- S(O)R77, -(C1-C6)-alkyl-S(O)2R77, -(C1-C6)-alkyl-NR77R’77, -(C1-C6)-alkyl-C(O)OR77, -(C1- C6)-alkyl-C(O)NR77R’77, -(C1-C6)-alkyl-heteroaryl, -(C1-C6)-alkyl-aryl or -(C1-C6)-alkyl- aryl-heteroaryl, wherein said aryl or heteroaryl in the -(C1-C6)-alkyl-heteroaryl, -(C1-C6)- alkyl-aryl or -(C1-C6)-alkyl-aryl-heteroaryl groups is optionally mono- or poly- substituted with –OH, -NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl, -(C1-C6)-alkyl-COOH, -(C1- C6)-hydroxyalkyl, -(C1-C6)-aminoalkyl, -(C1-C6)-alkyl-NHCONH2, -(C1-C3)-alkoxy, -(C1- C3)-alkoxy-COOH, or halogen, and wherein the heteroaryle is selected from the group consisting of pyridine, pyrimidine, pyrazine, pyridazine, quinoleine, isoquinoleine, quinoxaline, quinoxoline, imidazole, oxazole, thiazole, furane, pyrrole, thiophene, benzimidazole, benzoxazole, benzothiazole, benzothiophene, indole, isoindole, benzofurane and triazole and the aryle is phenyl or naphtyl, preferably phenyl. The heteroaryle is advantageously pyridine, indole, isoindole, benzothiophene, benzofurane, imidazole, oxazole, thiazole, furane, pyrrole, thiophene or triazole, more advantageously pyridine, isoindole, imidazole, thiophene. The aryl or heteroaryle is advantageously mono-, di- or tri- substituted with OH, NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -CH2-COOH, -O-CH2-COOH, -CH2-NH-CO-NH2, -CHCH3OH, -CH2-NH2 or halogen. In each of these embodiments, X7 is preferably NH or N(Me), more preferably NH.
Figure imgf000024_0001
In some embodiments, in the above formula (I), Xaa7 is , preferably
Figure imgf000024_0002
, wherein X7 is N(R77); R7 is -(C1-C6)-alkyl-heteroaryl or -(C1-C6)-alkyl-aryl, wherein said aryl or heteroaryl is optionally monosubstituted with –OH, -NH2, -COOH, - CONH2, -CN, -CF3, -(C1-C6)-alkyl or halogen, preferably -COOH; preferably the aryl is phenyl; preferably R7 is -CH2-heteroaryl or -CH2-phenyl, wherein said phenyl is optionally monosubstituted with –OH, -NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl or halogen, preferably COOH and R77 is H or Me. In some embodiments, in the above formula (I), Xaa7 is
Figure imgf000024_0003
, ,
Figure imgf000024_0004
Figure imgf000025_0001
Figure imgf000025_0002
, , , with R, R’ and R” being independently OH, NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C3)-alkyl, -(C1-C3)- alkoxy, -CH2-COOH, -O-CH2-COOH, -CH2-NH-CO-NH2, -CHCH3OH, -CH2-NH2 or halogen. In each of these embodiments, X7 is preferably NH or N(Me), more preferably NH. In all these embodiments, the configuration of the carbon atom linked to –X7 and to – (CO)- is advantageously S. In some embodiments, in the above formula (I), Xaa7 is
Figure imgf000026_0001
Figure imgf000026_0002
Figure imgf000027_0001
, with R, R’ and R” being independently OH, NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C3)-alkyl, -(C1-C3)- alkoxy, CH2-COOH, O-CH2-COOH, CH2-NH-CO-NH2, CHCH3OH, CH2-NH2 or halogen. In each of these embodiments, X7 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa7 is
Figure imgf000027_0002
Figure imgf000027_0003
Figure imgf000027_0004
with R, R’ and R” being independently OH, NH2, - COOH, -CONH2, -CN, -CF3, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -CH2-COOH, -O-CH2-COOH, -CH2-NH-CO-NH2, -CHCH3OH, -CH2-NH2 or halogen. In each of these embodiments, X7 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa8
Figure imgf000028_0001
Figure imgf000028_0002
, wherein X8 is NH or N(Me), preferably NH. In some other embodiments, X8 is O. R8, R’8, R88, R’88 , R’’88, R’’’88 and n8 are as disclosed herein. In some embodiment, Xaa8 is
Figure imgf000028_0004
In some embodiments, in the above formula (I), Xaa8 is or
Figure imgf000028_0005
Figure imgf000028_0003
. X8 is preferably NH or N(Me), more preferably NH. R8, R’’88 and n8 are as disclosed herein. In some embodiments, in the above formula (I), R8 is H, methyl, ethyl, -C(O)-(C1-C2)- alkyl, -C(=NH)-NH2 or -C(O)-(C1-C2)-haloalkyl; and n8 is an integer from 1-3. In some embodiments, R8 is H. In some embodiments, n8 is 1, 2 or 3, in particular 3. In some embodiments, in the above formula (I), Xaa8 is is
Figure imgf000029_0001
, preferably
Figure imgf000029_0002
, wherein X8 is NH or N(Me), preferably NH; R8 and R’8 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, -C(=NH)-NH2, or -C(O)-(C1-C3)-haloalkyl, preferably H and n8 is an integer from 1-4, preferably 3. In some embodiments, in the above formula (I), Xaa8 is
Figure imgf000029_0003
, O ,
Figure imgf000029_0004
In each of these embodiments, X8 is preferably NH or N(Me), more preferably NH. In some embodiments, Xaa8 is
Figure imgf000030_0005
Figure imgf000030_0006
Figure imgf000030_0007
. In each of these embodiments, X8 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa8 is
Figure imgf000030_0001
, preferably , with X8 being preferably NH or N(Me), more preferably NH.
Figure imgf000030_0003
In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000030_0004
,
Figure imgf000030_0002
, wherein X9 is NH or N(Me), preferably NH. In some other embodiments, X9 is O. R9, R’9, R99 and n9 are as disclosed herein. In some of these embodiments, R’9 is a hydrogen atom, R9 is H, -C(=NH)-NH2, -C(O)NH2, -(C1-C3)-alkyl, - (C1-C3)-haloalkyl, -C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R99 is H or -(C1-C3)- alkyl; and n9 is an integer from 1-4. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000031_0001
; R9
Figure imgf000031_0002
Figure imgf000031_0003
is H, -C(=NH)-NH2, -C(O)NH2, -(C1-C3)-alkyl, -(C1-C3)- haloalkyl, -C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R99 is H or -(C1-C3)-alkyl; and n9 is an integer from 1-4. X9 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000031_0004
Figure imgf000031_0005
; R9 is H, -C(=NH)-NH2, -C(O)NH2, or -(C1-C3)- alkyl; R99 is H, or -(C1-C3)-alkyl; and n9 is an integer from 1-2. In each of these embodiments, X9 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000031_0006
, ,
Figure imgf000031_0008
, , , , , , or
Figure imgf000031_0007
In each of these embodiments, X9 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000032_0001
, ,
Figure imgf000032_0006
, , , , , ,
Figure imgf000032_0005
, In each of these embodiments, X9 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000032_0002
wherein X9 is NH or N(Me), preferably NH; R9 and R’9 are as disclosed herein, preferably are independently H and -C(=NH)-NH2; and n9 is an integer from 1-4, preferably 2. In some embodiments, in the above formula (I), Xaa9 is
Figure imgf000032_0003
Figure imgf000032_0004
. In each of these embodiments, X9 is preferably NH or N(Me), more preferably NH. In some embodiments, in the above formula
Figure imgf000033_0001
Figure imgf000033_0002
wherein X10 is NH or N(Me), preferably NH. In some other embodiments, X10 is O. R10, R’10, R100 and n10 are as disclosed herein. In some embodiments, in the above formula (I), Xaa10 is
Figure imgf000033_0003
or
Figure imgf000033_0004
wherein R10 is H, -C(=NH)-NH2, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, -(C1-C6)-alkyl-NH2 or -C(O)-(C1-C3)-haloalkyl; R’10 is H; R100 is H, OH, NH2 -(C1-C3)-alkyl, -(C1-C3)-alkoxy or halogen; and n10 is independently an integer from 0-3. In each of these embodiments, X10 is preferably NH or N(Me), more preferably NH. In these embodiments, Xaa10 is preferably
Figure imgf000033_0005
.
In some embodiments, in the above formula
Figure imgf000034_0001
, preferably
Figure imgf000034_0002
, wherein X10 is NH; R10 and R’10 are independently H and n10 is an integer from 0-5, preferably 2 or 3.
Figure imgf000034_0003
In some embodiments, in the above formula (I), Xaa10 is , ,
Figure imgf000034_0004
, wherein R10 is H, -C(=NH)-NH2, -(C1-C2)-alkyl, -(C1-C3)-alkyl-NH2, acetyl, trifluoromethyl; R’10 is H and R100 is H, OH, NH2 or halogen. The compounds of formula (I) may comprise any combinations of the Xaa1 to Xaa10 groups disclosed herein. In some embodiments, the present invention relates to compounds of formula (I): Ra-Xaa1-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-(Xaa10)y-Rb (I) wherein: Ra is H or -(C1-C3)-alkyl; y is 0 or 1; Xaa1 is
Figure imgf000035_0001
wherein one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl, preferably when the chain bearing the NR11R’11 group is substituted, the carbon atom in alpha of the NR11R’11 group is substituted by - (C1-C3)-alkyl, preferably a methyl group; wherein: R11 and R’11 are independently H or -(C1-C3)-alkyl, preferably H; n1 is an integer from 1-4, preferably 3; Xaa2 is
Figure imgf000035_0002
wherein: X2 is NH or N(Me), preferably NH; R2 and R’2 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl or -C(O)-(C1- C3)-haloalkyl, preferably R2 is H or methyl, and R’2 is H, more preferably R2 and R’2 are H; R22 is OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -O-C(O)-(C1-C6)-alkyl or –O- C(O)-(C1-C6)- haloalkyl, preferably OH, fluorine, methyl, methoxy, -O-C(O)-CH3, -O- C(O)-CF3, more preferably OH; Xaa3 is
Figure imgf000035_0003
wherein: X3 is N(R33); R3 is -(C1-C6)-alkyl-NR33R’33, preferably -CH2-CH2-NR33R’33; R33 and R’33 are independently H or methyl, preferably H; Xaa4 is
Figure imgf000036_0001
wherein: X4 is NH or N(Me), preferably NH; R4 and R’4 are independently H; Xaa5 is
Figure imgf000036_0002
wherein: X5 is NH, N(Me), preferably NH; R5 and R’5 are independently H or -(C1-C3)-alkyl, preferably H; n5 is an integer from 1-3, preferably 2; Xaa6 is
Figure imgf000036_0003
wherein: R66 and R’66 are independently H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, - (C1-C3)-haloalkyl, -NH2, preferably H and -NH2; n6 is an integer from 0-3, preferably 1; Xaa7 is
Figure imgf000036_0004
wherein: X7 is N(R77); R7 is -(C1-C6)-alkyl-heteroaryl or -(C1-C6)-alkyl-aryl, wherein said aryl or heteroaryl is optionally monosubstituted with –OH, -NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl or halogen, preferably -COOH; preferably the aryl is phenyl; preferably R7 is -CH2- heteroaryl or -CH2-phenyl, wherein said phenyl is optionally monosubstituted with –OH, -NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl or halogen, preferably COOH; R77 is H or Me; Xaa8 is
Figure imgf000037_0001
wherein: X8 is NH or N(Me), preferably NH; R8 and R’8 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, -C(=NH)-NH2, or -C(O)-(C1-C3)-haloalkyl, preferably H; n8 is an integer from 1-4, preferably 3; Xaa9 is
Figure imgf000037_0002
wherein: X9 is NH or N(Me), preferably NH; R9 and R’9 are independently H and -C(=NH)-NH2; n9 is an integer from 1-4, preferably 2; Xaa10 is,
Figure imgf000037_0003
wherein: X10 is NH; R10 and R’10 are independently H; n10 is an integer from 0-5, preferably 3; Rb is -NR5aR6a with R5a and R6a as disclosed herein above or below (e.g. in connection with formula (Ia)), or hydrates, solvates, or salts thereof. In some embodiments, the compounds of the invention are compounds of formula (I) as disclosed herein above wherein: Ra is as disclosed herein; y is 0 or 1; Xaa1 is
Figure imgf000038_0001
, more specifically
Figure imgf000038_0002
, wherein one or more carbon atoms of the chain bearing the NH2 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl, preferably wherein the carbon atom in alpha of the NH2 group is substituted by–(C1-C3)-alkyl, preferably a methyl group; Xaa2 is , more specifically
Figure imgf000038_0003
Figure imgf000038_0004
wherein: X2 is NH or N(Me), preferably NH; R22 is OH, fluorine, methyl, methoxy or -O-C(O)-CH3, -O-C(O)-CF3, in particular OH, -O-C(O)-CH3 or -O-C(O)-CF3, preferably OH; and R2 and R’2 are as defined above, preferably R2 is H or methyl, more preferably H, and R’2 is H, more preferably R2 and R’2 are H; Xaa3 is
Figure imgf000038_0006
, more specifically
Figure imgf000038_0005
wherein: X3 is NH or N(Me), preferably NH; and R3 is as disclosed herein, more specifically R3 is -(C1-C4)-alkyl-NHR33 with R33 as disclosed herein, more specifically R33 is H; Xaa4 is
Figure imgf000039_0001
, wherein: X4 is NH or N(Me), preferably NH; R4 and R’4 are H; 5 Xaa5 is
Figure imgf000039_0003
, more specifically
Figure imgf000039_0002
wherein: X5 is NH or N(Me), preferably NH; and R5 is H or –(C1-C3)-alkyl, preferably methyl, more preferably H; 10 Xaa6 is
Figure imgf000039_0006
, more specifically
Figure imgf000039_0004
Xaa7 is
Figure imgf000039_0007
, , more specifically
Figure imgf000039_0005
Figure imgf000039_0008
15 wherein: X7 is NH or N(Me), preferably NH; and R is –OH, -NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl or halogen, preferably -COOH; Xaa8 is
Figure imgf000040_0001
, more specifically
Figure imgf000040_0002
wherein: X8 is NH or N(Me), preferably NH; Xaa9 is
Figure imgf000040_0003
wherein: X9 is NH or N(Me), preferably NH; Xaa10 is , more specifically
Figure imgf000040_0005
Figure imgf000040_0004
wherein: X10 is NH or N(Me), preferably NH; n10 is 2 or 3, preferably 3; R10 is H, -C(=NH)-NH2, -(C1-C2)-alkyl, -(C1-C3)-alkyl-NH2, acetyl, trifluoromethyl, preferably H; R’10 is H; Rb is -NR5aR6a with R5a and R6a as disclosed herein above or below (e.g. in connection with formula (Ia)), or hydrates, solvates, or salts thereof. In some embodiments, the compounds of the invention are compounds of formula (I) as disclosed herein above wherein: when R5a is: -A5-NHR6 A5 is an unsubstituted C2-C6-alkenediyl group, an unsubstituted C1-C6-alkanediyl or a C1- C6-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl, an amine and a –(C1-C6)-aminoalkyl; and Re is a hydrogen atom, a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl, provided that Re is a -(C1-C6)-hydroxyalkyl when A5 is an unsubstituted C1-C6-alkanediyl. In some embodiments, the compounds of the invention (compounds of formula (I)) are compounds of formula (Ia):
Figure imgf000041_0001
wherein: R1a and R2a are, independently of each other, a hydrogen atom or a -(C1-C3)-alkyl; R3a is H or NH2; R4a is
Figure imgf000041_0002
y is equal to 0 or 1; R5a and R6a are as disclosed herein in relation to formula (I); or hydrates, solvates, or salts thereof. In some embodiments, in formula (Ia): R5a is: -A1-NRa-A2-NHRb, -A3-CO-NRc-A4-NHRd, or -A5-NHRe wherein: A1 is an unsubstituted (C2-C3)-alkanediyl or a (C2-C3)-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl and a –(C1-C3)-hydroxyalkyl; A2 is an unsubstituted (C2-C4)-alkanediyl or a (C2-C4)-alkanediyl substituted by one or more hydroxyl; Ra is a hydrogen atom, a –(C1-C3)-hydroxyalkyl, a –(C1-C4)- hydroxyaminoalkyl or a methanediyl that forms a cycle with a carbon atom of A1 or A2 in position beta relative to the nitrogen atom bearing Ra; Rb is a hydrogen atom or a methanediyl that forms a cycle with a carbon atom of A2 in position beta relative to the nitrogen atom bearing Rb; A3 is an unsubstituted C1-C2-alkanediyl or a C1-C2-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl and a carbamoyl; A4 is an unsubstituted C2-C5-alkanediyl or a C2-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl and a carboxyl; Rc is a hydrogen atom; Rd is a hydrogen atom; A5 is an unsubstituted C2-C6-alkenediyl group, an unsubstituted C2-C4- alkanediyl or a C2-C4-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl and a –(C1-C3)-aminoalkyl, Re is a hydrogen atom or a -(C1-C3)-alkyl, provided that Re is a -(C1-C3)- alkyl when A5 is an unsubstituted C2-C4-alkanediyl; R6a is a hydrogen atom, a –(C1-C3)-hydroxyalkyl or a methanediyl that forms a cycle with a carbon atom of A1 or A5 in position beta relative to the nitrogen atom bearing R6a, or hydrates, solvates, or salts thereof. In some embodiments, in the above formula (Ia), R1a and R2a are a hydrogen atom. In some embodiments, in the above formula (Ia), R4a is
Figure imgf000043_0001
In some embodiments, in the above formula (Ia), R5a is -A1-NRa-A2-NHRb withA1,A2, Ra and Rb as disclosed herein above. More specifically, R5a may be one the following groups:
Figure imgf000043_0002
Even more specifically, R5a may be one the following groups:
Figure imgf000043_0003
Figure imgf000044_0002
In these embodiments, R6a is preferably H. In some embodiments, in the above formula (Ia), R5a is -A3-CO-NRc-A4-NHRd with A3, A4, Rc and Rd as disclosed herein above. More specifically, R5a may be one the following groups:
Figure imgf000044_0001
Figure imgf000045_0002
In these embodiments, R6a is preferably H. In some embodiments, in the above formula (Ia), R5a is -A5-NHRe with A5 and Re as disclosed herein above. More specifically, R5a may be one the following groups:
Figure imgf000045_0003
In these embodiments, R6a is preferably H. In embodiments, in the above formula (I), -NR5aR6a is one of the following groups:
Figure imgf000045_0001
The compound according to the invention may be selected from the group consisting of compounds 1 to 80, represented below, and the salts, e.g. pharmaceutically acceptable salts, hydrates and/or solvates thereof.
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
In particular, the compound of formula (I) may be selected from the group consisting of compounds 1 to 7 as disclosed hereinabove and salts, e.g. pharmaceutically acceptable salts, hydrates and/or solvates thereof. The compounds of formula (I) may be prepared by methods known to the skilled person. Exemplary methods are shown in the Examples. In the following, the expression “a compound of formula (I)” refers to a compound of formula (I) as described herein, including any compounds of formula (I) disclosed in the “Examples” section, as well as any salts, hydrates, solvates, isomers, mixtures of isomers in any ratio and any combinations thereof. The term “a compound of formula (I)” may refer to a single compound of formula (I) or a combination of two or more compounds of formula (I) or salts, hydrates, solvates, isomers or mixtures of isomers thereof. Therapeutic indications The compounds of formula (I) have been found to be effective against bacteria, in particular against multi-drug resistant bacteria. Resultantly, the compounds of formula (I) may be useful in medicine. Thus, in an aspect, the present invention relates to a compound of formula (I) for use in medicine, in particular for the treatment or prevention of a bacterial infection. In some embodiments, the bacterial infection is multi-drug resistant. In some embodiments, the bacterial strain is hospital-acquired. In some embodiments, the bacterial strain is nosocomial. In some embodiments, the bacterial infection comprises infection from Gram-negative bacteria. In some embodiments, the bacterial infection comprises infection from Gram-positive bacteria. In some embodiments, the bacterial infection comprises infection by more than one bacterial strain. Multiresistant bacterial pathogens that cause infection comprise methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended spectrum ß-lactamase formers (ESBL), carbapenem-resistant Enterobacteriaceae, multiresistant Pseudomonas and Acinetobacter species. In some embodiments, the bacterial or microbial infection is an infection caused or suspected to be caused in whole or in part by bacteria of the Achromobacter, Actinobacillus, Actinomyces, Acinetobacter, Aeromonas, Anaplasma, Bacillus, Bacteroides, Bartonella, Bdellovibrio, Bifidobacterium, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Hemobartonella, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria, Mannheimia, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Neorickettsia, Nocardia, Pasteurella, Peptostreptococcus, Photorhabdus, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Sphaerophorus, Spirillum, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Treponema, Tropheryma, Ureaplasma, Vibrio, or Yersinia families. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by bacteria of Acinetobacter sp., Bacillus sp., Burkholderia sp., Enterobacter sp., Enterococcus sp., Escherichia sp., Klebsiella sp., Staphylococcus sp., Stenotrophomonas sp., Serratia sp.and Pseudomonas sp.. In some embodiments, the bacteria is selected from the group consisting of Staphylococus sp., Escherichia sp., Klebsiella sp., Pseudomonas sp., or Acinetobacter sp. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by Acinetobacter baumannii, Bacillus subtilis, Burkholderia cepacia, Enterobacter clocae, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Serratia marcescens or Pseudomonas aeruginosa. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by A. baumannii, E. cloacae, E. coli, Klebsiella spp., M. morganii, P. mirabilis, S. marcescens, Staphylococcus spp., Enterococcus spp., S. pneumoniae. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa. In some embodiments, the bacterial infection is an infection caused or suspected to be caused in whole or in part by multidrug-resistant P. aeruginosa. In accordance with a further aspect, the present invention relates a method of treatment or prevention of a bacterial infection using a therapeutically effective amount of a compound of formula (I). In other words, the present invention relates to a method of treating or preventing a bacterial infection in a subject in need thereof comprising administering to the subject, that may be human or animal, a therapeutically effective amount of a compound of formula (I). More specifically, the present invention provides a method for treating a subject suffering from a multi-drug resistant bacterial infection comprising administering to said subject a therapeutically amount of a compound of formula (I). A “therapeutically effective amount” as used herein refers to an amount that (i) treats or prevents the infection, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the infection, or (iii) prevents or delays the onset of one or more symptoms of the infection described herein. The amount of a compound which constitutes a therapeutically effective amount will vary depending on many factors, such as for instance the compound and its biological activity, the composition used for administration, the route of administration, the type of disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds, and the age, body weight, general health, sex, and diet of the patient. Such an effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge. In another aspect, the present invention relates to a compound of formula (I) for use in a method of treating or preventing a bacterial infection, in particular a multi-drug resistant bacterial infection. In accordance with a further aspect, the present invention relates to the use of a compound of formula (I) for the preparation of a pharmaceutical composition, preferably a medicament, for the treatment or prevention of a bacterial infection, in particular a multi- drug resistant bacterial infection. Compositions The present invention also relates to pharmaceutical or veterinary compositions, in particular a medicament, comprising a compound of formula (I) and one or more excipients, in particular one or more pharmaceutically or veterinary acceptable excipient(s) and to their uses for the above-mentioned purpose. In one aspect, the present invention relates to a pharmaceutical or veterinary composition, in particular a medicament, comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically or veterinary acceptable excipient. The pharmaceutical or veterinary composition is particularly useful in the treatment of bacterial infections. Pharmaceutically or veterinary acceptable excipients include fillers and carriers, ointment bases, bases for suppositories, solvents, surfactants, emulsifiers, dispersants or wetting agents, buffers, acids and bases, isotonicity agents, adsorbent, viscosity- increasing agents, gel formers, thickeners and/or binders, disintegrants, coating materials and film formers for films or diffusion membranes, capsule materials, natural or synthetic polymers, plasticizers, penetration enhancers, stabilizers, preservatives, colourants, flavourings, sweeteners, flavour- and/or odour-masking agents. Combinations The compounds of formula (I) can be administered in therapeutically effective amounts in a combinational therapy with one or more other pharmaceutically active agents (pharmaceutical combinations). Therefore, the present invention also relates to such pharmaceutical combinations. For example, the compounds of the present invention can be combined with an antibiotic compound. The compounds can be administered simultaneously (as a single preparation or separate preparation), sequentially or separately. In an aspect of the invention, a compound of formula (I) is administered prior to the administration of one or more other pharmaceutically active agents. In another aspect of the invention, a compound of formula (I) is administered concomitantly with the administration of one or more other pharmaceutically active agents. In yet another aspect of the invention, a compound of formula (I) is administered immediately after administration of the other pharmaceutically active agent(s). The pharmaceutically active agents (compounds of formula (I) and other pharmaceutically active agents) may be packaged in a kit or separately. Thus, in an aspect, the present invention relates to a kit comprising: - a composition comprising a compound of formula (I) as described herein; - a second composition comprising one or more other pharmaceutically active agents, and - preferably instructions for using said kit. The other pharmaceutically active agent is preferably an antibiotic compound. The antibiotic compound is preferably selected from the group consisting of beta- lactams, aminoglycosides, tetracyclines, glycylcyclines, macrolides, azalides, ketolides, synergistins, lincosamides, fluoroquinolones, phenicols, rifamycins, sulfamides, trimethoprim, glycopeptides, oxazolidinones, nitromidazoles, fosfomycins, polymyxins and lipopeptides. Exemplary antibiotics are amoxicillin, amoxicillin-clavulanic acid, ampicillin, ampicillin-subactam, benzylpenicillin, cloxacillin, phenoxymethylpenicillin, piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin- clavulanic acid, cefalexin, cefazolin, cefuroxime, cefixime, cefotaxime, cefepime, cefoxitin, ceftazidime, ceftazidime-avibactam, ceftriaxone, cefiderocol, ceftolozane-tazobactam, imipenem, imipenem-relebactam, meropenem, meropenem-vaborbactam, ertapenem, aztreonam, spectinomycin, amikacin, gentamicin, tobramycin, plazomicin, doxycycline, minocycline, eravacycline, tigecycline, omadacycline, azithromycin, clarithromycin, clindamycin, ciprofloxacin, levofloxacin, chloramphenicol, rifampicin, sulfamethoxazole, trimethoprim, sulfamethoxazole-trimethoprim, vancomycin, linezolid, metronidazole, nitrofurantoin, Fosfomycin, colistin, polymyxin B and daptomycin. Administration routes The compounds of formula (I) and the compositions comprising thereof can have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, dermal, transdermal or parenteral route. Suitable administration forms for oral administration include for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. Suitable administration forms for parenteral administration are preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders. Suitable administration forms for the dermal or transdermal administration routes are, for example, pharmaceutical forms for aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (for example patches), milk, pastes, foams or dusting powders. Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure. EXAMPLES The following abbreviations have been used: AcCN: Acetonitrile AcOH: acetic acid Alloc: Allyloxycarbonyl Boc: tert-butyloxycarbonyl EDC.HCl: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride DCM: dichloromethane DIPEA: N,N-diisopropylethylamine DBF: dibenzofuran DMSO: dimethylsulfoxyde DMF: dimethylformamide EDTA: ethylenediaminetetraacetic acid EtOH : ethanol EtOAc: Ethyl acetate Et2O: ethoxyethane Fmoc: fluorenylmethoxycarbonyl Fmoc-O-Su: N-(9H-Fluoren-9-ylmethoxycarbonyloxy)succinimide HBTU: N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate HFBA: heptafluorobutyric acid HFIP: 1,1,1,3,3,3-hexafluoropropan-2-ol HOBt: 1H-1,2,3-Benzotriazol-1-ol HMBC: Heteronuclear Multiple Bond Correlation HSQC: Heteronuclear Single Quantum Coherence HPLC: high performance liquid chromatography iPrOH: isopropylalcohol LC-MS: liquid chromatography coupled with mass spectrometry MeOH: methanol PyBOP Rt: retention time TBDMS: ter-butyldimethylsilyl TBME: tert-butyl methylether t-Bu: tert-butyl THF: tetrahydrofuran TFA: trifluoroacetic acid I – Materials & Peptide synthesis Chemical analogs were obtained by solid-phase peptide synthesis (SPPS; Merrifield R. B. J. Am. Chem. Soc.1963, 85, 2149; herein incorporated by reference in its entirety) applying the orthogonal Fmoc/tBu strategy. Classic peptide couplings were carried out using the uronium reagent HBTU. For each coupling, 3.0 equivalents of amino-acid and 2.9 equivalents of HBTU were used. Each coupling was repeated twice. At the end of the coupling steps, Fmoc deprotection was carried out using a solution of DMF/piperidine, and the next amino acid was added using the same strategy. The last amino acid introduced was protected by a Boc group on its N-terminal position. At the end of the synthesis, the cleavage of peptide from the resin was performed. Depending on the protocol used, a fully deprotected peptide or peptide with all protecting groups was obtained. In the latter case, C-terminal position of the protected peptides were substituted during a peptide coupling in solution followed by a cleavage of the protecting groups. The final peptide was purified by preparative HPLC and salt exchange performed. Purity of the pure final peptide was checked by LC-MS analysis. Preparative HPLC purification method using TFA as additive: Flow: 20 mL/min Mobile phase A: water milliQ, TFA 0.1% Mobile phase B: acetonitrile Gradient: 2% to 30% of B in 20 minutes Run time: 29 minutes Preparative HPLC purification method using HFBA as additive: Flow: 2.5 mL/min Mobile phase A: water milliQ, HFBA 0.2% Mobile phase B: acetonitrile Gradient: 20% to 50% of B in 15 minutes Run time: 29 minutes HPLC-MS analytical method for final purity check (AM1): Flow: 0.7 mL/min Mobile phase A: milliQ water, TFA 0.1% Mobile phase B: acetonitrile Gradient: 2% to 30% of B in 15 minutes Run time: 31 minutes HPLC-MS analytical method for final purity check (AM2): Flow: 0.8 mL/min Mobile phase A: milliQ water, 0.1% formic acid, 0.1% acetic acid, 0.04% HFBA Mobile phase B: acetonitrile, 0.05% formic acid Gradient: 10% to 35% of B in 3 min then, 35 to 50 % of B in 2 min, then 50 to 95% in 2 min. Run time: 12.5 minutes HPLC-MS analytical method for final purity check (AM3): Flow: 0.8 mL/min Mobile phase A: milliQ water, HFBA 0.2% Mobile phase B: acetonitrile, 0.05 % formic acid Gradient: 20% to 50% of B in 8 min then, 50 to 95 % of B in 2 min. Run time: 16 minutes Solvents and amino-acid building blocks 1.5-diaminopentane trityl resin, 1.3-diaminopropane trityl resin, Fmoc-rink amide resin and N1,N4-bis-Boc-norspermidine [122248-82-2] were purchased from Chem Impex International Inc (Wood dale, IL, USA). 2-chlorotrityl resin, Fmoc-Lys(Boc)-OH [71989-26-9, Fmoc-Pro-OH [71989-31-6], Fmoc- Gly-OH [29022-11-5], Fmoc-Dab(Boc)-OH [125238-99-5], Boc-Lys(Boc)-OH [15098-69- 8], Fmoc-D-Orn(Boc)-OH [118476-89-4], Fmoc-His(Trt)-OH [109425-51-6], Fmoc-L- Orn(Boc)-OH [109425-55-0], Fmoc-β-Ala-OH [35737-10-1], Fmoc-(4R-NHBoc)Pro-OH [273222-06-3], Fmoc-(4S-NHBoc)Pro-OH [221352-74-5], Fmoc-β-HomoSer(tBu)-OH [203854-51-7], Fmoc-MeLys(Boc)-OH [197632-76-1], N-Boc-1,3-diaminopropane hydrochloride [127346-48-9] and 1,4-Bis-Boc-1,4,7-triazaheptane [120131-72-8] were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Fmoc-(4-CO2tBu)Phe-OH [183070-44-2], Fmoc-Asp-(Alloc)-OH [146982-24-3] and t- butyl (3-amino-2-hydroxypropyl)carbamate [14412-84-5] were purchased from Fluorochem (Hadfield, United Kingdom). tert-Butyl N-(3-amino-2,2-difluoropropyl)carbamate [1044675-84-4] was purchased from abcr Gmbh (Karlsruhe, Germany). Fmoc-β-(D)-Ser(Trt)-OH [1820583-73-0], Fmoc-(S)-3-NH2-2-OH propionic acid [172721- 23-2], tert-butyl (4-aminobut-2-en-1-yl)carbamate [146394-99-2], tert-butyl N-[(2S)-3- amino-2-hydroxypropyl]carbamate [853944-08-8], tert-butyl N-[(2R)-3-amino-2- hydroxypropyl]carbamate [1042665-83-5] and tert-butyl-N-(2-amino-3-{[(tert- butoxy)carbonyl]amino}propyl)carbamate [149876-86-8] were purchased from Enamine (Kiev, Ukraine). Fmoc-homoSer(Trt)-OH [111061-55-3], Fmoc-Ser(Trt)-OH [111061-56-4], N-Boc-N- methylethylenediamine [121492-06-6] and all other reagents and solvents, including of HPLC grade, were purchased from Merck (Darmstadt, Germany). Synthesis of non-commercial amino-acid building blocks Example 1: Synthesis of non-commercial amino-acid building blocks: aminothreonine Aminothreonine building block was prepared following the procedure described in patent application FR 1451623. Step 1: Hydroxyectoin (50.0 g, 316.4 mmol) was dissolved in water (260 mL). NaOH (2.0 eq., 632.9 mmol, 25.3 g) was added portion wise and the mixture was stirred at room temperature until dissolution of all the NaOH. The solution obtained was heated at 50 °C for 6 hours then was cooled down to room temperature then to 5 °C with an ice bath. Aqueous HCl (6 N) was added carefully (~100 mL) until pH = 4. The solution obtained was frozen to -80 °C then lyophilized. The white solid obtained was dissolved in aqueous HCl (6 N, 300 mL) and the mixture was heated at 110 °C for 3 hours. The solution obtained was diluted with water (300 mL), frozen to -80 °C then lyophilized to give (2S,3S)-2,4-diamino-3-hydroxy-butanoic acid (1-1) as a pale yellow solid (107 g, contains 2.0 eq. of NaCl, purity >90%). LC-MS (0.7 mL/min; 100:0 to 90:10 of water (0.1% TFA)/AcCN in 10 min): Rt = 2.30 min, [M+H]+ = 135. 1H NMR (D2O, 600 MHz, mixture of 2 diastereomers 70:30):δ (ppm) 3.22 (dd, J = 10.2 and 13.2 Hz, 0.3 H), 3.34- 3.47 (m, 1.7H), 4.08 (d, J = 4.8 Hz, 0.3H), 4.24 (d, J = 3.0 Hz, 0.7H), 4.46 (td, J = 3.0 and 10.2 Hz, 0.7H), 4.48-4.52 (m, 0.3H). 13C NMR (D2O, 150 MHz, mixture of 2 diastereomers): δ (ppm) 42.65, 43.38, 57.70, 66.92, 67.45, 170.45. Marfey’s analysis: 2S,3S/2R,3S = 73:27 (2S,3S: Rt = 96.01 min, 73%; 2R,3S: Rt = 97.84 min, 27%). Step 2: (2S,3S)-2,4-diamino-3-hydroxy-butanoic acid (1-1) (53 g, ~160 mmol) was put in a 2 L round bottom flask and dissolved in water (250 mL). NaOH (3.0 eq., 480 mmol, 19.0 g) was added portion wise (slightly exothermic). The mixture was stirred until dissolution of the solids then a solution of CuSO4.5H2O (0.5 eq., 80 mmol, 20.0 g) in water (125 mL) was added slowly. The dark blue solution obtained was put in an oil bath at room temperature. The system was heated at 110 °C for 30 minutes then was cooled down slowly to room temperature for 4 hours. A solution of Boc2O (2.0 eq., 320 mmol, 52.0 g) in dioxane (275 mL) was added and the reaction stirred at room temperature for 70 hours. A solution of Boc2O (0.5 eq., 80 mmol, 13.0 g) in dioxane (60 mL) was added slowly and the mixture was stirred at room temperature for 24 hours. The suspension obtained was filtered. The pale blue solid obtained was rinsed with water (~700 mL), Et2O (~300 mL) then dried to give ((2S,3S)-2-amino-4-(tert-butoxycarbonylamino)-3- hydroxy-butanoic acid)2Cu (1-2) as a pale blue solid (17.1 g, 40% yield over 2 steps). The product was used as a crude in the next step without further purification. Step 3: ((2S,3S)-2-amino-4-(tert-butoxycarbonylamino)-3-hydroxy-butanoic acid)2Cu (1- 2) (17.1 g, 32.0 mmol) was suspended in water (300 mL). A solution of Na2EDTA (1.5 eq., 48.0 mmol, 15.9 g) and NaOH (3.0 eq., 96.0 mmol, 3.84 g) in water (300 mL) was added. The mixture was stirred at room temperature for 4 hours until full dissolution of the suspension. The solution obtained was cooled down in an ice bath then a solution of FmocOSu (2.5 eq., 80.0 mmol, 35.7 g) in dioxane (500 mL) was added slowly. At the end of the addition, Na2CO3 (2.5 eq., 80.0 mmol, 8.5 g) was added and the mixture was risen to room temperature then stirred at room temperature for 18 hours. The limpid blue solution obtained was washed with Et2O (4*200 mL) then cooled down in an ice bath. Aqueous 1 N HCl was added slowly until pH = 3-4 (~250 mL). This aqueous phase was extracted with EtOAc (5*200 mL). Organic phases were combined, washed with brine (2*150 mL), dried over MgSO4, filtered and concentrated down to give pale yellow oil. Acetonitrile (200 mL) was added and the mixture stirred at room temperature for 70 hours. The suspension was filtered, the solid rinsed with AcCN (100 mL) then dried to give (2S,3S)-4-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3- hydroxy-butanoic acid (1-3) as white powder (29.1 g, quant. yield, 95% purity by LC-MS (5% of DBF, no diastereomer observed)). LC-MS (0.7 mL/min; 100:0 to 70:30 of water (0.1% TFA)/AcCN in 15 min): Rt = 13.96 min, 95% (254 nm), [M+H-Boc]+ = 357. 1H NMR(DMSO-d6, 600 MHz, 343 K): δ (ppm) 1.39 (s, 9H), 3.00-3.04 (m, 1H), 3.12-3.20 (m, 1H), 3.85-3.88 (m, 1H), 4.00-4.13 (m, 1H), 4.22-4.25 (m, 1H), 4.28-4.31 (m, 2H), 6.61 (br s, 0.8H), 7.33 (t, J = 7.2 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.87 (d, J = 7.2 Hz, 2H). 13C NMR (DMSO-d6, 150 MHz, 343 K) : δ (ppm) 27.93, 42.88, 46.48, 57.26, 65.69, 69.96, 77.56, 119.65, 124.87, 126.70, 127.24, 140.40, 143.50, 143.54, 151.30, 155.41, 155.65, 171.06. Marfey’s analysis : Rt = 95.60 min (2S, 3S), 100% (340 nm), [M+H]+ = 695. Step 4: (2S,3S)-4-(tert-butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-3-hydroxy-butanoic acid (1-3) (29.1 g, 63.6 mmol) was suspended in a mixture of acetone and 2,2-dimethoxypropane (1:1, 480 mL). The suspension was cooled down with an ice bath then BF3.OEt2 (catalytic, 900 µL) was added drop wise. The reaction was stirred in the melting ice bath until obtaining a limpid orange/brown solution (about 2.5 hours, completion of the reaction was checked by LC- MS). An aqueous saturated solution of NaHCO3 (200 mL), AcOEt (400 mL) then water (300 mL) were added and the phases were separated. The aqueous phase was extracted with AcOEt (2*200 mL). The organic phases were combined, washed with aqueous 0.1 N HCl (200 mL), brine (200 mL), dried over MgSO4, filtered and concentrated down. The pale yellow oil obtained was dissolved in Et2O (100 mL), the solution was cooled down in an ice bath then hexane (400 mL) was added. Formation of a suspension was observed while adding hexane. At the end of the addition a sticky solid was observed at the bottom of the flask. Et2O was added at room temperature and the mixture was triturated to obtain a white solid which was triturated for 18 hours. The suspension obtained was filtered off to give (2S)-2-[(5S)-3-tert-butoxycarbonyl-2,2- dimethyl-oxazolidin-5-yl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (1-4) as white powder (22.9 g, 73% yield, 96% purity by LC-MS (2% of DBF and 1.5% of starting material were observed, no diastereomer was observed)). LC-MS (0.7 mL/min; 100:0 to 70:30 of water (0.1% TFA)/AcCN in 15 min): Rt = 19.66 min, 96% (254 nm), [M+H-Boc- CH(CH3)2]+ = 357. 1H NMR (DMSO-d6, 600 MHz, 343 K) : δ (ppm) 1.43 (s, 12H), 1.47 (s, 3H), 3.36-3.41 (m, 1H), 3.54-3.59 (m, 1H), 4.22-4.25 (m, 2H), 4.30-4.33 (m, 2H), 4.38 (br s, 1H), 7.30-7.34 (m, 2H), 7.39-7.44 (m, 2H), 7.58 (br s, 1H), 7.69-7.72 (m, 2H), 7.87 (d, J = 7.8, 2H). 13C NMR (DMSO-d6, 150 MHz, 343 K) : δ (ppm) 27.78, 46.48, 46.84, 65.72, 72.81, 78.86, 92.94, 119.65, 124.83, 126.66, 127.25, 140.41, 143.48, 151.04, 155.51, 170.37. The regioselectivity of protections was determined using HMBC and HSQC analyses. A clear signal was observed in HMBC between the CHα (4.25 ppm) and the CO of the Fmoc protecting the amine in the alpha position (155.5 ppm) proving the regioselectivity of the protections. Marfey’s analysis : Rt = 96.19 min (2S, 3S), 100% (340 nm), [M+H]+ = 695. Example 2: Synthesis of non-commercial amino-acid building blocks: lysine- dehydroarginine dipeptide. Lysine-dehydroarginine dipeptide building block was prepared in accordance with the procedures described in Schmidt, U. and Wild, J. Ang. Chem. Int. Ed.1984, 23, 991 and applied in Berwe, M. et al., Org. Process Res. Dev.2011, 15, 1348; and Freeman, N. S. et al., J. Org. Chem.2011, 76, 3078. Scheme 1 is representative of the synthesis. Scheme 1: Synthesis of lysine-dehydroarginine dipeptide building block
Figure imgf000074_0001
Step 1: A solution of benzyl carbamate (90.0 g, 0.59 mol, 1.0 eq.) and monohydrate dihydroxyacetic acid 1 (60.3 g, 1.1 mol, 1.1 eq.) in toluene (840 mL) was introduced in a 2 L flask and the solution was heated at 40°C for 1.5 hours. Half of the solvent was concentrated down under reduce pressure. Toluene (540 mL) was added and half of the solvent was concentrated down under reduce pressure. Toluene (540 mL) was added and the reaction was stirred at 40°C for 2 hours and then cooled down to 20°C. The white solid was filtered, rinsed with toluene and dried under vacuum. The expected compound 2 of Scheme 1 (133.0 g, quant. yield, 95% purity (1H NMR)) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) : δ (ppm) : 5.05 (s, 2H), 5.21 (d, J = 8.8 Hz, 1H), 6.62-6.80 (br s, 1H), 7.26-7.44 (m, 5H), 8.14 (d, J = 8.8 Hz, 1H), 12.10-13.60 (br s, 1H). Step 2: Step 2-1: 2-(((benzyloxy)carbonyl)amino)-2-hydroxyacetic acid 2 (133.0 g, 0.59 mol, 1.0 eq.) was diluted in methanol (480 mL). Trimethyl orthoformate (TMOF, 130.2 mL, 1.11 mol, 2.0 eq.) and hydrochloric acid in methanol (1.25 M, 24 mL, 0.03 mol, 0.05 eq.) were added successively. The mixture was stirred at 56 °C for 40 min. Solvent was concentrated down under reduce pressure and 600 mL of Et2O were added. If necessary starting material was filtered off. The filtrate was concentrated down and dried under reduce pressure. The expected intermediate (149.6 g) was obtained as a white solid. Purity was assessed by NMR, and was >95%. 1H NMR (400 MHz, DMSO- d6) δ (ppm) : 3.26 (s, 3H), 3.66 (s, 3H), 5.08 (s, 2H), 5.16 (d, J = 9,2 Hz, 1H), 7.28-7.44 (m, 5H), 8.49 (d, J = 8.8 Hz, 1H). Step 2-2: A 2 L three-necked round bottom flask equipped with a dropping funnel and a condenser was dried by three heating+vacuum/argon cycles. The previous intermediate (149.5 g, 0.59 mol, 1.0 eq.) was introduced under argon followed by anhydrous toluene (720 mL). Three drops of concentrated sulfuric acid were added. Phosphorus trichloride (80 mL, 0.69 mol, 1.2 eq.) was introduced in the dropping funnel. The mixture was heated at 75°C and PCl3 was added over 1 h at this temperature. At the end of the addition, the mixture was stirred at 75°C for 13 h. After cooling down to room temperature, the solid was filtered off and the filtrate was concentrated down under vacuum to remove excess of PCl3. The crude mixture was diluted in 720 mL of anhydrous toluene under argon. Triethyl phosphite (100 mL, 0.65 mol, 1.1 eq.) was then added and the mixture was stirred at 75°C for 2 h and then at 90°C for 30 min. The reaction mixture was cooled down to room temperature; solvent and excess of triethyl phosphite were removed under vacuum. The crude mixture was dissolved in EtOAc. Organic phase was washed twice with saturated Na2CO3, dried over MgSO4, filtered and concentrated down under vacuum. The crude mixture was precipitated in Et2O (1 h at 5 °C). The suspension was filtered off and the solid was dried under vacuum to give compound 3 of Scheme 1 as a white solid (165.1 g, 70% yield over 2 steps, 90% purity (1H NMR)). 1H NMR (400 MHz, CDCl3) : δ (ppm) 3.74-3.87 (m, 9H), 4.93 (dd, J = 22.4 and 9.6 Hz, 1H), 5.13-5.14 (m, 2H), 5.60 (d, J = 8.4 Hz, 1H), 7.28-7.43 (m, 5H). 31P NMR (400 MHz, CDCl3) : δ (ppm) 18.45. Step 3: Methyl 2-(((benzyloxy)carbonyl)amino)-2-(diethoxyphosphoryl)acetate 3 (20.1 g, 55.7 mmol, 1.0 eq.) was dissolved in 600 mL of EtOH.10% palladium on charcoal (2.0 g, cat.) was added and the reaction mixture was stirred under H2 atmosphere for 8 to 14 h. Deprotection was monitored by 31P NMR. After completion the mixture was filtered through celite. The filtrate was concentrated down under vacuum. The crude was dissolved in DCM and concentrated down under reduce pressure. The operation was repeated three times in order to remove traces of EtOH. The free amine was used directly in the next step. The crude product was dissolved in 60 mL of DCM. Fmoc-Lys(Boc)-OH (26.0 g, 55.7 mmol, 1.0 eq.) was added followed by PyBOP (29.0 g, 55.7 mmol, 1.0 eq.). The reaction mixture was cooled down to 0°C before adding diisopropylethylamine drop wise (28.0 mL, 160.7 mmol, 3.0 eq.). The reaction mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with EtOAc. The organic phase was washed twice with a 5% KHSO4 aqueous solution, twice with a saturated aqueous solution of NaHCO3 and once with brine. Organic phases were dried over MgSO4, filtered and concentrated down under vacuum. The crude product was purified by column chromatography over silica (40-63 µm, pore 60 Å, 1.4 kg, 100% EtOAc). In order to remove traces of EtOAc, the crude mixture was dissolved in CHCl3 and concentrated under reduce pressure to give compound 4 of Scheme 1 as a white foam (33.4 g, 83% yield, 95% purity (1H NMR)). 1H NMR (400 MHz, DMSO-d6) : δ (ppm) 1.15-1.62 (m, 21 H), 2.87-2.90 (m, 2H), 3.70 (d, J = 4.8 Hz, 3H), 4.03-4.25 (m, 8H), 4.90-5.15 (m, 1H), 6.75 (br s, 1H), 7.32 (t, J = 7.2 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.50-7.58 (m, 1H), 7.72 (d, J = 7.2 Hz, 2H), 7.89 (d, J = 7.6 Hz, 2H), 8.77-8.85 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 16.08, 22.76, 28.24, 29.18, 31.54, 31.73, 46.60, 49.43, 50.88, 52.67, 52.71, 54.16, 63.06, 63.11, 63.28, 63.35, 65.63, 77.32, 120.07, 125.26, 127.03, 127.60, 140.66, 143.74, 143.83, 155.52, 155.89, 167.11, 172.71. 31P NMR (400 MHz, CDCl3) : δ (ppm) 18.25. Step 4: 1.3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (50.0 g, 172.1 mmol, 1.0 eq.) was dissolved in 400 mL of DMF. Aminopropan-3-ol (52.5 mL, 688.0 mmol, 4.0 eq.) was added drop wise followed by dimethylaminopyridine (2.1 g, 17.2 mmol, 0.1 eq.). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was dissolved in 4 L of Et2O. The organic phase was washed with 800 mL of aqueous 0.1 M AcOH, 800 mL of saturated aqueous NaHCO3, 800 mL of H2O and 800 mL of brine. The organic phase was dried over MgSO4, filtered and concentrated down under vacuum. Compound 5 of Scheme 1 was obtained as a white solid (51.1 g, quant. yield, 95% purity (1H NMR)). 1H NMR (250 MHz, CDCl3): δ (ppm) 1.47 (s, 9H), 1.50 (s, 9H), 1.64-1.76 (m, 2H), 3.52-3.63 (m, 4H), 8.42-8.61 (br s, 1H), 11.44 (s, 1H). Step 5 and 5’: 1.2 eq. of the aldehyde was prepared for the Horner-Wadsworth-Emmons (1.0 eq. of compound 4 of Scheme 1). Step 5: In a 2 L flask, compound 5 of Scheme 1 (20.5 g, 64.6 mmol, 1.2 eq.) was dissolved in DCM (stabilized over amylene, 410 mL). Pyridine (30.9 mL, 465.1 mmol, 7.2 eq.) was added followed by Dess-Martin periodinane (29.7 g, 70 mmol, 1.3 eq.). The reaction was stirred at room temperature for 3 h. A saturated aqueous solution of Na2CO3 (600 ml) and 300 mL of Et2O were added. The mixture was stirred at room temperature for 10 min. The suspension obtained was filtered through celite.1.1 L of Et2O was added and the organic phase was washed with water (3 x 1 L). The organic phase was dried over MgSO4, filtered and concentrated down under vacuum. The aldehyde 6 was obtained as yellow oil (22.1 g, quant.) and used directly in the next step without purification.1H NMR (400 MHz, CDCl3) : δ (ppm) 1.48 (s, 9H), 1.50 (s, 9H), 2.79 (t, J = 6.0 Hz, 2H), 3.74 (q, J = 6.4 Hz, 2H), 8.56-8.64 (m, 1H), 9.83 (s, 1H), 11.44 (s, 1H). Step 5’: In a 500 mL flask under argon compound 4 of Scheme 1 (36.1 g, 53.4 mmol, 1.0 eq.) was dissolved with 300 mL of anhydrous CH3CN. Dry lithium chloride (2.73 g, 64.1 mmol, 1.2 eq.) was added and the reaction mixture was stirred at room temperature for 30 min. The aldehyde 6 of Scheme 1 (22.1 g) was dissolved in 40 mL of anhydrous CH3CN. The solution was added to the reaction mixture then diisopropylethyl-amine (10.83 ml, 64.1 mmol, 1.2 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 3 to 4 days. The reaction was monitored by 31P NMR. After completion, 1 L of EtOAc was added and the organic phase was washed with 100 mL of H2O. The organic phase was dried over MgSO4, filtered and concentrated down under vacuum. After HPLC analysis, the Z/E ratio was determined as 86/14. The crude product was purified by column chromatography on silica (40-63 µm, pore 60 Å, 1.5 kg, 3/2 petroleum ether/EtOAc ≈ 2 L, then 1/1 petroleum ether/EtOAc ≈ 3 L then 2/3 petroleum ether/EtOAc).3 fractions were obtained, the first one containing the alkene E, the second a mixture of Z and E alkene and the third the alkene Z with a good purity (> 95% by HPLC). The second fraction was purified again by column chromatography. After combining the various fractions, compound (Z)-7 of Scheme 1 was obtained as a white foam (30.0 g, 71% yield, 90% purity (1H NMR)). (Z)-7: 1H NMR (400 MHz, DMSO-d6) : δ (ppm) 1.23-1.46 (m, 31H), 1.53-1.68 (m, 2H), 2.31-2.36 (m, 2H), 2.90 (br s, 2H), 3.37 (br s, 2H), 3.64 (s, 3H), 4.08-4.26 (m, 4H), 6.41-6.43 (m, 1H), 6.77-6.79 (m, 1H), 7.31- 7.34 (m, 2H), 7.39-7.42 (m, 2H), 7.52-7.54 (m, 1H), 7.70-7.73 (m, 2H), 7.87-7.90 (m, 2H), 8.37-8.41 (m, 1H), 9.32 (br s, 1H), 11.47 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 22.74, 27.34, 27.55, 27.95, 28.25, 29.23, 31.51, 46.63, 51.87, 54.42, 65.61, 77.30, 78.12, 82.80, 120.06, 125.28, 127.01, 127.59, 128.04, 132.93, 140.67, 143.73, 143.84, 151.85, 155.27, 155.51, 155.93, 163.04, 164.39, 171.31. NOESY experiment (2D NMR) showed a clear interaction between the CH2 vicinal to the vinylic CH and the NH of the amide bond proving the Z stereochemistry of the double bond. To confirm it, NOESY experiment was done on the other isomer and showed a clear interaction between the vinylic CH and the NH of the amide bond proving the E stereochemistry of the double bond. Step 6: 1 L of 0.8 M CaCl2 solution in a mixture of iPrOH/H2O (7/3) was prepared. Compound (Z)-7 of Scheme 1 (30.0 g, 35.8 mmol, 1.0 eq.) was dissolved in 580 mL of the 0.8 M CaCl2 solution. The mixture was stirred at room temperature for 20 min then was cooled down to 0°C and aqueous NaOH (1 M, 71.6 mL, 71.6 mmol, 2.0 eq.) was added dropwise. The mixture was stirred at room temperature for 16 h then EtOAc and saturated NH4Cl aqueous solution were added. Aqueous phase was extracted twice with EtOAc. The combined organic phases were washed with brine. Organic phase was concentrated down under vacuum. The crude mixture was purified by column chromatography on silica (40-63 µm, pore 60 Å, 1 kg, crude absorbed on silica, 100% DCM, then MeOH/DCM 2/98, 4/96, 6/94, 8/9210/90). Compound (Z)-8 of Scheme 1 was obtained as a white foam (17.0 g, 55% yield, >95% purity (LC-MS)). 1H NMR (400 MHz, DMSO-d6) : δ (ppm) 1.23-1.46 (m, 31H), 1.55-1.71 (m, 2H), 2.22-2.26 (m, 2H), 2.90 (br s, 2H), 3.37 (br s, 2H), 4.08-4.29 (m, 4H), 6.26-6.34 (m, 1H), 6.75-6.78 (m, 1H), 7.29- 7.33 (m, 2H), 7.38-7.42 (m, 2H), 7.57-7.60 (m, 1H), 7.70-7.74 (m, 2H), 7.84-7.89 (m, 2H), 8.32-8.34 (m, 1H), 8.94 (br s, 1H), 11.49 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 22.91, 27.54, 27.95, 28.23, 29.20, 31.60, 46.64, 54.92, 65.66, 77.26, 78.09, 82.77, 120.03, 121.34, 125.28, 127.01, 127.24, 127.56, 128.87, 140.66, 143.72, 143.84, 151.94, 155.22, 155.50, 155.99, 163.04, 170.19. LC-MS (0.7 mL/min; 60:40 to 10:90 of water (0.1% TFA)/AcCN in 25 min): Rt = 17.30 min, 90% (254 nm), [M+H]+ = 823. The same reaction was done starting from (E)-7 to give the corresponding (E)-8: 1H NMR (400 MHz, DMSO-d6) : δ (ppm) 1.23-1.46 (m, 31H), 1.55-1.71 (m, 2H), 2.60-2.68 (m, 2H), 2.90 (br s, 2H), 3.37 (br s, 2H), 3.96-4.02 (br s, 1H), 4.08-4.29 (m, 3H), 6.12-6.18 (m, 1H), 6.75-6.78 (m, 1H), 7.29-7.33 (m, 2H), 7.38-7.42 (m, 2H), 7.60-7.68 (m, 1H), 7.70-7.74 (m, 2H), 7.84-7.89 (m, 2H), 8.32-8.34 (m, 1H), 9.20 (br s, 1H), 11.49 (br s, 1H). LC-MS (0.7 mL/min; 60:40 to 10:90 of water (0.1% TFA)/AcCN in 25 min): Rt = 20.97 min, 96% (254 nm), [M+H]+ = 823. Example 3: Synthesis of non-commercial building-blocks for C-terminal derivatization Example 3.1
Figure imgf000079_0001
Step 1: Under inert atmosphere and magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (1.19 g, 6.65 mmol, 1.0 eq.) was dissolved in 12 mL of anhydrous 2-propanol. The mixture was heated at 82 °C then a solution of benzyl N- (oxiran-2-ylmethyl)carbamate (1.45 g, 6.65 mmol, 1.0 eq.) in 5 mL of anhydrous 2- propanol was added dropwise. The reaction mixture was stirred 1 h at 82 °C, cooled down at room temperature and concentrated under vacuum. tert-Butyl N-[3-[[3- (benzyloxycarbonylamino)-2-hydroxypropyl] amino]propyl]carbamate was obtained (3.2 g). Step 2: To a solution of tert-butyl N-[3-[[3-(benzyloxycarbonylamino)-2-hydroxy- propyl]amino]propyl]carbamate (3.20 g, 6.29 mmol, 1.0 eq.) in 63 mL of methanol, at room temperature, was added tert-butoxycarbonyl tert-butyl carbonate (2.06 g, 9.44 mmol, 1.5 eq.) and N-ethyl-N-(propan-2-yl)propan-2-amine (2.2 mL, 12.6 mmol, 2.0 eq.). The reaction mixture was stirred 1 h at 65 °C then concentrated under vacuum. The residue was taken up with water and 6 mL of 0.5 M citric acid. This aqueous phase was extracted with EtOAc. The organic phases were combined and washed with saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by flash column chromatography (EtOAc/heptane 30/70 to 50/50). tert-Butyl N-[3-(benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate was obtained as a gum (1.98 g). Step 3: At room temperature, under inert atmosphere and magnetic stirring, tert-butyl N- [3-(benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)propyl] carbamate (1.98 g, 3.70 mmol, 1.0 eq.) was dissolved in 75 mL of methanol. Palladium (787 mg, 0.740 mmol, 2.0 eq.) and cyclohexene (10 mL, 92.5 mmol, 25 eq.) were added. The mixture was stirred for 2 h at 70 °C then filtered through a Millipore filter. The remaining filtrate was concentrated under vacuum to give tert-butyl N-(3-amino-2- hydroxy-propyl)-N-[3-(tertbutoxycarbonylamino)propyl]carbamate (1.6 g). Example 3.2
Figure imgf000080_0001
Step 1:To (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (10 g, 68.5 mmol, 1.0 eq.) in CH2Cl2 (40 mL) at 0 °C, was added prop-2-en-1-yl carbonochloridate (7.7 mL, 71.9 mmol, 1.05 eq.) then dropwise N,N-diethylethanamine (20 mL, 0.144 mol, 2.1 eq.) (slightly exothermic). The reaction was stirred 2 h at room temperature then concentrated. The residue was taken up in EtOAc and washed with water and brine. The organic phase was dried over Na2SO4 then concentrated under vacuum. Allyl N-[(2S)-3-chloro-2- hydroxy-propyl]carbamate was obtained as a colorless liquid (11 g). Step 2: To allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (11.70 g, 60.4 mmol, 1.0 eq.) in MeOH (90 mL) was added dropwise 4.3 M sodium methanolate (22 mL, 96.7 mmol, 1.6 eq.). The reaction was stirred 2 h at room temperature then partially concentrated. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over Na2SO4 and concentrated under vacuum. The crude was purified by flash column chromatography using CH2Cl2/MeOH (98/2) as eluent. Allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a colorless liquid (4.8 g). Step 3: To a solution of allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate (744 mg, 4.73 mmol, 1.0 eq.) in 12 mL of 2-propanol, at 70 °C, was added dropwise over 15 min tert-butyl N- [(2S)-3-amino-2-hydroxypropyl]carbamate (900 mg, 4.73 mmol, 1.0 eq.). The mixture was stirred for 1 h at 70 °C then concentrated under vacuum. tert-Butyl N-[(2S)-3-[[(2R)- 3-(allyloxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained (1.6 g). Step 4: At 5 °C and under magnetic stirring, tert-butyl N-[(2S)-3-[[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]amino]-2-hydroxy-propyl]carbamate (1.60 g, 4.61 mmol, 1.0 eq.) was dissolved in 30 mL of CH2Cl2. Prop-2-en-1-yl carbonochloridate (0.54 mL, 5.07 mmol, 1.1 eq.) and N-ethyl-N-isopropyl-propan-2-amine (1.4 mL, 7.83 mmol, 1.7 eq.) were added. The reaction mixture was risen at room temperature, stirred overnight and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/methanol 95/5). Allyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2- hydroxy-propyl]carbamate was obtained as a colourless oil (1.48 g). Step 5: To allyl-N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.48 g, 3.43 mmol, 1.0 eq.) in CH2Cl2 (60 mL) at 0 °C, was added dropwise 2,2,2-trifluoroacetic acid (5.5 mL, 71.9 mmol, 21 eq.). The mixture was risen to room temperature, stirred 3 h then concentrated under vacuum. The crude product was dried overnight under vacuum. Allyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-amino-2-hydroxy- propyl]carbamate was obtained (1.5 g). Example 3.3
Figure imgf000081_0001
Step 1: To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (15.00 g, 0.103 mol, 1.0 eq.) in 60 mL of CH2Cl2 at 5 °C, was added N,N-diethylethanamine (36 mL, 0.257 mol , 2.5 eq.) then prop-2-en-1-yl carbonochloridate (12 mL, 0.108 mol, 1.05 eq.). The mixture was stirred for 20 min at 5 °C then risen at room temperature. The mixture was stirred for additional 1 h then washed with water and brine. The organic layers were combined, dried over MgSO4 and concentrated under vacuum. Allyl N-[(2S)-3-chloro-2- hydroxy-propyl]carbamate was obtained (10.6 g). Step 2: To a solution of allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (10.6 g, 54.74 mmol, 1.0 eq.) in 100 mL of MeOH was added dropwise sodium methanolate (20 mL, 87.6 mmol, 1.6 eq.). The reaction mixture was stirred for 1.5 h then partially concentrated under vacuum. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by flash column chromatography. Allyl N-[[(2S)-oxiran-2- yl]methyl]carbamate was obtained (4.97 g). [α]D = -10.9° (c = 10.2 mg/1.05 mL; MeOH) Step 3: To a solution of tert-butyl N-[(2R)-3-amino-2-hydroxypropyl]carbamate (1.5 g, 7.49 mmol, 1.0 eq.) in 20 mL of 2-propanol at 70 °C, was added dropwise over 25 min a solution of allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate (1.0 g, 6.36 mmol, 0.85 eq.) in 4 mL of 2-propanol. The mixture was stirred for 1 h at 70 °C. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-methanol-7 N NH3 in MeOH 90-9-1) 100/0 to 0/100). tert-Butyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained (1.47 g). Step 4: To a solution of tert-butyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]-2-hydroxy-propyl]carbamate (1.47 g, 4.15 mmol, 1.0 eq.) in 43 mL of ethanol, was added by portion tert-butoxycarbonyl tert-butyl carbonate (1.02 g, 4.51 mmol, 1.01 eq.). The mixture was stirred for 1.5 h at room temperature then concentrated under vacuum. tert-butyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2S)-3- (tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (1.73 g). Step 5: Under inert atmosphere and magnetic stirring, tert-butyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2S)-3-(tert-butoxycarbonylamino)-2- hydroxy-propyl]carbamate (1.73 g, 3.83 mmol, 1.0 eq.) was dissolved in 32 mL of anhydrous CH2Cl2. Phenylsilane (2.4 mL, 19.1 mmol, 5.0 eq.) was added. The mixture was stirred for 15 min under argon bubbling, then palladium-tetrakis(triphenylphosphine) (221 mg, 0.191 mmol, 0.05 eq.) was added. The mixture was stirred for 1.5 h at room temperature then concentrated under vacuum. The product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-MeOH-7N NH3 in MeOH 90-9-1) 100/0 to 0/100 then CH2Cl2-MeOH-7N NH3 in MeOH 80-19-1). tert-Butyl N-[(2S)-3-amino-2- hydroxy-propyl]-N-[(2S)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a beige solid (888 mg). Example 3.4
Figure imgf000082_0001
Step 1: Under inert atmosphere and magnetic stirring, (2R)-1-amino-3-chloropropan-2- ol hydrochloride (39.5 g, 270 mmol, 1.0 eq.) was dissolved in 778 mL of CH2Cl2. Triethylamine (151 mL, 1.08 mol, 4.0 eq.) was added, followed by a dropwise addition of prop-2-en-1-yl carbonochloridate (58 mL, 540 mmol, 2.0 eq.). The reaction mixture was stirred for 1 h at room temperature. Water was added to the reaction mixture. The phases were separated and the organic layer was washed with brine. The phases were separated and the organic phase was dried over MgSO4, filtered and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min). Allyl N-[(2R)-3-chloro-2- hydroxy-propyl]carbamate was obtained as a yellow oil (33.6 g). Step 2: Under inert atmosphere and magnetic stirring, allyl N-[(2R)-3-chloro-2-hydroxy- propyl]carbamate (33.6 g, 174 mmol, 1.0 eq.) was dissolved in 850 mL of methanol. Sodium methanolate (79 mL, 347 mmol, 2.0 eq.) was added dropwise. The reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated to dryness and the residue was taken up in EtOAc (500 ml). The organic phase was washed with 250 mL of saturated NH4Cl then 150 mL of brine, dried over MgSO4, filtered and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min) to give two batches of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate. The first batch was obtained as a yellow oil with a purity of 98% (9.55 g, [α]D = +0.10° (c = 10 mg/mL; MeOH)) and the second batch was obtained as a colourless oil with a purity of 95% (3.05 g). Step 3: To a solution of tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]carbamate (2.5 g, 12.5 mmol, 1.0 eq.) in 2-propanol (34 mL), stirred at 70 °C, was added dropwise over 25 min a solution of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate (2.07 g, 12.5 mmol, 1.0 eq.) in 2-propanol (6 mL). The mixture was stirred for 1 h at 70 °C. The mixture was concentrated under vacuum and purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-MeOH-(NH37N in MeOH) 90-9-1) 100/0 to 0/100). Tert-butyl N-[(2S)-3- [[(2S)-3-(allyloxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained as a gum (738 mg) and allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2- hydroxypropyl]-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy- propyl]carbamate was obtained as a white solid (2.78 g; [α]D = -13.7° (c = 0.42 mg/mL; MeOH)). Step 4: To a solution of tert-butyl N-[(2S)-3-[[(2S)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]-2-hydroxy-propyl]carbamate (2.7 g, 7.84 mmol, 1.0 eq.) in ethanol (80 mL), stirred at room temperature, was added, by portion, tert-butoxycarbonyl tert-butyl carbonate (1.9 g, 8.44 mmol, 1.1 eq.). The mixture was stirred for 1h30 at room temperature then concentrated under vacuum. Tert-butyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxy carbonylamino)-2- hydroxy-propyl]carbamate was obtained (3.49 g). Step 5: Under inert atmosphere and magnetic stirring, tert-butyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2- hydroxy-propyl]carbamate (3.49 g, 7.64 mmol, 1.0 eq.) was dissolved in 64 mL of anhydrous CH2Cl2. Phenylsilane (4.9 mL, 38.2 mmol, 5.0 eq.) was added and the mixture was stirred 15 min under argon bubbling. Then, palladium- tetrakis(triphenylphosphine) (442 mg, 0.382 mmol, 0.05 eq.) was added. The reaction mixture was stirred for 6 h. Additional phenylsilane (2.4 mL) and palladium- tetrakis(triphenylphosphine) (220 mg) were added to the reaction. The mixture was stirred overnight then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2 then CH2Cl2/MeOH 95/5 then CH2Cl2/MeOH/(NH3 7M in MeOH) 90/9/1 then CH2Cl2/MeOH/(NH3 7M in MeOH) 80/19/1). tert-Butyl N-[(2R)-3-amino-2-hydroxy-propyl]-N-[(2R)-3- (tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a brown solid foam (1.82 g). Step 6: Under inert atmosphere and magnetic stirring, tert-butyl N-[(2R)-3-amino-2- hydroxy-propyl]-N-[(2R)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.82 g, 4.91 mmol, 1.0 eq.) was dissolved in 7 mL of anhydrous pyridine and 21 mL of anhydrous CH2Cl2. Fmoc-Lys(Boc)-OH (2.37 g, 4.91 mmol, 1.0 eq.) was added. The mixture was cooled at 0-5 °C then, 3-(ethyliminomethyleneamino)-N,N-dimethylpropan- 1-amine hydrochloride (1.04 g, 5.40 mmol, 1.1 eq.) was added. The reaction mixture was risen to room temperature and stirred overnight then concentrated to dryness. The residue was taken up in EtOAc and a small volume of MeOH. The organic phase was washed with water acidified with citric acid, saturated NaHCO3 then brine. The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (heptane/(EtOAc-MeOH 9-1) 70/30 to 60/40). Tert-butyl N-[(2R)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9Hfluoren-9-ylmethoxycarbonylamino) hexanoyl]amino]-2-hydroxypropyl]-N-[(2R)-3- (tert-butoxycarbonylamino)-2-hydroxypropyl] carbamate was obtained as a white solid foam (2.9 g). Step 7: To a solution of tert-butyl N-[(2R)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxypropyl]-N-[(2R)-3- (tert-butoxycarbonylamino)-2-hydroxypropyl]carbamate (2.9 g, 3.49 mmol, 1.0 eq.) in THF (66 mL) was added N-ethylethanamine (9.2 mL, 88.8 mmol, 25 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/[CH2Cl2-MeOH- (NH4OH 7M in MeOH) 90-10-1] 100/0 to 0/100). Tert-butyl N-[(2R)-3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]-N-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a white solid foam (1.92 g). [α]D = -1.1° (c = 0.62 mg/mL; MeOH) Example 3.5
Figure imgf000085_0001
Step 1: Allyl N-[(2R)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a yellow oil (33.6 g) following the step 1 described in example 3.4. Step 2: Two batches of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate were obtained as a yellow oil (9.55 g, [α]D = +0.10° (c = 10 mg/mL; MeOH)) and a colourless oil (3.05 g) following the step 2 described in example 3.4. Step 3: Allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxypropyl]-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained as a white solid (2.78 g; [α]D = -13.7° (c = 0.42 mg/mL; MeOH)) following the step 3 described in example 3.4. Step 4: To a solution of allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]amino]-2-hydroxy- propyl]carbamate (738 mg, 1.39 mmol, 1.0 eq.) in CH2Cl2 (14 mL) was added 2,2,2- trifluoroacetic acid (0.53 mL, 6.95 mmol, 5.0 eq.). The mixture was stirred overnight at room temperature. THF (5 mL) and 4 M hydrogen chloride in dioxane (7.0 mL, 28.0 mmol, 20 eq.) were added and the reaction mixture was stirred 3 days at room temperature. The reaction was not complete: additional 4 M hydrogen chloride in dioxane (7.0 mL, 28.0 mmol, 20 eq.) was added and the mixture was stirred overnight at room temperature. The mixture was co-evaporated with toluene to give allyl N-[(2R)-3-[[(2R)- 3-(allyloxycarbonylamino)-2-hydroxypropyl]-[(2R)-3-amino-2-hydroxy-propyl]amino]-2- hydroxypropyl]carbamate dihydrochloride (720 mg). Step 5: Under inert atmosphere, allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-[(2R)-3-amino-2-hydroxy-propyl]amino]-2-hydroxy-propyl]carbamate dihydrochloride (720 mg, 1.36 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (8 mL). Anhydrous pyridine (6 mL) was added followed by Fmoc-Lys(Boc)-OH (670 mg, 1.39 mmol, 1.02 eq.). The mixture was stirred at room temperature and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (286 mg, 1.49 mmol, 1.1 eq.) was added. The reaction mixture was stirred for 5 h at room temperature. The expected product was not detected. N-ethyl-N-isopropyl-propan-2-amine (1.2 mL, 6.79 mmol, 5.0 eq.), Fmoc-Lys(Boc)-OH (670 mg, 1.39 mmol, 1.02 eq.) and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine;hydrochloride (286 mg, 1.49 mmol, 1.1 eq.) were added. The mixture was stirred overnight at room temperature then concentrated (co-evaporation with toluene). The residue was taken up in a small volume of MeOH/EtOAc and washed with water, 50 mL citric acid, saturated NaHCO3 then brine. The organic phase was dried over MgSO4 and concentrated under vacuum. The crude product was purified through silica gel flash column chromatography (CH2Cl2/MeOH 100/0 to 95/5). Allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxypropyl]-[(2R)- 3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxypropyl]amino]-2-hydroxy- propyl]carbamate was obtained in mixture with Fmoc-Lys(Boc)-OH with a purity of 50% (1.09 g). Step 6: To a solution of allyl N-[(2R)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-[(2R)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]amino]-2-hydroxy- propyl]carbamate (1200 mg, 0.70 mmol, 1.0 eq.) in THF (25 mL) was added N- ethylethanamine (2.5 mL, 24.2 mmol, 34 eq.). The mixture was stirred overnight then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2 then [CH2Cl2/MeOH/(NH37M in MeOH) 90/9/1]. Allyl N-[(2R)- 3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxypropyl]-[(2R)-3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]amino]-2-hydroxy- propyl]carbamate was obtained as a gum (378 mg). Example 3.6
Figure imgf000086_0001
Step 1: To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (20 g, 0.14 mol, 1.0 eq.) in CH2Cl2 (100 mL) at -5 °C, were added N,N-diethylethanamine (40 mL, 0.29 mol, 2.1 eq.) and dropwise prop-2-en-1-yl carbonochloridate (15 mL, 0.14 mol, 1.05 eq.). The reaction was stirred for 2 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water and 1 M HCl. The organic phase was dried over Na2SO4, filtered then concentrated under vacuum. Allyl N- [(2S)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a colourless liquid (12.6 g). Step 2:To a solution of allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (12.6 g, 65.1 mmol, 1.0 eq.) in MeOH (97 mL) was added dropwise 4.3 M sodium methanolate (25% w/w, 24 mL, 0.104 mol, 1.6 eq.). The reaction was stirred for 2 h at room temperature then partially concentrated. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over Na2SO4, filtered then concentrated to dryness. The crude was then purified by flash column chromatography using CH2Cl2/MeOH (98/2). Allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a colorless liquid (5.1 g). Step 3: To allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate (1.0 g, 6.36 mmol, 1.0 eq.) in 2- propanol (15 mL) at 70 °C, was added dropwise (15 min) a solution of tert-butyl 3- (aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.33 g, 6.36 mmol, 1.0 eq.) in 2- propanol (2 mL). The reaction was stirred 45 min at 70 °C then concentrated under vacuum. Tert-butyl 3-[[[(2R)-3-(allyloxycarbonylamino)-2-hydroxypropyl]amino]methyl]- 3-hydroxy-azetidine-1-carboxylate was obtained (2.3 g). Step 4: To tert-butyl 3-[[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (2.2 g, 6.12 mmol, 1.0 eq) in ethanol (60 mL) was added tert-butoxycarbonyl tert-butyl carbonate (1.47 g, 6.73 mmol, 1.1 eq.). The reaction was stirred 2 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash chromatography (CH2Cl2/MeOH 94/6). Tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]- tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained as a colourless oil (1.75 g). [α]D = + 7.1° (c =0.21, MeOH) Step 5: Under inert atmosphere, to tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.25 g, 2.72 mmol, 1.0 eq.) in anhydrous CH2Cl2 (40 mL) were added phenylsilane (1.0 mL, 8.16 mmol, 3.0 eq.) and palladium-tetrakis(triphenylphosphine) (220 mg, 0.19 mmol, 0.07 eq.). The reaction was stirred 3 h at room temperature then concentrated to dryness. Tert-butyl 3-[[[(2S)-3-amino-2-hydroxy-propyl]-tert- butoxycarbonylamino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained (940 mg). Step 6: To tert-butyl 3-[[[(2S)-3-amino-2-hydroxy-propyl]-tert- butoxycarbonylamino]methyl]-3-hydroxy-azetidine-1-carboxylate (0.94 g, 2.50 mmol, 1.0 eq.) in CH2Cl2 (15 mL) and pyridine (8 mL) at 0 °C, were added Fmoc-Lys(Boc)-OH (1.21 g, 2.50 mmol, 1.0 eq.) then N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (388 mg, 2.50 mmol, 1.0 eq.). The reaction was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water. The organic phase was dried over Na2SO4 then concentrated to dryness. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 94/6). Tert-butyl 3-[[tert-butoxycarbonyl-[(2S)-3-[[(2S)-6-(tertbutoxycarbonylamino)-2- (9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2- hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained as a colorless oil (1.12 g). Step 7: To tert-butyl 3-[[tert-butoxycarbonyl-[(2S)-3-[[(2S)-6-(tert-butoxycarbonylamino)- 2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy- propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.12 g, 1.36 mmol, 1.0 eq.) in THF (30 mL) was added N-ethylethanamine (5.0 mL, 47.6 mmol, 35 eq.). The reaction mixture was stirred 4 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 90/10). Tert- butyl 3-[[[(2S)-3-[[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]-2- hydroxy-propyl]-tertbutoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained as a colorless oil (470 mg). [α]D = +6.25° (c=0.16, MeOH) Example 3.7
Figure imgf000088_0001
Step 1: Under inert atmosphere, at 0 °C and under magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (10.0 g, 55.7 mmol, 1.0 eq.) was dissolved in 100 mL of anhydrous CH2Cl2. N-ethyl-N-(propan-2-yl)propan-2-amine (21 mL, 0.12 mol, 2.2 eq.) was added followed by a dropwise addition (over 15 min) of prop-2-en-1-yl carbonochloridate (7.1 mL, 66.8 mmol, 1.2 eq.). The mixture was stirred for 1 h at room temperature. The mixture was washed with water (2x100 ml). The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The residue obtained was crystallized on cooling. The crude product was then purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 100% of EtOAc in 25 min). Tert-butyl N-[3- (allyloxycarbonylamino)propyl]carbamate was obtained (13.1 g). Step 2: Under inert atmosphere, at 0 °C and under magnetic stirring, tert-butyl N-[3- (allyloxycarbonylamino)propyl]carbamate (13.1 g, 50.7 mmol, 1.0 eq.) was dissolved in 128 mL of anhydrous 1,4-dioxane. A solution of 4 M HCl (127 mL, 0.51 mol, 10 eq.) was added dropwise in 30 min. The mixture was risen to room temperature, stirred overnight then concentrated under vacuum. Allyl N-(3-aminopropyl)carbamate hydrochloride was obtained (9.5 g). Step 3: Under inert atmosphere and magnetic stirring, (2R)-3-aminopropane-1,2-diol (24.08 g, 0.256 mol, 1.0 eq.) and N,N-diethylethanamine (52.6 mL, 384.6 mmol, 1.5 eq.) were dissolved in 723 mL of anhydrous MeOH. The mixture was cooled down to 0 °C then tert-butoxycarbonyl tert-butyl carbonate (83.94 g, 0.385 mol, 1.5 eq.) was added by portion. The resulting mixture was risen to room temperature, stirred 2 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min). Tert- butyl N-[(2R)-2,3-dihydroxypropyl]carbamate was obtained as a white solid (33.36 g). Step 4: Under inert atmosphere and magnetic stirring, tert-butyl N-[(2R)-2,3- dihydroxypropyl]carbamate (33.36 g, 0.174 mol, 1.0 eq.) was suspended in 200 mL of anhydrous pyridine. The mixture was cooled down to 0 °C and methanesulfonyl chloride (15 mL, 0.192 mol, 1.1 eq.) was added dropwise. The mixture was stirred at 0 °C for 10 min and was added dropwise to a solution of NaOH (20.94 g, 0.523 mol, 3.0 eq.) in water (200 mL) and DMSO (133 mL). The resulting solution was stirred for 10 min at 0 °C and was added to 2 L of iced water. A solution of 534 mL of heptane and 2.1 L of EtOAc was added and the phases were separated. The organic phase was washed with water then brine. The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 100 % of EtOAc in 30 min). Tert-butyl N-[[(2R)-oxiran-2- yl]methyl]carbamate was obtained as a white solid (17.42 g). [α]D = +0.061° (c = 12.5 mg/mL) Step 5: To allyl N-(3-aminopropyl)carbamate hydrochloride (3.37 g, 17.3 mmol, 1.0 eq.) in suspension in 30 mL of isopropanol, was added N-ethyl-N-(propan-2-yl)propan-2- amine (6.0 mL, 34.6 mmol, 2.0 eq.). The mixture was warmed to 70 °C and a solution of tert-butyl [(2R)-oxiran-2-ylmethyl]carbamate (3.00 g, 17.3 mmol, 1.0 eq.) in isopropanol (10 mL) was added dropwise. The resulting mixture was stirred for 1 h at 70 °C then concentrated under reduced pressure. The residue was taken up with CH2Cl2 (75 mL) then N,N-dimethylpyridin-4-amine (0.21 g, 1.73 mmol, 0.1 eq.) and tert-butoxycarbonyl tert-butyl carbonate (5.67 g, 26.0 mmol, 1.5 eq.) were added. The mixture was stirred for 12 h at room temperature under N2 then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 100% EtOAc in 20 min). Tert-butyl N-[3-(allyloxycarbonylamino)propyl]-N-[(2R)-3- (tertbutoxycarbonylamino)-2-hydroxypropyl]carbamate was obtained (1.25 g). Step 6: Under inert atmosphere, tert-butyl N-[3-(allyloxycarbonylamino)propyl]-N-[(2R)- 3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.25 g, 2.90 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (25 mL). Phenylsilane (0.74 mL, 5.79 mmol, 2.0 eq.) then palladium-tetrakis(triphenylphosphine) (335 mg, 0.290 mmol, 0.1 eq.) were added under argon bubbling. The mixture was stirred for 3 h then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-MeOH 90-10), 0 to 100% of CH2Cl2-MeOH 90/10 in 18 min then CH2Cl2/MeOH/NH4OH 80/19/1 for 10 min). Tert-butyl N-(3-aminopropyl)-N-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (680 mg). Step 7: Under inert atmosphere, Fmoc-Lys(Boc)-OH (0.95 g, 1.96 mmol, 1.0 eq.) was dissolved in pyridine (15 mL). 3-(Ethyliminomethyleneamino)-N,N-dimethyl-propan-1- amine hydrochloride (0.41 g, 2.15 mmol, 1.1 eq.) then tert-butyl N-(3-aminopropyl)-N- [(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (0.68 g, 1.96 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was co-evaporated with toluene (3x25 ml), then taken up with EtOAc (50 ml), washed with brine (50 ml). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-MeOH 90/10), 0 to 50% of CH2Cl2-MeOH 90/10 in 20 min). Tert-butyl N-[3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]propyl]-N-[(2R)-3-(tert-butoxycarbonyl amino)-2-hydroxy-propyl]carbamate was obtained (1.35 g). Step 8: Under inert atmosphere, tert-butyl N-[3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]propyl]-N-[(2R)-3-(tert- butoxycarbonyl amino)-2-hydroxy-propyl]carbamate (1.35 g, 1.69 mmol, 1.0 eq.) was dissolved in anhydrous THF (15 mL). N-ethylethanamine (3.5 mL, 33.8 mmol, 20 eq.) was added. The mixture was stirred for 12 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2-MeOH-NH4OH 80-19-1), 0 to 50% of CH2Cl2-MeOH-NH4OH 80-19-1 for 18 min, then 100% of CH2Cl2-MeOH-NH4OH 80-19-1 for 10 min). tert-Butyl N-[3-[[(2S)- 2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]propyl]-N-[(2R)-3- (tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (840 mg). Example 3.8
Figure imgf000091_0001
Step 1: Under inert atmosphere and magnetic stirring, tert-butyl N-(3-amino-2-hydroxy- propyl)carbamate (0.39 g, 2.05 mmol, 1.0 eq.) was dissolved in 4.5 mL of isopropanol. The mixture was heated under reflux (90 °C) then benzyl N-(oxiran-2- ylmethyl)carbamate (0.50 g, 2.05 mmol, 1.0 eq.) was added dropwise. The reaction mixture was stirred for 1 h under reflux then cooled down to room temperature and concentrated under vacuum. Tert-butyl N-[3-[[3-(benzyloxycarbonylamino)-2- hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained as a yellow oil (1.6 g). Step 2: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]amino]-2-hydroxy-propyl]carbamate (815 mg, 2.05 mmol, 1.0 eq.) in CH2Cl2 (15.8 mL), N,N-diethylethanamine (0.62 mL, 4.51 mmol, 2.2 eq.) and 20 mL of CH2Cl2 were mixed. The reaction mixture was cooled down at 0 °C then a solution of tert-butoxycarbonyl tert-butyl carbonate (895 mg, 4.1 mmol, 2.0 eq.) in 5 mL of CH2Cl2 was added dropwise. The mixture was risen to room temperature, stirred for 1 h then washed with water. The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min). Tert-butyl N-[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)-2- hydroxy-propyl] carbamate was obtained as a white solid (677 mg). Step 3: Under inert atmosphere and magnetic stirring, tert-butyl N-[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)-2- hydroxy-propyl]carbamate (677 mg, 1.36 mmol, 1.0 eq.) was dissolved in MeOH (40.5 mL). Cyclohexene (3.4 mL, 34.0 mmol, 25 eq.) was added and the mixture was stirred for 5 min under bubbling of nitrogen. Palladium (10%, 290 mg, 0.272 mmol, 0.2 eq.) was added and the mixture was heated at 70 °C for 2 h. The reaction mixture was filtered on celite. The filtrate was concentrated under vacuum to give tert-butyl N-(3-amino-2- hydroxy-propyl)-N-[3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate as a white solid (322 mg). Step 4: Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (498 mg, 1.06 mmol, 1.2 eq.) was dissolved in 10 mL of CH2Cl2. 3-(ethyliminomethyleneamino)-N,N- dimethyl-propan-1-amine hydrochloride (204 mg, 1.06 mmol, 1.2 eq.) and pyridine (5.1 mL, 0.063 mol, 71 eq.) were added. The mixture was cooled down at 0 °C and tert-butyl N-(3-amino-2-hydroxy-propyl)-N-[3-(tert-butoxycarbonylamino)-2-hydroxy- propyl]carbamate (322 mg, 0.886 mmol, 1.0 eq.) was added by portion. The reaction mixture was risen up to room temperature and stirred overnight.20 mL of CH2Cl2 were added and the mixture was washed with 10 mL of water. The organic phases was dried over MgSO4, filtered and concentrated under vacuum to give a crude yellow oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 5 % of MeOH in 30 min). Tert-butyl N-[3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H- fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a white solid (567 mg). Step 5: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2- hydroxy-propyl]-N-[3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (245 mg, 0.301 mmol, 1.0 eq.) was dissolved in anhydrous THF (4 mL). N-ethylethanamine (22 mg, 0.301 mmol, 1.0 eq.) was added. The mixture was stirred for 3 h at room temperature then concentrated under vacuum. Tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as an oil (265 mg). Example 3.9
Figure imgf000093_0001
Step 1: To tert-butyl N-(3-amino-2-hydroxypropyl)carbamate (5.0 g, 25.0 mmol, 1.0 eq.) solubilized in anhydrous CH2Cl2 (100 mL) under inert atmosphere, was added N,N- diethylethanamine (3.5 mL, 25.0 mmol, 1.0 eq.). The mixture was cooled down at 0°C and tert-butyl(chloro)dimethylsilane (4.52 g, 30.0 mmol, 1.2 eq.) was added. The mixture was risen up to room temperature, stirred for 4 h then concentrated under vacuum. The residue was taken up with EtOAc (200 ml). The mixture was poured in a solution of water (100 ml) and saturated NaHCO3 (100 ml). The phases were separated and the organic layer was washed with brine, dried over MgSO4, filtered then concentrated under vacuum. Tert-butyl N-[3-amino-2-[tert-butyl(dimethyl)silyl]oxypropyl]carbamate was obtained (7.3 g). Step 2: To a solution of 9H-fluoren-9-ylmethyl (3-hydroxypropyl)carbamate (5.0 g, 16.0 mmol, 1.0 eq.) in CH2Cl2 (50 mL) at 0 °C, was added 1,1,1-tris(acetyloxy)-1,1-dihydro- 1,2-benziodoxol-3-(1H)-one (7.13 g, 16.0 mmol, 1.0 eq.). The mixture was stirred for 10 min at 0 °C then risen to room temperature and stirred for 1 h. The mixture was concentrated, and the residue was taken up in EtOAc (100 mL) and washed with saturated NaHCO3 (30 mL). The phases were separated and the organic phase was washed with brine, dried over MgSO4 then concentrated under vacuum. The residue was taken up with CH2Cl2, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2/MeOH 90/10), 0 to 100% of CH2Cl2/MeOH 90/10 in 30 min).9H-fluoren- 9-ylmethyl N-(3-oxopropyl)carbamate was obtained (4.5 g) and stored at 4 °C. Step 3: Under inert atmosphere, tert-butyl N-[3-amino-2-[tert-butyl(dimethyl)silyl]oxy- propyl]carbamate (3.0 g, 9.85 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane (125 mL).9H-fluoren-9-ylmethyl N-(3-oxopropyl)carbamate (2.91 g, 9.85 mmol, 1.0 eq.) and acetic acid (0.96 mL, 16.7 mmol, 1.7 eq.) were added. The mixture was stirred for 10 min then sodium triacetoxyborohydride (3.55 g, 16.7 mmol, 1.7 eq.) was added. The reaction mixture was stirred for 12 h then concentrated under reduced pressure. The residue was taken up with EtOAc (200 mL) and the resulting solution was washed with brine (200 mL). The phases were separated and the organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2/MeOH 90/10), 0 to 100% of CH2Cl2/MeOH 90/10 in 30 min). 9H-fluoren-9-ylmethyl N-[3-[[3-(tert-butoxycarbonylamino)-2-[tert- butyl(dimethyl)silyl]oxy-propyl]amino]propyl]carbamate was obtained (4.1 g). Step 4: To a solution of 9H-fluoren-9-ylmethyl N-[3-[[3-(tert-butoxycarbonylamino)-2- [tert-butyl(dimethyl)silyl]oxy-propyl]amino]propyl]carbamate (1.50 g, 2.57 mmol, 1.0 eq.) in CH2Cl2 (37.5 mL) were added N,N-diethylethanamine (0.54 mL, 3.85 mmol, 1.5 eq.), DMAP (31 mg, 0.257 mmol, 0.1 eq.) and di-tert-butyl dicarbonate (0.84 g, 3.85 mmol, 1.5 eq.). The mixture was stirred overnight then concentrated under vacuum. The residue was taken up with EtOAc (100 mL), washed with brine (100 mL). The phases were separated, and the organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2/MeOH 90/10), 0 to 30% of CH2Cl2/MeOH 90/10 in 30 min). Tert-butyl N-[3-(tert-butoxycarbonylamino)-2-[tertbutyl(dimethyl)silyl]oxy-propyl]-N-[3-(9H-fluoren- 9-ylmethoxycarbonylamino)propyl]carbamate was obtained (1.1 g). Step 5: Under inert atmosphere, tert-butyl N-[3-(tert-butoxycarbonylamino)-2-[tert- butyl(dimethyl)silyl]oxy-propyl]-N-[3-(9H-fluoren-9- ylmethoxycarbonylamino)propyl]carbamate (1.1 g, 1.61 mmol, 1.0 eq.) was dissolved in anhydrous THF (20 mL). N-ethylethanamine (5.0 mL, 48.3 mmol, 30 eq.) was added. The mixture was stirred for 12 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(CH2Cl2/MeOH 90/10), 0 to 100% of CH2Cl2/MeOH 90/10 in 12 min then 100% of CH2Cl2/MeOH 90/10 for 10 min). Tert-butyl N-(3-aminopropyl)-N-[3-(tert- butoxycarbonylamino)-2-[tert-butyl(dimethyl)silyl]oxy-propyl]carbamate was obtained (220 mg). Example 3.10
Figure imgf000094_0001
Step 1: Tert-butyl N-[(2R)-2,3-dihydroxypropyl]carbamate was obtained as a white solid (33.36 g) following the step 3 described in example 3.7. Step 2: Tert-butyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained as a white solid (17.42 g) following the step 4 described in example 3.7. [α]D = +0.061° (c = 12.5 mg/mL) Step 3: To a solution of Cbz-Dab(Boc)-OH (5.6 g, 15.9 mmol, 1.0 eq.) in THF (91 mL) at -20 °C, were added N,N-diethylethanamine (2.4 mL, 17.5 mmol, 1.1 eq.) and isobutyl chloroformate (2.5 mL, 19.1 mmol, 1.2 eq.). The mixture was stirred for 20 min at -20 °C then 2.4 M lithium aluminium hydride (11 mL, 26.9 mmol, 1.7 eq.) was added. The reaction mixture was risen to room temperature and stirred for 2 h. The mixture was hydrolysed with ice, filtered on decalite and extracted with EtOAc. The phases were separated and the organic phase was washed with brine, dried over MgSO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography. Tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4- hydroxybutyl]carbamate was obtained (1.2 g). Step 4: Tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-hydroxybutyl]carbamate (1.2 g, 3.55 mmol, 1.0 eq.) was dissolved in CH2Cl2 (20 mL). A solution of 4 M hydrogen chloride in dioxane (9.0 mL, 36.0 mmol, 10 eq.) was added. The mixture was stirred overnight at room temperature then concentrated under vacuum. The crude product was triturated with diisopropyl ether and the precipitate was filtered and dried under vacuum. Benzyl N-[(1S)-3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride was obtained as a white solid (900 mg). Step 5: To a solution of benzyl N-[(1S)-3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride (900 mg, 3.28 mmol, 1.0 eq.) in CH2Cl2 (30 mL) were added N,N- diethylethanamine (1.6 mL, 11.5 mmol, 3.5 eq.) and a solution of tert-butyl-chloro- dimethylsilane (dropwise , 987 mg, 6.55 mmol, 2.0 eq.) in CH2Cl2 (10 mL). The reaction mixture turned pink. It was stirred for 3 h then concentrated under vacuum. The crude product was purified by silica gel column chromatography to give two batches (1.2 g and 600 mg) of benzyl N-[(1S)-3-amino-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl]carbamate. Step 6: Benzyl N-[(1S)-3-amino-1-[[tertbutyl(dimethyl)silyl]oxymethyl]propyl] carbamate (600 mg, 1.7 mmol, 1.0 eq.) was suspended in isopropanol (3.4 mL). The mixture was stirred until complete dissolution of benzyl N-[(1S)-3-amino-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl] carbamate, then it was heated at 82 °C. A solution of tert-butyl [(2R)-oxiran-2-ylmethyl]carbamate (322 mg, 1.7 mmol, 1.0 eq.) in isopropanol (4 mL) was added dropwise over 20 min. After 3 h of stirring at 82 °C, the reaction was not complete: an additional solution of tert-butyl [(2R)-oxiran-2- ylmethyl]carbamate (150 mg, 0.8 mmol, 0.47 eq.) in isopropanol (2 mL) was added dropwise. The reaction mixture was stirred for 1 h under reflux then cooled down to room temperature and stirred overnight at room temperature. The crude product was purified by silica gel column chromatography. Tert-butyl N-[(2S)-3-[[(3S)-3- (benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]amino]-2- hydroxypropyl]carbamate was obtained. Step 7: To a solution of tert-butyl N-[(2S)-3-[[(3S)-3-(benzyloxycarbonylamino)-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]-2-hydroxypropyl]carbamate (590 mg, 1.12 mmol, 1.0 eq.) in CH2Cl2 (50 mL) were added tert-butoxycarbonyl tert-butyl carbonate (0.49 g, 2.24 mmol, 2.0 eq.), N,N-diethylethanamine (0.39 mL, 2.81 mmol 2.5 eq.) and N,N-dimethylpyridin-4-amine (27 mg, 0.22 mmol, 0.2 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over MgSO4, filtered then concentrated under vacuum. The crude product was purified by flash column chromatography to give 500 mg of tert-butyl N-[(3S)-3- (benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N-[(2R)-3- (tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate and 140 mg of [(1R)-1-[[[(3S)-3- (benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-tert-butoxycarbonyl- amino]methyl]-2-(tert-butoxycarbonylamino)ethyl] tert-butyl carbonate. Step 8: Tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy- butyl]-N-[(2R)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate and [(1R)-1- [[[(3S)-3-(benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-tert- butoxycarbonyl-amino]methyl]-2-(tert-butoxycarbonylamino)ethyl] tert-butyl carbonate (640 mg, 0.88 mmol, 1.0 eq.) were dissolved in MeOH (50 mL) and hydrogenated with a H-cube® hydrogenation reactor (1 mL/min, room temperature and full H2) on a Pd/C (0.88 mmol, 1.0 eq.) at 10% cartridge. The mixture was concentrated under vacuum. Tert-butyl N-[(3S)-3-amino-4-[(tert- butyldimethylsilyl)oxy]butyl]-N-[(2R)-3-{[(tert-butoxy)carbonyl]amino}-2- hydroxypropyl]carbamate and [(1R)-1-[[[(3S)-3-amino-4-[tert-butyl(dimethyl)silyl]oxy- butyl]-tertbutoxycarbonyl-amino]methyl]-2-(tertbutoxycarbonylamino)ethyl] tert-butyl carbonate were obtained in mixture (470 mg). Step 9: Under inert atmosphere, Fmoc-Lys(Boc)-OH (375 mg, 0.78 mmol, 1.0 eq.) and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (164 mg, 0.85 mmol, 1.1 eq.) was dissolved in dry pyridine (9.9 mL, 0.12 mol). The mixture was stirred and a solution of tert-butyl N-[(3S)-3-amino-4-[(tert-butyldimethylsilyl)oxy]butyl]- N-[(2R)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxypropyl]carbamate and [(1R)-1-[[[(3S)- 3-amino-4-[tert-butyl(dimethyl)silyl]oxy-butyl]-tertbutoxycarbonyl-amino]methyl]-2- (tertbutoxycarbonylamino)ethyl] tert-butyl carbonate (459 mg, 0.78 mmol, 1.0 eq.) in ahydrous CH2Cl2 was added. The resulting mixture was stirred overnight then concentrated under vacuum. The residue was taken-up in EtOActhen washed with a solution of 2% citric acid, NaHCO3 then brine. The organic phase was dried over MgSO4, filtered then concentrated under vacuum. The crude product was purified by flash column chromatography. Tert-butyl N-[(3S)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N-[(2R)- 3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate (75 mg) and [(1R)-1-[(tert- butoxycarbonylamino)methyl]-2-[tertbutoxycarbonyl-[(3S)-3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]ethyl] tert-butyl carbonate (390 mg) were obtained. Step 10: Tert-butyl N-[(3S)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N-[(2R)- 3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate and [(1R)-1-[(tert- butoxycarbonylamino)methyl]-2-[tertbutoxycarbonyl-[(3S)-3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]ethyl] tert-butyl carbonate (459, 0.44 mmol, 1.0 eq.) was dissolved in a solution of N-ethylethanamine at 20% in THF (5 mL). The mixture was stirred for 5 h then concentrated under vacuum. The crude product was purified by flash column chromatography. tert-butyl N-[(3S)-3-[(2S)-2-amino-6-{[(tert- butoxy)carbonyl]amino}hexanamido]-4-[(tert-butyldimethylsilyl)oxy]butyl]-N-[(2R)-3- {[(tert-butoxy)carbonyl]amino}-2-hydroxypropyl]carbamate (50 mg) and [(1R)-1-[[[(3S)-3- [[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]-4- [tertbutyl(dimethyl)silyl]oxy-butyl]-tert-butoxycarbonyl-amino]methyl]-2-(tert- butoxycarbonylamino)ethyl] tert-butyl carbonate (370 mg) were obtained. Example 3.11
Figure imgf000098_0001
Step 1: Allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a colourless liquid (11 g) following the step 1 described in example 3.2. Step 2: Allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a colourless liquid (4.8 g) following the step 2 described in example 3.2. Step 3: To tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]carbamate (1.50 g, 7.88 mmol, 1.0 eq.) in isopropanol (20 mL) at 70 °C was added dropwise over 15 min allyl N-[[(2S)- oxiran-2-yl]methyl]carbamate (1.24 g, 7.88 mmol, 1.0 eq.). The reaction was stirred for 40 min at 70 °C then concentrated under vacuum. Tert-butyl N-[(2S)-3-[[(2R)-3- (allyloxycarbonylamino)-2-hydroxypropyl]amino]-2-hydroxy-propyl]carbamate was obtained (2.6 g). Step 4: To tert-butyl N-[(2S)-3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]-2-hydroxy-propyl]carbamate (2.7 g, 7.80 mmol, 1.0 eq.) in CH2Cl2 (35 mL) were added tert-butoxycarbonyl tert-butyl carbonate (2.04 g, 9.36 mmol, 1.2 eq.) and N,N-diethylethanamine (1.3 mL, 9.36 mmol, 1.2 eq.). The reaction mixture was stirred overnight at room temperature. The reaction was taken up in CH2Cl2 and washed with water then citric acid solution (2%). The organic phase was dried over Na2SO4 then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography with CH2Cl2/MeOH 94/6. Tert-butyl N-[(2S)-3-(allyloxycarbonylamino)- 2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a colourless oil (1.6 g). Step 5: Under inert atmosphere, to tert-butyl N-[(2S)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.6 g, 3.58 mmol, 1.0 eq.) in anhydrous CH2Cl2 (40 mL) was added phenylsilane (1.4 mL, 10.7 mmol, 3.0 eq.) and tetrakis(triphenylphosphine)-palladium (289 mg, 0.25 mmol, 0.07 eq.). The reaction was stirred 3 h at room temperature then concentrated under vacuum. Tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]-N-[(2R)-3- (tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (1.27 g). Step 6: To a solution of Fmoc-Lys(Boc)-OH (1.69 g, 3.50 mmol, 1.0 eq.) in CH2Cl2 (16 mL) and pyridine (8 mL) at 5 °C were added N-[3-(dimethylamino)propyl]-N'- ethylcarbodiimide (652 mg, 4.20 mmol, 1.2 eq.) then after 5 min tert-butyl N-[(2S)-3- amino-2-hydroxy-propyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy- propyl]carbamate (1.27 g, 3.50 mmol, 1.0 eq.). The reaction was stirred for 18 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water then saturated NaHCO3. The organic phase was dried over Na2SO4 before concentration to dryness. The crude was purified by silica gel flash column chromatography (CH2Cl2/MeOH 96/4). Tert-butyl N-[(2S)-3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2- hydroxypropyl]-N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxypropyl]carbamate was obtained as a colourless oil (1.28 g). [α]D = -5.4° (c=0.24, MeOH) Step 7: To a solution of tert-butyl N-[(2S)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2-(9H- fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]-N-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate (1.28 g, 1.57 mmol, 1.0 eq.) in THF (30 mL) was added N-ethylethanamine (6.0 mL, 57.1 mmol, 36 eq.). The reaction was stirred 3 h at room temperature then concentrated under vacuum. The crude was purified by silica gel flash column chromatography (CH2Cl2/MeOH/NH4OH 90/10/1). Tert-butyl N- [(2S)-3-[[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]-2-hydroxy-propyl]- N-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a beige foam (670 mg). Example 3.12
Figure imgf000099_0001
Step 1: To a solution of tert-butyl N-(3-aminopropyl)carbamate (2.0 g, 11.5 mmol, 1.0 eq.) in MeOH (50 mL) was added prop-2-enenitrile (0.76 mL, 11.5 mmol, 1.0 eq.). The reaction mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water. The organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 97/3). Tert- butyl N-[3-(2-cyanoethylamino)propyl]carbamate was obtained as a colorless oil (2 g). Step 2: To a solution of tert-butyl N-[3-(2-cyanoethylamino)propyl]carbamate (6.40 g, 28.2 mmol, 1.0 eq.) in acetonitrile (56 mL) were added ethyl bromoacetate (3.9 mL, 35.2 mmol, 1.25 eq.), dipotassium carbonate (5.84 g, 42.2 mmol, 1.5 eq.) and sodium iodide (844 mg, 5.63 mmol, 0.2 eq.). The mixture was heated to 70 °C and stirred for 18 h. The reaction was diluted with EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 97/3). Ethyl 2-[3-(tert- butoxycarbonylamino)propyl-(2-cyanoethyl)amino]acetate was obtained as a colorless oil (8.1 g). Step 3: To a solution of ethyl 2-[3-(tert-butoxycarbonylamino)propyl-(2- cyanoethyl)amino]acetate (3.75 g, 12 mmol, 1.0 eq.) in ethanol (50 mL) was added by portion sodium borohydride (760 mg, 20 mmol, 1.7 eq.). The reaction mixture was heated to 60 °C and stirred for 2 h. After 2 h, the reaction was not complete: sodium borohydride (300 mg, 7.9 mmol, 0.7 eq.) was added and the mixture was stirred at 70 °C for 1.5 h. The starting material was detected and additional sodium borohydride (300 mg, 7.9 mmol, 0.7 eq.) was added and the mixture was stirred 4 h at 75 °C. The reaction was partially concentrated, taken up in EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 97/3). Tert-butyl N-[3-[2- cyanoethyl(2-hydroxyethyl)amino]propyl]carbamate was obtained as a colorless oil (1.2 g). Step 4: To a solution of tert-butyl N-[3-[2-cyanoethyl(2- hydroxyethyl)amino]propyl]carbamate (1.20 g, 4.42 mmol, 1.0 eq.) in CH2Cl2 (56 mL) was added tert-butyl-chloro-dimethyl-silane (1.67 g, 11.1 mmol , 2.5 eq.), triethylamine (1.6 mL, 11.5 mmol, 2.6 eq.) and N,N-dimethylpyridin-4-amine (0.11 g, 0.884 mmol, 0.2 eq.). The mixture was stirred for 18 h at room temperature. The reaction mixture was diluted with CH2Cl2 and washed with water. The phases were separated and the organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 96/4). tert-butyl N-[3-[2-[tert-butyl(dimethyl)silyl]oxyethyl-(2-cyanoethyl)amino]propyl]carbamate was obtained as a colourless oil (0.93 g). Step 5: Tert-butyl N-[3-[2-[tert-butyl(dimethyl)silyl]oxyethyl-(2- cyanoethyl)amino]propyl]carbamate (0.93 g, 2.41 mmol, 1.0 eq.) was dissolved in MeOH (15 mL). The mixture was cooled down to 0 °C. Nickel(II) chloride hexahydrate (250 mg, 1.05 mmol, 0.4 eq.) was added followed by an addition by portion, over 40 min, of sodium borohydride (630 mg, 16.7 mmol, 6.9 eq.). The mixture was risen to 10 °C, stirred for 1 h then hydrolysed with ice water. The aqueous phase was extracted with EtOAc. The combined organic phases were washed with water, dried over Na2SO4, filtered and concentrated under vacuum. Tert-butyl N-[3-[3-aminopropyl-[2- [tertbutyl(dimethyl)silyl]oxyethyl] amino]propyl]carbamate was obtained (770 mg). Step 6: To a solution of tert-butyl N-[3-[3-aminopropyl-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]propyl]carbamate (770 mg, 1.98 mmol, 1.0 eq.) in CH2Cl2 (8 mL) and pyridine (4 mL), were added Fmoc-Lys(Boc)-OH (1.05 g, 2.17 mmol, 1.1 eq.) and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (337 mg, 2.17 mmol, 1.1 eq.). The reaction mixture was stirred for 18 h at room temperature then concentrated. The residue was taken up with EtOAc and washed with water. The phases were separated and the organic layer was dried over Na2SO4, filtered then concentrated under vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH 95/5). 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1-[3- [3-(tert-butoxycarbonylamino)propyl-[2- [tertbutyl(dimethyl)silyl]oxyethyl]amino]propylcarbamoyl]pentyl]carbamate was obtained as a colourless oil (450 mg). Step 7: To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1- [3-[3-(tert-butoxycarbonylamino)propyl-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]propylcarbamoyl]pentyl]carbamate (450 mg, 0.536 mmol, 1.0 eq.) in THF (15 mL) was added N-ethylethanamine (3.0 mL, 28.7 mmol, 54 eq.). The reaction was stirred overnight at room temperature then concentrated under vacuum. Tert-butyl N-[3-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]amino]propyl-[2- [tertbutyl(dimethyl)silyl]oxyethyl]amino]propyl]carbamate was obtained (430 mg). Example 3.13
Figure imgf000102_0001
Step 1: To a solution at 0-5 °C of tert-butyl N-(4-amino-3-hydroxybutyl)carbamate (1000 mg, 4.90 mmol, 1.0 eq.) in CH2Cl2 (20 mL), were added triethylamine (0.8 mL, 5.88 mmol, 1.2 eq.) and chloro(triethyl)silane (0.9 mL, 5.39 mmol, 1.1 eq.). The mixture was risen to room temperature, stirred overnight then concentrated under reduced pressure. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over MgSO4, filtered then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography. tert-Butyl N-(4-amino-3- triethylsilyloxy-butyl)carbamate was obtained (1.36 g). Step 2: tert-Butyl N-(4-amino-3-triethylsilyloxy-butyl)carbamate (860 mg, 2.7 mmol, 1.0 eq.) was dissolved in 5 mL of isopropanol. The mixture was heated to reflux. A solution of benzyl N-(oxiran-2-ylmethyl)carbamate (0.66 g, 2.70 mmol, 1.0 eq.) in isopropanol (2 mL) was added dropwise. The mixture was stirred under reflux for 1 h then cooled down at room temperature and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography. Tert-butyl N-[4-[[3- (benzyloxycarbonylamino)-2-hydroxypropyl]amino]-3-triethylsilyloxy-butyl]carbamate was obtained (800 mg). Step 3: Under inert atmosphere, tert-butyl N-[4-[[3-(benzyloxycarbonylamino)-2-hydroxy- propyl]amino]-3-triethylsilyloxy-butyl]carbamate (1.25 g, 2.38 mmol, 1.0 eq.) was dissolved in 9.5 mL of anhydrous CH2Cl2. The mixture was cooled down at 0-5 °C and triethylamine (1.17 mL, 8.56 mmol, 3.6 eq.) then chloro(triethyl)silane (1.2 mL, 7.13 mmol, 3.0 eq.) were added. The mixture was risen to room temperature, stirred overnight then concentrated under vacuum. The residue was suspended in CH2Cl2 and washed with water. The organic phase was dried over MgSO4, filtered then concentrated under vacuum. tert-Butyl N-[4-[[3-(benzyloxycarbonylamino)-2-triethylsilyloxypropyl]amino]-3- triethylsilyloxy-butyl]carbamate was obtained (1.7g). Step 4: tert-Butyl N-[4-[[3-(benzyloxycarbonylamino)-2-triethylsilyloxypropyl] amino]-3-triethylsilyloxy-butyl]carbamate (1.7 g, 2.66 mmol, 1.0 eq.) and tert- butoxycarbonyl tert-butyl carbonate (1159 mg, 5.31 mmol, 2.0 eq.) were dissolved in CH2Cl2 (13 mL). The mixture was cooled down at 0-5 °C and N,N-diethylethanamine (0.80 mL, 5.84 mmol, 2.2 eq.) was added. The mixture was risen to room temperature, stirred overnight then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography. Tert-butyl N-[3-(benzyloxycarbonylamino)-2- triethylsilyloxypropyl]-N-[4-(tert-butoxycarbonylamino)-2-triethylsilyloxybutyl]carbamate was obtained (1.4 g). Step 5: To a solution of tert-butyl N-[3-(benzyloxycarbonylamino)-2- triethylsilyloxypropyl]-N-[4-(tert-butoxycarbonylamino)-2-triethylsilyloxybutyl]carbamate (1.4 g, 1.89 mmol, 1.0 eq.) in 50 mL of MeOH, were added cyclohexene (4.8 mL, 47.3 mmol, 25 eq.) then palladium (10%, 403 mg, 0.378 mmol, 0.2 eq.). The mixture was heated to reflux, stirred for 1 h then cooled down to room temperature and stirred for 2 h. The mixture was filtered on a Millipore filter. The filtrate was concentrated under vacuum. Tert-butyl N-(3-amino-2-triethylsilyloxy-propyl)-N-[4-(tertbutoxycarbonylamino)- 2-triethylsilyloxy-butyl]carbamate was obtained as an oil (1.1 g). Step 6: Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (399 mg, 0.825 mmol, 1.0 eq.) and 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (174 mg, 0.908 mmol, 1.1 eq.) were dissolved in anhydrous pyridine (10 mL). Tert-butyl N-(3-amino-2-triethylsilyloxy-propyl)-N-[4-(tertbutoxycarbonylamino)-2- triethylsilyloxy-butyl]carbamate (500 mg, 0.825 mmol, 1.0 eq.) was added. The mixture was stirred for 4 h at room temperature then concentrated under vacuum. The residue was taken up with EtOAc then washed with water, saturated NaHCO3 and brine. The organic phase was dried over MgSO4, filtered and dried under vacuum. The crude product was purified by silica gel flash column chromatography. tert-butyl N-[4-(tert- butoxycarbonylamino)-2-triethylsilyloxybutyl]-N-[3-[[rac-(2S)-6-(tert- butoxycarbonylamino)-2-(9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-2- triethylsilyloxy-propyl]carbamate was obtained (660 mg). Step 7: Under inert atmosphere, tert-butyl N-[4-(tert-butoxycarbonylamino)-2- triethylsilyloxybutyl]-N-[3-[[rac-(2S)-6-(tert-butoxycarbonylamino)-2-(9Hfluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-triethylsilyloxy-propyl]carbamate (660 mg, 0.62 mmol, 1.0 eq.) was dissolved in 10 mL of a solution of diethylamine at 20% in anhydrous THF. The mixture was stirred overnight then concentrated under vacuum. Tert-butyl N-[3-[[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]-2- triethylsilyloxy-propyl]-N-[4-(tert-butoxycarbonylamino)-2-triethylsilyloxybutyl]carbamate was obtained (584 mg). Example 3.14
Figure imgf000104_0001
Step 1: Under inert atmosphere and magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (2.21 g, 12.3 mmol, 1.0 eq.) was dissolved in 27 mL of anhydrous isopropanol. The mixture was heated to reflux then benzyl N-(oxiran-2- ylmethyl)carbamate (3.0 g, 12.3 mmol, 1.0 eq.) was added dropwise. The mixture was stirred for 1 h under reflux then concentrated under vacuum. tert-butyl N-[3-[[3- (benzyloxycarbonylamino)-2-hydroxypropyl]amino]propyl]carbamate was obtained as a yellow oil (5.1 g). Step 2: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]amino]propyl]carbamate (4.70 g, 12.3 mmol, 1.0 eq.) and N, N-diisopropylethylamine (4.7 mL, 27.1 mmol, 2.2 eq.) were dissolved in anhydrous CH2Cl2 (47 mL). The mixture was cooled down to 0 °C and a solution of tert-butoxycarbonyl tert-butyl carbonate (5.4 g, 24.6 mmol, 2.0 eq.) in CH2Cl2 (10 mL) was added dropwise. The mixture was risen to room temperature, stirred for 1 h then washed with water. The organic layer was dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 100 % of EtOAc in 30 min). Tert-butyl N-[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate was obtained as a white solid (3.15 g). Step 3: Under inert atmosphere and magnetic stirring, tert-butyl N-[3- (benzyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (3.15 g, 6.54 mmol, 1.0 eq.) was dissolved in MeOH (194 mL) then cyclohexene (17 mL, 0.164 mol, 25 eq.) was added. The mixture was stirred for 5 min then palladium (139 mg, 1.31 mmol, 0.2 eq.) was added. The mixture was heated at 70 °C, stirred for 4 h then concentrated under vacuum. The crude was purified by silica gel flash column chromatography (CH2Cl2/MeOH + 1% NH3, 0 to 10 % of MeOH in 30 min). Tert-butyl N-(3-amino-2-hydroxy-propyl)-N-[3- (tertbutoxycarbonylamino)propyl]carbamate was obtained as a yellow oil (1.2 g). Step 4: Under inert atmosphere and magnetic stirring, tert-butyl N-(3-amino-2-hydroxy- propyl)-N-[3-(tert-butoxycarbonylamino)propyl]carbamate (1.00 g, 2.88 mmol, 1.0 eq.) was dissolved in anhydrous dichloroethane (31 mL). Ethyl oxoacetate (0.67 mL, 3.17 mmol, 1.1 eq.) and acetic acid (0.28 mL, 4.89 mmol, 1.7 eq.) were added and the reaction mixture was stirred 30 min. Sodium triacetoxyborohydride (1037 mg, 4.89 mmol, 1.7 eq.) was added and the resulting solution was stirred for 6 h. Then ethanol (20 mL) and sodium borohydride (871 mg, 23.0 mmol, 8.0 eq.) were added (by portion for sodium borohydride). The mixture was stirred overnight at room temperature then heated at 50 °C. Additional sodium borohydride (327 mg, 8.63 mmol, 3.0 eq.) was added and the mixture was stirred for 30 min at 50 °C before to be concentrated under vacuum. The residue was taken up in 200 mL of EtOAc and washed with water (50 mL). The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give tert-butyl N-[3-(tert-butoxycarbonylamino)propyl]-N-[2-hydroxy-3-(2- hydroxyethylamino)propyl]carbamate as a white solid (1.24 g). Step 5: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-(tert- butoxycarbonylamino)propyl]-N-[2-hydroxy-3-(2-hydroxyethylamino)propyl]carbamate (1.24 g, 3.17 mmol, 1.0 eq.) and N-ethyl-N-isopropyl-propan-2-amine (5.5 mL, 31.7 mmol, 10 eq.) were dissolved in anhydrous CH2Cl2 (15 mL). The mixture was cooled down to 0 °C then tert-butyl-chloro-dimethyl-silane (2.51 g, 15.8 mmol, 5.0 eq.) was added by portion. The mixture was risen to room temperature, stirred overnight and washed with water. The organic layer was dried with MgSO4, filtered and concentrated under vacuum to give a crude orange oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min). Tert-butyl N-[3-(tert-butoxycarbonylamino)propyl]-N-[2-[tertbutyl(dimethyl)silyl]oxy-3-[2- [tertbutyl(dimethyl)silyl]oxyethylamino]propyl]carbamate was obtained as a colourless oil (772 mg). Step 6: Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (301 mg, 0.629 mmol, 1.2 eq.), tert-butyl N-[3-(tert-butoxycarbonylamino)propyl]-N-[2-[tert- butyl(dimethyl)silyl]oxy-3-[2-[tert-butyl(dimethyl)silyl]oxyethylamino]propyl]carbamate (365 mg, 0.524 mmol, 1.0 eq.) and anhydrous pyridine (5 mL) were dissolved in anhydrous CH2Cl2 (10 mL). The reaction mixture was cooled down at 0°C and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (121 mg, 0.629 mmol, 1.2 eq.) was added by portion. The mixture was risen to room temperature, stirred overnight and concentrated under vacuum to give a crude yellow oil. The crude product was purified by silica gel flash column chromatography. Tert-butyl N-[3-[[(2S)-6- (tert-butoxycarbonylamino)-2-(9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]-[2- [tertbutyl(dimethyl)silyl]oxyethyl]amino]-2-[tertbutyl(dimethyl)silyl]oxy-propyl]-N-[3- (tertbutoxycarbonylamino)propyl]carbamate was obtained as a white solid (424 mg). Step 7: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-[2-[tert- butyl(dimethyl)silyl]oxyethyl]amino]-2-[tert-butyl(dimethyl)silyl]oxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (424 mg, 0.392 mmol, 1.0 eq.) and N- ethylethanamine (2.0 mL, 19.3 mmol, 49 eq.) were dissolved in anhydrous THF (8 mL). The mixture was stirred for 2 h at room temperature then concentrated under vacuum to give a crude oil. Tert-butyl N-[3-[[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]-[2- [tertbutyl(dimethyl)silyl]oxyethyl]amino]-2-[tertbutyl(dimethyl)silyl]oxy-propyl]-N-[3- (tertbutoxycarbonylamino)propyl]carbamate was obtained (425 mg). Example 3.15
Figure imgf000106_0001
Step 1: To a solution of Cbz-Dab(Boc)-OH (5.0 g, 14.2 mmol, 1.0 eq.) in THF (81.2 mL) at -20 °C, were added N,N-diethylethanamine (2.2 mL, 15.6 mmol, 1.1 eq.) and isobutyl carbonochloridate (2.2 mL, 17 mmol, 1.2 eq.). The reaction was stirred for 20 min at -20 °C then 2.4 M lithium aluminum hydride (10 mL, 24 mmol, 1.7 eq.) was added (temperature -12 °C to -3 °C). The reaction mixture was raised to room temperature then stirred for 2 h. The mixture was hydrolysed with ice, filtered on decalite and washed with EtOAc. The filtrate was evaporated to dryness. The crude was purified by silica gel flash column chromatography (CH2Cl2/MeOH (95/5). Tert-butyl N-[(3S)-3- (benzyloxycarbonylamino)-4-hydroxybutyl] carbamate was obtained as a colourless oil (1.9 g). Step 2: To a solution of tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-hydroxy- butyl]carbamate (1.9 g, 5.61 mmol, 1.0 eq.) in CH2Cl2 (60 mL) was added 4 M HCl (16 mL, 64.0 mmol, 11 eq.). The reaction was stirred for 18 h at room temperature then concentrated under vacuum. The residue was triturated with diisopropylether then the resulting mixture was filtered. The solid residue was dried under vacuum. Benzyl N-[(1S)- 3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride was obtained as a white solid (1.32 g). Step 3: To benzyl N-[(1S)-3-amino-1-(hydroxymethyl)propyl]carbamate hydrochloride (920 mg, 3.35 mmol, 1.0 eq.) in CH2Cl2 (30 mL) were added N,N-diethylethanamine (1.6 mL, 11.7 mmol, 3.5 eq.), a solution of tert-butyl-chloro-dimethyl-silane (dropwise , 1.01 g, 6.70 mmol, 2.0 eq.) in CH2Cl2 (4 mL) and N,N-dimethylpyridin-4-amine (82 mg, 0.670 mmol, 0.2 eq.). The reaction mixture was stirred 4 h at room temperature then concentrated under vacuum. Benzyl N-[(1S)-3-amino-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl]carbamate was obtained as a brown oil (1.2 g). Step 4: To benzyl N-[(1S)-3-amino-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl]carbamate (814 mg, 2.31 mmol, 1.0 eq.) in isopropanol (10 mL) at 70 °C, was added dropwise over 15 min a solution of tert-butyl N-[[(2S)-oxiran-2-ylmethyl]carbamate (400 mg, 2.31 mmol, 1.0 eq.) in isopropanol (2 mL). The reaction mixture was stirred 1 h at 70 °C then concentrated under vacuum. tert- butyl N-[(2R)-3-[[(3S)-3-(benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy- butyl]amino]-2-hydroxypropyl]carbamate was obtained. Step 5: To a solution of tert-butyl N-[(2R)-3-[[(3S)-3-(benzyloxycarbonylamino)-4- [tertbutyl(dimethyl)silyl]oxy-butyl]amino]-2-hydroxypropyl]carbamate (1.21 g, 2.30 mmol, 1.0 eq.) in CH2Cl2 (30 mL) were added tert-butoxycarbonyl tert-butyl carbonate (1.00 g, 4.60 mmol, 2.0 eq.), N,N-diethylethanamine (0.80 mL, 5.75 mmol, 2.5 eq.) and N,N- dimethylpyridin-4-amine (56 mg, 0.460 mmol, 0.2 eq).The mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over Na2SO4 before concentration to dryness. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 97/3 to 95/5). tert-butyl N-[(3S)-3- (benzyloxycarbonylamino)-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N-[(2S)-3- (tertbutoxycarbonylamino)-2-hydroxypropyl]carbamate was obtained as a beige oil (1.26 g). Step 6: A solution tert-butyl N-[(3S)-3-(benzyloxycarbonylamino)-4-[tert- butyl(dimethyl)silyl]oxy-butyl]-N-[(2S)-3-(tert-butoxycarbonylamino)-2- hydroxypropyl]carbamate (0.63 g, 1 mmol, 1.0 eq.) in ethanol (350 mL) was hydrogenated with a H-cube® hydrogenation reactor (P = 1 Bar at 30 °C) on a Pd/C (106 mg, 1 mmol, 1.0 eq.) at 10% cartridge. The reaction mixture was concentrated under vacuum to tert-butyl N-[(3S)-3-amino-4-[tert-butyl(dimethyl)silyl]oxy-butyl]-N-[(2S)-3- (tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate as a beige oil (480 mg). Step 7: To a solution of tert-butyl N-[(3S)-3-amino-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N- [(2S)-3-(tertbutoxycarbonylamino)-2-hydroxypropyl]carbamate (480 mg, 0.976 mmol, 1.0 eq.) in CH2Cl2 (8 mL) and pyridine (3 mL), were added Fmoc-Lys(Boc)-OH (471 mg, 0.976 mmol, 1.0 eq.) and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (167 mg, 1.07 mmol, 1.1 eq.). The reaction mixture was stirred overnight at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water and brine. The organic phase was dried over Na2SO4 before concentration to dryness. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 9/1). tert-butyl N-[(3S)-3-[[(2S)-6-(tert-butoxycarbonylamino)-2- (9Hfluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4-[tertbutyl(dimethyl)silyl]oxy- butyl]-N-[(2S)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a colourless oil (700 mg). Step 8: To a solution of tert-butyl N-[(3S)-3-[[(2S)-6-(tertbutoxycarbonylamino)- 2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-4-[tert- butyl(dimethyl)silyl]oxy-butyl]-N-[(2S)-3-(tert-butoxycarbonylamino)-2- hydroxypropyl]carbamate (700 mg, 0.743 mmol, 1.0 eq.) in THF (30 mL) was added N- ethylethanamine (8.0 mL, 76.6 mmol, 103 eq.). The reaction mixture was stirred 3 h at room temperature then concentrated under vacuum. tert-butyl N-[(3S)-3-[[(2S)-2-amino- 6-(tertbutoxycarbonylamino)hexanoyl]amino]-4-[tertbutyl(dimethyl)silyl]oxy-butyl]-N- [(2S)-3-(tertbutoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained as a white wax (600 mg). [α]D = + 12.8 ° (c = 0.49, MeOH) Example 3.16
Figure imgf000108_0001
Step 1: Allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a colorless liquid (11 g) following the step 1 described in example 3.2. Step 2: Allyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a colorless liquid (4.8 g) following the step 2 described in example 3.2. Step 3: To tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.00 g, 4.94 mmol, 1.0 eq.) in 2-propanol (12 mL) at 70 °C was added dropwise over 15 min allyl N- [[rac-(2S)-oxiran-2-yl]methyl]carbamate (777 mg, 4.94 mmol, 1.0 eq.). The reaction was stirred 50 min at 70 °C then concentrated under vacuum. The crude was then purified by silica gel flash column chromatography (CH2Cl2/MeOH 94/6 to 90/10). Tert-butyl 3- hydroxy-3-[[[rac-(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]methyl]azetidine-1-carboxylate was obtained as a colourless oil (1.08 g). [α]D= +2.4° (c = 0.21 mg/mL; MeOH) Step 4: To tert-butyl 3-hydroxy-3-[[[rac-(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]methyl]azetidine-1-carboxylate (1.08 g, 3.00 mmol, 1.0 eq.) in CH2Cl2 (20 mL) were added prop-2-en-1-yl carbonochloridate (0.35 mL, 3.31 mmol, 1.1 eq.) and N- ethyl-N-isopropyl-propan-2-amine (0.73 mL, 4.21 mmol, 1.4 eq.). The reaction was stirred 18 h at room temperature then concentrated under vacuum. The reaction was taken up in EtOAc and washed with water. The organic phase was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 95/5). Tert-butyl 3-[[allyloxycarbonyl-[rac- (2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]amino]methyl]-3-hydroxy-azetidine-1- carboxylate was obtained as a colourless oil (1.02 g). [α]D = +5.4° (c = 0.24 mg/mL; MeOH) Step 5: To tert-butyl 3-[[allyloxycarbonyl-[rac-(2S)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.02 g, 2.30 mmol, 1.0 eq.) in CH2Cl2 (25 mL) was added trifluoroacetic acid (2.6 mL, 34.5 mmol, 15 eq.). The reaction mixture was stirred 3 h at room temperature then concentrated under vacuum to give allyl N-[(3-hydroxyazetidin-3-yl)methyl]-N-[rac-(2S)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]carbamate;2,2,2-trifluoroacetic acid (1.0 g). [α]D = +1.1° (c = 0.26 mg/mL; MeOH) Step 6: Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (1.17 g, 2.42 mmol, 1.05 eq.) was dissolved in anhydrous DMF (20 mL). The mixture was cooled down to 5 °C and 108enzotriazole-1-yloxy(tripyrrolidin-1- yl)phosphonium;hexafluorophosphate (1.20 g, 2.30 mmol, 1.0 eq.), N-ethyl-N-isopropyl- propan-2-amine (1.0 mL, 5.75 mmol, 2.5 eq.) and N-[(3-hydroxyazetidin-3-yl)methyl]-N- [rac-(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]carbamate;2,2,2-trifluoroacetic acid (1.05 g, 2.30 mmol, 1.0 eq.) were added. The reaction was risen to room temperature, stirred for 18 h then diluted with EtOAc and washed twice with water. The organic phase was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 95/5). Allyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate was obtained as a colourless oil (1.12 g). Step 7: To allyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate (1.12 g, 1.41 mmol, 1.0 eq.) in THF (35 mL) was added N-ethylethanamine (6.0 mL, 57.7 mmol, 41 eq.). The reaction mixture was stirred for 4 h at room temperature then concentrated under vacuum. Allyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-2-amino-6-(tert- butoxycarbonylamino)hexanoyl]-3-hydroxyazetidin-3-yl]methyl]carbamate was obtained (1.15 g). Example 3.17
Figure imgf000110_0001
Step 1: Allyl N-[(2R)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a yellow oil (33.6 g) following the step 1 described in Example 3.4. Step 2: Allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained as a yellow oil (9.55 g) following the step 2 described in Example 3.4. [α]D= +0.10° (c = 10 mg/mL; MeOH) Step 3: Tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (2.0 g, 9.39 mmol, 1.0 eq.) was dissolved in isopropanol (15 mL). The mixture was heated to reflux and a solution of allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate (1476 mg, 9.39 mmol, 1.0 eq.) in isopropanol (5 mL) was added dropwise. The mixture was heated at 70 °C, stirred for 30 min and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography. Tert-butyl 3-[[[(2S)-3- (allyloxycarbonylamino)-2-hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1- carboxylate was obtained (2.0 g). Step 4: To a solution of tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (2.0 g, 5.56 mmol, 1.0 eq.) in ethanol (60 mL) was added tert-butoxycarbonyl tert-butyl carbonate (1.34 g, 6.12 mmol, 1.1 eq.). The reaction mixture was stirred for 3 h at room temperature then concentrated under vacuum. Tert-butyl 3-[[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained (2.8 g). Step 5: Under inert atmosphere, tert-butyl 3-[[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]-tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate (2.3 g, 5.01 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (83 mL). Phenylsilane (1.91 mL, 15.01 mmol, 3.0 eq.) was added then the mixture was stirred for 20 min. Palladium - triphenylphosphine (1:4) (405 mg, 0.350 mmol, 0.07 eq.) was added. The reaction mixture was stirred for 1 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash chromatography to give tert-butyl 3-[[[(2R)- 3-amino-2-hydroxy-propyl]-tert-butoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1- carboxylate (1.4 g). [α]D = -6.1° (c = 0.52 mg/mL; MeOH) Step 6: Under inert atmosphere, Fmoc-Lys(Boc)-OH (1.29 g, 2.66 mmol, 1.0 eq.), 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (562 mg, 2.93 mmol, 1.1 eq.) and pyridine (33 mL) were dissolved in anhydrous CH2Cl2 (23.5 mL). A solution of tert-butyl 3-[[[(2R)-3-amino-2-hydroxy-propyl]-tert-butoxycarbonyl- amino]methyl]-3-hydroxy-azetidine-1-carboxylate (990 mg, 2.64 mmol, 1.0 eq.) in anhydrous CH2Cl2 (10 mL) was added. The mixture was stirred overnight then concentrated under vacuum. The residue was taken up with EtOAc and washed with 2% aqueous citric acid, saturated NaHCO3 and brine. The organic phase was dried over MgSO4, filtered then concentrated under vacuum. The crude product was purified on silica gel flash column chromatography. Tert-butyl 3-[[tert-butoxycarbonyl-[(2R)-3-[[(2S)- 6-(tertbutoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoyl]amino]-2-hydroxypropyl]amino]methyl]-3-hydroxy- azetidine-1-carboxylate was obtained (1.5 g). Step 7: Under inert atmosphere, tert-butyl 3-[[tert-butoxycarbonyl-[(2R)-3-[[(2S)-6- (tertbutoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]- 2-hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.5 g, 1.82 mmol, 1.0 eq.) was dissolved in a solution of N-ethylethanamine in anhydrous THF (solution at 20% in THF). The mixture was stirred overnight then concentrated under vacuum. The crude product was purified on silica gel flash column chromatography. Tert-butyl 3- [[[(2R)-3-[[(2S)-2-amino-6-(tertbutoxycarbonylamino)hexanoyl]amino]-2-hydroxy- propyl]-tertbutoxycarbonyl-amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained (990 mg). Example 3.18
Figure imgf000112_0001
Step 1: Allyl N-[(2R)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a yellow oil (33.6 g) following the step 1 described in Example 3.4. Step 2: Allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained as a yellow oil (9.55 g) following the step 2 described in Example 3.4. [α]D = +0.10° (c = 10 mg/mL; MeOH) Step 3: Under inert atmosphere and magnetic stirring, allyl N-[[(2R)-oxiran-2- yl]methyl]carbamate (1.50 g, 9.54 mmol, 1.0 eq.) was dissolved in anhydrous isopropanol (19 mL). The solution was heated at 90 °C (reflux) and tert-butyl N-(3- aminopropyl)carbamate (1.70 g, 9.54 mmol, 1.0 eq.) was added by portion. The reaction mixture was stirred under reflux for 1 h then concentrated under vacuum. Tert-butyl N- [3-[[(2S)-3-(allyloxycarbonylamino)-2-hydroxypropyl]amino]propyl]carbamate was obtained as a yellow oil (3.9 g). Step 4: Under inert atmosphere and magnetic stirring, tert-butyl N-[3-[[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]amino]propyl]carbamate (3.16 g, 9.54 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (50 mL) then triethylamine (2.9 mL, 21.0 mmol, 2.2 eq.) was added. The reaction mixture was a yellow solution. Tert- butoxycarbonyl tert-butyl carbonate (4166 mg, 19.1 mmol, 2.0 eq.) was added by portion. The mixture was stirred at room temperature for 2 h then washed with water. The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(MeOH + 1% NH3), 0 to 10 % of MeOH + 1 % NH3 in 30 min). Tert-butyl N-[(2R)- 3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate was obtained as a colourless oil (2.22 g). Step 5: Under N2 atmosphere and magnetic stirring, tert-butyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (2.20 g, 5.09 mmol, 1.0 eq.) and phenylsilane (3.1 mL, 25.5 mmol, 5.0 eq.) were dissolved in anhydrous THF (25 mL). This solution was stirred under argon bubbling for 15 min then palladium-tetrakis(triphenylphosphine) (1.18 g, 1.02 mmol, 0.2 eq.) was added. The reaction mixture was stirred at room temperature for 2 h then concentrated under vacuum to give a crude dark oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/(MeOH + 1% NH3), 0 to 10 % of MeOH + 1 % NH3 in 10 min then 40 min at 10 % of MeOH + 1 % NH3). Tert-butyl N-[(2R)-3-amino-2-hydroxy-propyl]-N-[3- (tertbutoxycarbonylamino)propyl]carbamate was obtained as a brown oil (940 mg). Example 3.19
Figure imgf000113_0001
Step 1: Under inert atmosphere and magnetic stirring, tert-butyl N-(3- aminopropyl)carbamate (5.36 g, 29.8 mmol, 1.0 eq.) and N-ethyl-N-(propan-2-yl)propan- 2-amine (11 mL, 65.6 mmol, 2.2 eq.) were dissolved in anhydrous CH2Cl2 (54 mL). The solution was cooled down to 0 °C then prop-2-en-1-yl carbonochloridate (3.8 mL, 35.8 mmol, 1.2 eq.) was added dropwise. The reaction mixture was risen to room temperature, stirred for 1 h then washed with water. The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a crude orange solid. The crude was purified by silica gel flash column chromatography (heptane/ EtOAc, 0 to 100 % of EtOAc in 30 min). Tert-butyl N-[3-(allyloxycarbonylamino)propyl]carbamate was obtained as a white solid (1.13 g). Step 2: Under inert atmosphere and magnetic stirring, tert-butyl N-[3- (allyloxycarbonylamino)propyl]carbamate (7.35 g, 28.5 mmol, 1.0 eq.) was dissolved in anhydrous 1,4-dioxane (72 mL) then a solution of 4 M HCl in 1,4-dioxane (41 mL, 0.285 mol, 10 eq.) in 1,4-dioxane was added dropwise. The mixture was stirred overnight at room temperature then concentrated under vacuum to give allyl N-(3- aminopropyl)carbamate hydrochloride as a white solid (5.5 g). Step 3: To a solution of allyl N-(3-aminopropyl)carbamate hydrochloride (3.37 g, 17.3 mmol, 1.0 eq.) in isopropanol (30 mL) was added N-ethyl-N-(propan-2-yl)propan-2- amine (6.0 mL, 34.6 mmol, 2.0 eq.). The mixture was heated to 70 °C and a solution of tert-butyl [(2S)-oxiran-2-ylmethyl]carbamate (3.0 g, 17.3 mmol, 1.0 eq.) in isopropanol (9 mL) was added dropwise. The mixture was stirred for 1 h, cooled down to room temperature then concentrated under reduced pressure. Under inert atmosphere, the residue was taken up with anhydrous CH2Cl2 (60 mL) then N,N-dimethylpyridin-4-amine (0.21 g, 1.73 mmol, 0.5 eq.) and tert-butoxycarbonyl tert-butylcarbonate (5.67 g, 26.0 mmol, 1.5 eq.) was added. The resulting mixture was stirred for 12 h at room temperature then concentrated under vacuum. The crude product was purified by flash column chromatography (heptane/EtOAc 0 to 100% EtOAc in 20 min). Tert-butyl N-[3- (allyloxycarbonylamino)propyl]-N-[(2S)-3-(tertbutoxycarbonylamino)-2-hydroxy- propyl]carbamate was obtained (2.88 g). Step 4: Under inert atmosphere, tert-butyl N-[3-(allyloxycarbonylamino)propyl]-N-[(2S)- 3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]carbamate (2.88 g, 6.67 mmol, 1.0 eq.) was dissolved in CH2Cl2 (90 mL). This solution was stirred under argon bubbling for 10 min. Phenylsilane (1.7 mL, 13.3 mmol, 2.0 eq.) was added then the mixture was stirred under argon bubbling for 10 min. Palladium-tetrakis(triphenylphosphine) (0.77 g, 0.667 mmol, 0.1 eq.) was added and the mixture was stirred 4 h at room temperature. The mixture was concentrated under vacuum. The crude product was purified by silica gel flash column chromatography: CH2Cl2/(CH2Cl2/MeOH 9/1), 0 to 100% of (CH2Cl2/MeOH 9/1) in 25 min, then 100% CH2Cl2/MeOH/NH4OH 90/9/1 15 min, then 100% CH2Cl2/MeOH/NH4OH 80/19/1 for 10 min. Tert-butyl N-(3-aminopropyl)-N-[(2S)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]carbamate was obtained (2.05 g). Example 3.20
Figure imgf000114_0001
Step 1: To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (50.0 g, 0.342 mol, 1.0 eq.) in CH2Cl2 (500 mL) at -5 °C, were added N,N-diethylethanamine (143 mL, 1.03 mol, 3.0 eq.) and prop-2-en-1-yl carbonochloridate (dropwise, 44 mL, 0.411 mol, 1.2 eq.). The reaction was risen to room temperature, stirred for 2 h then concentrated under vacuum. The residue was taken up in EtOAc and washed with water (2 x 100 mL) then 1 M HCl. The organic phase was dried over Na2SO4, filtered and concentrated under vacuum. Allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate was obtained as a colourless liquid (36.45 g). Step 2: To a solution of allyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (36.45 g, 0.188 mol, 1.0 eq.) in MeOH (290 mL) was added dropwise sodium methanolate (25%, 86 mL, 0.376 mol, 2.0 eq.) 25% w/w. The reaction was stirred for 2 h at room temperature then partially concentrated. The residue was taken up in EtOAc and washed with water then brine. The organic phase was dried over Na2SO4, filtered and concentrated under vacuum. The crude product was then purified by silica gel flash column chromatography (heptane/EtOAc 0 to 100 % of EtOAc in 30 min). Allyl N-[[(2S)-oxiran-2- yl]methyl]carbamate was obtained as a colourless liquid (4.87 g). Step 3: A solution of tert-butyl N-(3-aminopropyl)carbamate (1.0 g, 5.74 mmol, 1.0 eq.) in isopropanol (8 mL) was heated to 70 °C, then a solution of allyl N-[[(2S)-oxiran-2- yl]methyl]carbamate (0.90 g, 5.74 mmol, 1.0 eq.) in isopropanol (2 mL) was added dropwise over 15 min. The reaction mixture was stirred 1 h at 70 °C then concentrated under vacuum. Tert-butyl N-[3-[[(2R)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]propyl]carbamate was obtained. Step 4: To a solution of tert-butyl N-[3-[[(2R)-3-(allyloxycarbonylamino)-2-hydroxy- propyl]amino]propyl]carbamate (1.90 g, 5.73 mmol, 1.0 eq.) in ethanol (35 mL) was added tert-butoxycarbonyl tert-butyl carbonate (1.25 g, 5.73 mmol, 1.0 eq.). The reaction mixture was stirred 18 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 96/4). Tert-butyl N-[(2S)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate was obtained as a colourless oil (1.58 g). Step 5: Under inert atmosphere, to a solution of tert-butyl N-[(2S)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[3-(tert- butoxycarbonylamino)propyl]carbamate (1.6 g, 3.71 mmol, 1.0 eq.) in anhydrous CH2Cl2 (74 mL) was added phenylsilane (2.4 mL, 18.5 mmol, 5.0 eq.). The mixture was stirred under bubbling of N2 for 20 min then palladium-tetrakis(triphenylphosphine) (300 mg, 0.260 mmol, 0.07 eq.) was added. The reaction mixture was stirred for 2 h at room temperature, then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography. Tert-butyl N-[(2S)-3-amino-2-hydroxy-propyl]-N-[3- (tertbutoxycarbonylamino)propyl]carbamate was obtained (1.25 g). Example 3.21
Figure imgf000116_0001
Step 1: A solution of tert-butyl (oxiran-2-ylmethyl)carbamate (0.96 mL, 5.77 mmol, 1.0 eq.) in 5 mL of isopropanol was heated to reflux. Then a solution of (2-aminoethoxy)(tert- butyl)dimethylsilane (1.07 g, 5.77 mmol, 1.0 eq.) in 2 mL of isopropanol was added dropwise. The mixture was stirred for 1 h at 82 °C then concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography to give 260 mg of pure tert-butyl N-[3-[2-[tert- butyl(dimethyl)silyl]oxyethylamino]-2-hydroxy-propyl]carbamate. A second sample was obtained containing the expected product and starting material. The residue of this batch was suspended in EtOAc and washed with 0.5 N HCl then brine. The monitoring by TLC and LCMS showed only one product. The organic phase was dried over MgSO4 and concentrated to dryness to give 420 mg of tert-butyl N-[3-[2-[tert- butyl(dimethyl)silyl]oxyethylamino]-2-hydroxy-propyl]carbamate in mixture with a by- product of TBDMS protecting group. Step 2: Under inert atmosphere, Fmoc-Lys(Boc)-OH (582 mg, 1.2 mmol, 1.0 eq.) and 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine;hydrochloride (254 mg, 1.33 mmol, 1.33 eq.) were dissolved in anhydrous pyridine (10 mL). Tert-butyl N-[3-[2-[tert- butyl(dimethyl)silyl]oxyethylamino]-2-hydroxy-propyl]carbamate (420 mg, 1.20 mmol, 1.2 eq.) was added. The mixture was stirred overnight at room temperature then concentrated to dryness. The residue was suspended in EtOAc then washed with 1% citric acid, saturated NaHCO3 then brine. The organic layer was dried over MgSO4 then concentrated under vacuum. The crude product was purified by flash column chromatography.9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1-[[3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]-[2- [tertbutyl(dimethyl)silyl]oxyethyl]carbamoyl]pentyl]carbamate was obtained (580 mg). Step 3: 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1-[[3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]-[2- [tertbutyl(dimethyl)silyl]oxyethyl]carbamoyl]pentyl]carbamate (580 mg, 0.73 mmol, 1.0 eq.) was suspended in 15 mL of a solution of N-ethylethanamine at 20% in THF. The mixture was stirred overnight then concentrated to dryness to give 498 mg of a crude product. The expected product tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-[2-[tertbutyl(dimethyl)silyl]oxyethyl]amino]-2- hydroxy-propyl]carbamate was obtained in mixture with tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-(2-hydroxyethyl)amino]-2-hydroxy- propyl]carbamate. This crude mixture was used without purification in the next step. Step 4: Under inert atmosphere, the mixture of tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-[2-[tertbutyl(dimethyl)silyl]oxyethyl]amino]-2- hydroxy-propyl]carbamate and tert-butyl N-[3-[[(2S)-2-amino-6-(tert- butoxycarbonylamino)hexanoyl]-(2-hydroxyethyl)amino]-2-hydroxy-propyl]carbamate (300 mg, 0.65 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (2.6 mL). This solution was cooled down to 5 °C and triethylamine (107 µL, 0.78 mmol, 1.2 eq.) then chloro(triethyl)silane (119 µL, 0.72 mmol, 1.1 eq.) were added. The reaction mixture was stirred overnight then concentrated under vacuum. The crude product was purified by silica gel column chromatography. A mixture of tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-(2-triethylsilyloxyethyl)amino]-2-triethylsilyloxy- propyl]carbamate and tert-butyl N-[3-[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]-[2-[tertbutyl(dimethyl)silyl]oxyethyl]amino]-2- triethylsilyloxypropyl]carbamate was obtained (200 mg). Example 3.22
Figure imgf000117_0001
Step 1: Under inert atmosphere and magnetic stirring, (2R)-1-amino-3-chloropropan-2- ol hydrochloride (3.82 g, 26.2 mmol, 1.0 eq.) and triethylamine (15 mL, 0.105 mol, 4.0 eq.) were dissolved in anhydrous CH2Cl2 (75 mL) then prop-2-en-1-yl carbonochloridate (4.2 mL, 39.3 mmol, 1.5 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 1 h then washed with water (15 mL) and brine (15 mL). The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH for 30 min). Allyl N-[(2R)-3-chloro-2-hydroxy- propyl]carbamate was obtained as a yellow oil (1.95 g). Step 2: Under inert atmosphere and magnetic stirring, allyl N-[(2R)-3-chloro-2-hydroxy- propyl]carbamate (1.93 g, 9.97 mmol, 1.0 eq.) was dissolved in anhydrous MeOH (50 mL). Sodium methanolate (25%, 4.6 mL, 19.9 mmol, 2.0 eq.) in MeOH was added dropwise to this solution. The reaction mixture was stirred for 2 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc (100 mL) and washed with 25 mL of saturated NH4Cl then brine (15 mL). The organic layer was dried with MgSO4, filtered and concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography: CH2Cl2/MeOH, 0 to 10 % of MeOH for 30 min. Allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained as a colourless oil (1.0 g). [α]D = +0.079° (c = 7.3 mg/mL; MeOH) Step 3: Allyl N-[[(2R)-oxiran-2-yl]methyl]carbamate (886 mg, 5.64 mmol, 1.0 eq.) was dissolved in isopropanol (15 mL). The solution was heated to reflux and a solution of tert- butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.2 g, 5.64 mmol, 1.0 eq.) in isopropanol (5 mL) was added dropwise. The reaction mixture was stirred for 1 h under reflux then concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography to give tert-butyl 3-[[[(2S)-3- (allyloxycarbonylamino)-2-hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1- carboxylate (1.0 g). Step 4: To a solution at 0-5 °C of tert-butyl 3-[[[(2S)-3-(allyloxycarbonylamino)-2- hydroxypropyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (1.0 g, 2.78 mmol, 1.0 eq.) in CH2Cl2 (20 mL) were added prop-2-en-1-yl carbonochloridate (0.32 mL, 3.00 mmol, 1.1 eq.) then N,N-diethylethanamine (0.97 mL, 6.96 mmol, 2.5 eq.). The mixture was stirred for 20 min at 0-5 °C then risen to room temperature for 1 h. The mixture was washed with water and brine. The organic layer was dried over MgSO4 and concentrated under vacuum. Tert-butyl 3-[[allyloxycarbonyl-[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate was obtained (880 mg). Step 5: To a solution of tert-butyl 3-[[allyloxycarbonyl-[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]amino]methyl]-3-hydroxy-azetidine-1-carboxylate (880 mg, 1.98 mmol, 1.0 eq.) in dioxane (4 mL) was added a solution of 4 N HCl in dioxane (10 mL, 39.7 mmol, 20 eq.). The mixture was stirred for 3 h at room temperature, concentrated under vacuum then triturated with diisopropylether. Allyl N-[(2R)-3-(allyloxycarbonylamino)-2- hydroxy-propyl]-N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained (775 mg). Step 6: Under inert atmosphere, Fmoc-Lys(Boc)-OH (1.04 g, 2.14 mmol, 1.05 eq.) was dissolved in anhydrous DMF (18 mL). The solution was cooled down to 0-5°C and N- ethyl-N-isopropyl-propan-2-amine (0.89 mL, 5.10 mmol, 2.5 eq.), allyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (775 mg, 2.04 mmol, 1.0 eq.) then benzotriazol-1-yloxy(tripyrrolidin-1- yl)phosphonium hexafluorophosphate (1.06 g, 2.04 mmol, 1.0 eq.) were added. The reaction mixture was stirred for 15 min at 0-5 °C then risen to room temperature and stirred overnight. The reaction was diluted with EtOAc and washed twice with water. The organic layer was dried over MgSO4 and concentrated under vacuum. Allyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert-butoxycarbonylamino)-2- (9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy-azetidin-3- yl]methyl]carbamate was obtained as an oil (626 mg). Step 7: Allyl N-[(2R)-3-(allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-6-(tert- butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate (626 mg, 0.79 mmol, 1.0 eq.) was dissolved in 10 mL of a 20% solution of N-ethylethanamine in THF (2 mL, 19.3 mmol, 25 eq.). The mixture was stirred for 2 h then concentrated under vacuum. Allyl N-[(2R)-3- (allyloxycarbonylamino)-2-hydroxy-propyl]-N-[[1-[(2S)-2-amino-6-(tert- butoxycarbonylamino)hexanoyl]-3-hydroxyazetidin-3-yl]methyl]carbamate was obtained (270 mg). Example 3.23
Figure imgf000119_0001
Step 1: To a solution of tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-1-carboxylate (1.35 g, 6.47 mmol, 1.0 eq.) in CH2Cl2 (35 mL) at 5 °C, were added N-ethyl-N-isopropyl- propan-2-amine (1.6 mL, 9.06 mmol, 1.4 eq.) and allyl chloroformate (0.76 mL, 7.12 mmol, 1.1 eq.). The reaction mixture was stirred for 3 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed with water then with saturated NaHCO3. The organic phase was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 96/4). Tert-butyl 3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidine-1-carboxylate was obtained as a colourless oil (1.7 g). Step 2: To a solution of tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine- 1-carboxylate (1.74 g, 6.08 mmol, 1.0 eq.) in CH2Cl2 (45 mL) was added 4 M hydrogen chloride (15 mL, 60.0 mmol, 10 eq.). The reaction mixture was stirred for 36 h at room temperature then concentrated under vacuum. The crude product was triturated twice with diisopropyl ether. Allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained as a beige solid (1.26 g). Step 3:To a solution of Fmoc-Lys(Boc)-OH (2.73 g, 5.66 mmol, 1.0 eq.) in DMF (15 mL) at 5 °C, were added allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1.26 g, 5.66 mmol, 1.0 eq.), N-ethyl-N-isopropyl-propan-2-amine (2.5 mL, 14.1 mmol, 2.5 eq.) and benzotriazol-1-yloxy(tripyrrolidin-1-yl)phosphonium;hexafluorophosphate (3.24 g, 6.22 mmol, 1.1 eq.). The mixture was stirred for 18 h at room temperature then concentrated under vacuum. The residue was taken up in EtOAc and washed twice with water. The organic phase was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 96/4). 9H-fluoren-9-ylmethyl N-[(1S)-1-[3-[(allyloxycarbonylamino)methyl]-3-hydroxy- azetidine-1-carbonyl]-5-(tert-butoxycarbonylamino)pentyl]carbamate was obtained as a colourless oil (1.19 g). [α]D = +1.3° (c=0.31, MeOH) Step 4: To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[3- [(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine-1-carbonyl]-5-(tert- butoxycarbonylamino)pentyl]carbamate (1.19 g, 1.87 mmol, 1.0 eq.) in THF (30 mL) was added N-ethylethanamine (4.0 mL, 38.1 mmol, 20 eq.). The mixture was stirred for 3 h at room temperature then concentrated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 90/10). Tert-butyl N-[(5S)-6-[3- [(allyloxycarbonylamino)methyl]-3-hydroxyazetidin-1-yl]-5-amino-6-oxo- hexyl]carbamate was obtained as a colourless oil (300 mg). [α]D = +5.5 (c=0.38, MeOH) Example 3.24
Figure imgf000121_0001
Step 1: Under inert atmosphere and magnetic stirring, Fmoc-(D)-Dab(Boc)-OH (10 g, 22.0 mmol, 1.0 eq.) and N,N-diethylethanamine (3.7 mL, 26.4 mmol, 1.2 eq.) were dissolved in anhydrous THF (188 mL). This solution was cooled down at -5 °C and isobutyl carbonochloridate (3.1 mL, 24.2 mmol, 1.1 eq.) was added dropwise. The mixture turned white heterogeneous. The mixture was stirred for 30 min. A solution of 2.4 M lithium aluminium hydride in THF (14 mL, 33.0 mmol, 1.5 eq.) was added dropwise at -5 °C. A gaz release and an exothermic reaction was observed. The reaction mixture was risen to 0 °C and stirred for 3 h. 500 mL of EtOAc were added, the mixture was hydrolysed with 100 mL of water then risen to room temperature and stirred for 30 min. Aluminium salts were removed by filtration then the phases were separated. The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a crude white solid. The solid was triturated with diisopropyl ether. The suspension was filtered to give a first batch of product as white powder (2.8 g). The filtrate was concentrated to dryness. The residue was triturated with diisopropyl ether and the suspension obtained was filtered to give a second batch of product as a white powder (3.66 g). The two batches of white solid were mixed to give 9H-fluoren-9-ylmethyl N-[(1R)-3-(tert- butoxycarbonylamino)-1-(hydroxymethyl)propyl]carbamate as a white solid (6.46 g). Step 2: Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1R)-3- (tert-butoxycarbonylamino)-1-(hydroxymethyl)propyl]carbamate (6.46 g, 15.1 mmol, 1.0 eq.), N-ethyl-N-isopropyl-propan-2-amine (16 mL, 90.9 mmol, 6.0 eq.) and N,N- dimethylpyridin-4-amine (185 mg, 1.51 mmol, 0.1 eq.) were dissolved in anhydrous CH2Cl2 (70 mL). The mixture was cooled down to 0 °C and a solution of tert-butyl-chloro- dimethyl-silane (7.21 g, 45.4 mmol, 3.0 eq.) in anhydrous CH2Cl2 (10 mL) was added dropwise. The mixture was stirred overnight at 0 °C then washed with water. The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a crude brown oil. The crude was purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 50 % of EtOAc in 30 min).9H-fluoren-9-ylmethyl N-[(1R)-3-(tert- butoxycarbonylamino)-1-[[tert-butyl(dimethyl)silyl]oxymethyl]propyl]carbamate was obtained as an oil (5.55 g). Step 3: Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1R)-3- (tert-butoxycarbonylamino)-1-[[tert-butyl(dimethyl)silyl]oxymethyl]propyl]carbamate (5.55 g, 10.3 mmol, 1.0 eq.) and N-ethylethanamine (12 mL, 10.3 mmol, 1 eq.) were dissolved in anhydrous THF (48 mL). The reaction mixture was stirred overnight at room temperature then concentrated under vacuum to give a crude oil. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH, 0 to 10 % of MeOH in 30 min). Tert-butyl N-[(3R)-3-amino-4-[tert-butyl(dimethyl)silyl]oxybutyl]carbamate was obtained as a yellow oil (2.94 g). Step 4: Under inert atmosphere and magnetic stirring, Fmoc-Lys(Boc)-OH (0.92 g, 1.92 mmol, 1.2 eq.) and tert-butyl N-[(3R)-3-amino-4-[tert- butyl(dimethyl)silyl]oxybutyl]carbamate (510 mg, 1.60 mmol, 1.0 eq.) were dissolved in anhydrous CH2Cl2 (10 mL) then pyridine (5 mL) was added. A yellow solution was obtained. The reaction mixture was cooled down to 0 °C then a solution of propylphosphonic anhydride at 50 % in DMF (1.9 mL, 3.20 mmol, 2.0 eq.) was added dropwise. The reaction mixture was risen to room temperature and stirred overnight. The mixture was diluted in 50 mL of CH2Cl2 and washed with 10 mL of water. The organic layer was dried over MgSO4, filtered and concentrated under vacuum to give a yellow oil. The crude product was purified by silica gel flash column chromatography (heptane/EtOAc, 0 to 100 % of EtOAc in 30 min).9H-fluoren-9-ylmethyl N-[(1S)-5-(tert- butoxycarbonylamino)-1-[[(1R)-3-(tert-butoxycarbonylamino)-1- [[tertbutyl(dimethyl)silyl]oxymethyl]propyl]carbamoyl]pentyl]carbamate was obtained as a white gum (1.07 g). Step 5: Under inert atmosphere and magnetic stirring, 9H-fluoren-9-ylmethyl N-[(1S)-5- (tert-butoxycarbonylamino)-1-[[(1R)-3-(tert-butoxycarbonylamino)-1-[[tert- butyl(dimethyl)silyl]oxymethyl]propyl]carbamoyl]pentyl]carbamate (1.07 g, 1.39 mmol, 1.0 eq.) and N-ethylethanamine (4.0 mL, 1.39 mmol, 1.0 eq.) were dissolved in anhydrous THF (16 mL). The reaction mixture was stirred for 4 h then concentrated under vacuum. Tert-butyl N-[(5S)-5-amino-6-[[(1R)-3-(tert-butoxycarbonylamino)-1- [[tert-butyl(dimethyl)silyl]oxymethyl]propyl]amino]-6-oxohexyl]carbamate was obtained as an oil (775 mg). Example 3.25
Figure imgf000123_0001
Step 1: To a solution of (2R)-3-aminopropane-1,2-diol (12.70 g, 0.139 mol, 1.0 eq.) in ethanol (400 mL) was added slowly tert-butoxycarbonyl tert-butyl carbonate (33.46 g, 0.153 mol, 1.1 eq.). The mixture was stirred at room temperature for 1h40 then concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 100/0 to 90/10). Tert-butyl N-[rac-(2R)-2,3- dihydroxypropyl]carbamate was obtained as a white solid (30.3 g). Step 2: To a solution of tert-butyl N-[rac-(2R)-2,3-dihydroxypropyl]carbamate (30 g, 0.149 mol, 1.0 eq.) in pyridine (200 mL) at 0-5 °C, was added dropwise (30 min) methanesulfonyl chloride (13 mL, 0.164 mol, 1.1 eq.). The mixture was stirred for 10 min. This solution was added dropwise (30 min) to a solution of sodium hydroxide (17.89 g, 0.447 mol, 3.0 eq.) in water (200 mL) and DMSO (132 mL), stirred at 0-5 °C. The resulting reaction mixture was stirred for 10 min at 0 °C, poured into ice water (1.6 L) then extracted with a mixture of heptane (400 mL) and EtOAc (1.6 L). The organic layers were combined, washed with water and brine then dried over MgSO4, filtered and co- evaporated thrice with toluene. The crude product was purified by silica gel flash column chromatography (heptane/EtOAc 100/0 to 70/30). Tert-butyl N-[[(2R)-oxiran-2- yl]methyl]carbamate was obtained (11.0 g). [α]D = +4.4° (c = 0.84 mg/mL; MeOH) Step 3: Under inert atmosphere and at 0-5 °C, tert-butyl 3-(aminomethyl)-3- hydroxyazetidine-1-carboxylate (6 g, 28.2 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (100 mL) and THF (100 mL). N,N-diethylethanamine (8.0 mL, 57.4 mmol, 2.04 eq.) and prop-2-en-1-ylcarbonochloridate (3.1 mL, 28.7 mmol, 1.02 eq.) were added. The mixture was stirred at 0-5 °C for 20 min, risen to room temperature, stirred for 5 h then concentrated under vacuum. The residue was taken up with EtOAc and washed with water and brine. The organic layer was dried over MgSO4 before concentration under vacuum. The crude product was purified through silica gel column chromatography (heptane/EtOAc 50/50). Tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy- azetidine-1-carboxylate was obtained as a gum (6.96 g). Step 4: To a solution of tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine- 1-carboxylate (6.96 g, 23.8 mmol, 1.0 eq.) in CH2Cl2 (48 mL) was added 4 M hydrogen chloride (15 mL, 60.0 mmol, 2.5 eq.). The mixture was stirred for 5 h then 24 mL of MeOH were added to homogenize the mixture. The mixture was stirred for 1 h then concentrated under vacuum. The crude product was purified by silica gel column chromatography to give allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride as a light brown gum (5.36 g). Step 5: To a suspension of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1000 mg, 3.59 mmol, 1.0 eq.) in 2-propanol (20 mL) at 70 °C was added dropwise, during 15 min, a solution of tert-butyl[(2R)-oxiran-2-ylmethyl]carbamate (635 mg, 3.59 mmol, 1.0 eq.) and N,N-diethylethanamine (0.76 mL, 5.39 mmol, 1.5 eq.) in 2- propanol (5 mL). The mixture was stirred at 70 °C for 1h10 then concentrated under vacuum. The residue was suspended in CH2Cl2. A suspension was obtained then filtered. The filtrate was purified by silica gel flash column chromatography (CH2Cl2/MeOH 100/0 to 90/10). Tert-butyl N-[(2R)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxyazetidin-1-yl]-2-hydroxy-propyl]carbamate was obtained (440 mg). Step 6: Under inert atmosphere, at room temperature, tert-butyl N-[(2R)-3-[3- [(allyloxycarbonylamino)methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (1100 mg, 2.97 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (40 mL). Phenylsilane (1.8 mL, 14.8 mmol, 5.0 eq.) was added and the resulting mixture was stirred under argon bubbling for 15 min, then palladium-tetrakis(triphenylphosphine) (172 mg, 0.148 mmol, 0.05 eq.) was added. The reaction mixture was stirred for 3 h then concentrated under vacuum. The crude material was purified by silica gel flash column chromatography (100% CH2Cl2 then CH2Cl2/MeOH 90/10 then CH2Cl2/MeOH/(7 N NH3 in MeOH) 80/19/1). Tert-butyl N-[(2R)-3-[3-(aminomethyl)-3-hydroxy-azetidin-1-yl]-2- hydroxy-propyl]carbamate was obtained (370 mg). Step 7: To a solution of tert-butyl N-[(2R)-3-[3-(aminomethyl)-3-hydroxy-azetidin-1-yl]-2- hydroxy-propyl]carbamate (370 mg, 1.26 mmol, 1.0 eq.) in CH2Cl2 (13 mL) and pyridine (6.5 mL) was added Fmoc-Lys(Boc)-OH (610 mg, 1.26 mmol, 1.0 eq.). The mixture was cooled down to 0-5 °C and stirred at this temperature. Then 3- (ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (267 mg, 1.39 mmol, 1.1 eq.) was added. The reaction mixture was risen to room temperature, stirred overnight and concentrated under vacuum. The residue was taken up in a small volume of MeOH and EtOAc, washed with water acidified with citric acid, saturated NaHCO3 then brine. The organic phase was dried over MgSO4 then concentrated under vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH 100/0 to 90/10). 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1-[[1-[(2R)-3-(tert- butoxycarbonylamino)-2-hydroxy-propyl]-3-hydroxyazetidin-3- yl]methylcarbamoyl]pentyl]carbamate was obtained as a white powder (315 mg). Step 8: To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-5-(tert-butoxycarbonylamino)-1- [[1-[(2R)-3-(tert-butoxycarbonylamino)-2-hydroxy-propyl]-3-hydroxy-azetidin-3- yl]methylcarbamoyl]pentyl]carbamate (315 mg, 0.434 mmol, 1.0 eq.) in THF (15 mL) was added N-ethylethanamine (1.5 mL, 14.1 mmol, 32 eq.). The mixture was stirred at room temperature for 4 h then concentrated under vacuum. The crude product was purified by flash column chromatography (CH2Cl2/(CH2Cl2-MeOH-NH4OH 25% in H2O 50-45-5) 100- /0 to 50/50). Tert-butyl N-[(2R)-3-[3-[[[(2S)-2-amino-6- (tertbutoxycarbonylamino)hexanoyl]amino]methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy- propyl]carbamate was obtained as a white sticky foam (214 mg). [α]D= +5.0° (c = 0.44 mg/mL; MeOH) Example 3.26
Figure imgf000125_0001
Step 1: Tert-butyl N-[rac-(2R)-2,3-dihydroxypropyl]carbamate was obtained as a white solid (30.3 g) following the step 1 described in example 3.25. Step 2: Tert-butyl N-[[(2R)-oxiran-2-yl]methyl]carbamate was obtained (11 g) following the step 2 described in example 3.25. Step 3: Tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine-1-carboxylate was obtained as a gum (6.96 g) following the step 3 described in example 3.25. Step 4: To a solution of tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine- 1-carboxylate (4.3 g, 14.3 mmol, 1 eq.) in CH2Cl2 (30 mL) at room temperature was added 4M hydrogen chloride (9.0 mL, 36.0 mmol, 2.5 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum. Allyl N-[(3- hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained as a light brown gum (3.2 g). Step 5: To a solution of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (1.5 g, 6.60 mmol, 1.0 eq.) in 2-propanol (39 mL) in MeOH (13 mL) at 70 °C, was added dropwise (during 30 min) a solution of tert-butyl [(2R)-oxiran-2-ylmethyl]carbamate (1.174 mg, 6.64 mmol, 1.1 eq.) and N,N-diethylethanamine (1.4 mL, 9.90 mmol, 1.5 eq.) in 2-propanol (13 mL). The mixture was stirred at 70 °C for 1h20 then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 100/0 to 90/10). Tert-butyl N-[(2R)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate was obtained (1.61 g). Step 6: To a solution of tert-butyl N-[(2R)-3-[3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (1.0 g, 2.78 mmol, 1.0 eq.) in CH2Cl2 (30 mL) and MeOH (10 mL) stirred at room temperature, was added 4 M hydrogen chloride (4.2 mL, 16.7 mmol, 6.0 eq.). The mixture was stirred overnight then concentrated under vacuum. Allyl N-[[1-[(2R)-3-amino-2-hydroxy-propyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate dihydrochloride was obtained as a foaming sticky gum (883 mg). [α]D = -5.3° (c=0.43 mg/mL, MeOH) Step 7: To a solution of allyl N-[[1-[(2R)-3-amino-2-hydroxy-propyl]-3-hydroxy-azetidin- 3-yl]methyl]carbamate dihydrochloride (880 mg, 2.60 mmol, 1.0 eq.) in CH2Cl2 (26 mL) and anhydrous pyridine (13 mL) were added N,N-diethylethanamine (1.1 mL, 7.79 mmol, 3.0 eq.) and Fmoc-Lys(Boc)-OH (1.25 g, 2.60 mmol, 1.0 eq.). The mixture was cooled down and stirred at 0-5 °C, then 3-(ethyliminomethyleneamino)-N,N-dimethyl- propan-1-amine hydrochloride (548 mg, 2.86 mmol, 1.1 eq.) was added. The mixture was risen to room temperature, stirred overnight then concentrated under vacuum. The residue was taken up in a small volume of MeOH and EtOAc then washed with an aqueous solution of citric acid, saturated NaHCO3 then brine. The organic phase was dried over MgSO4 before concentration to dryness. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH 100/0 to 90/10). Tert-butyl N- [(5S)-6-[[(2R)-3-[3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy- propyl]amino]-5-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate was obtained (815 mg). Step 8: To a solution of tert-butyl N-[(5S)-6-[[(2R)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5-(9H-fluoren-9- ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate (815 mg, 1.10 mmol, 1.0 eq.) in THF (37 mL) was added N-ethylethanamine (3.7 mL, 35.8 mmol, 32.5 eq.). The mixture was stirred at room temperature for 4 h then concentrated under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/[CH2Cl2-MeOH-NH4OH 25% in water) 50-45-5] 100/0 to 50/50). Tert-butyl N-[(5S)-6-[[(2R)-3-[3- [(allyloxycarbonylamino)methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5- amino-6-oxohexyl]carbamate was obtained as a colourless gum (525 mg). [α]D = + 7.05° (c=0.67 mg/mL, MeOH) Example 3.27
Figure imgf000127_0001
Step 1: To a solution of (2S)-1-amino-3-chloro-propan-2-ol hydrochloride (15 g, 0.103 mol) in CH2Cl2 (60 mL) and MeOH (10 mL) at 0°C, were added N,N-diethylethanamine (16 mL, 0.113 mol) and dropwise a solution of tert-butoxycarbonyl tert-butyl carbonate (23 g, 0.105 mol) in CH2Cl2 (10 mL). The reaction mixture was stirred 2h at room temperature then diluted in CH2Cl2 and washed with water then brine. The organic phase was dried over Na2SO4 before concentration to dryness. Tert-butyl N-[(2S)-3-chloro-2- hydroxy-propyl]carbamate was obtained as a colourless oil (21 g). [α]D = +8.7° (c=0.31, MeOH) Step 2: To tert-butyl N-[(2S)-3-chloro-2-hydroxy-propyl]carbamate (10.0 g, 47.7 mmol, 1.0 eq.) in MeOH (90 mL) at room temperature, was added dropwise 4.3 M sodium methanolate (18 mL, 77.4 mmol, 1.62 eq.). The reaction mixture was stirred 2 h at room temperature then partially concentrated. The residue was taken up with EtOAc and washed with water and brine. The organic phase was dried over Na2SO4 and filtered before concentration under vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH 98/2). Tert-butyl N-[[(2S)-oxiran-2-yl]methyl]carbamate was obtained as a white solid (4.8 g). Step 3: Tert-butyl 3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidine-1-carboxylate was obtained as a gum (6.96 g) following the step 3 described in example 3.25. Step 4: Allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride was obtained as a light brown gum (5.36 g) following step 4 described in example 3.25. Step 5: To a solution of allyl N-[(3-hydroxyazetidin-3-yl)methyl]carbamate hydrochloride (3.37 g, 12.1 mmol, 1.0 eq.) in 2-propanol (70 mL) and MeOH (20 mL) at 70 °C, was added dropwise (during 15 min) a solution of tert-butyl [(2S)-oxiran-2- ylmethyl]carbamate (2.1 g, 12.1 mmol, 1.0 eq.) and N,N-diethylethanamine (2.6 mL, 18.2 mmol, 1.5 eq.) in isopropanol (20 mL). The mixture was stirred at 72 °C for 1 h then the mixture was stirred at 85 °C for 20 min to complete the reaction. The mixture was concentrated and coevaporated with toluene. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH from 100/0 to 90/10). Tert-butyl N-[(2S)-3-[3-[(allyloxycarbonylamino)methyl]-3-hydroxy- azetidin-1-yl]-2-hydroxy-propyl]carbamate was obtained (1.63 g). Step 6: To a solution of tert-butyl N-[(2S)-3-[3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidin-1-yl]-2-hydroxy-propyl]carbamate (500 mg, 1.39 mmol, 1.0 eq.) in CH2Cl2 (14 mL) was added 4 M hydrogen chloride (1.7 mL, 6.96 mmol, 5.0 eq.). The mixture was stirred at room temperature. After 5 min, a precipitate appeared.1,4-dioxane (10 mL) was added and the mixture was stirred for 30 min. MeOH (5 mL) was added to homogenize the solution. The mixture was stirred for 2h40 and 4 M hydrogen chloride (1.0 mL, 4.00 mmol, 2.9 eq.) was added to complete the reaction. It was stirring for additional 1 h and concentrated under vacuum. Allyl N-[[1-[(2S)-3-amino-2-hydroxy- propyl]-3-hydroxy-azetidin-3-yl]methyl]carbamate dihydrochloride was obtained as a foaming sticky gum (500 mg). Step 7: Under inert atmosphere, allyl N-[[1-[(2S)-3-amino-2-hydroxy-propyl]-3-hydroxy- azetidin-3-yl]methyl]carbamate dihydrochloride (500 mg, 1.46 mmol, 1.0 eq.) was dissolved in anhydrous CH2Cl2 (15 mL). N,N-diethylethanamine (0.62 mL, 4.38 mmol, 3.0 eq.), anhydrous pyridine (7.5 mL) and Fmoc-Lys(Boc)-OH (705 mg, 1.46 mmol, 1.0 eq.) were added. The mixture was cooled down to room temperature and stirred at 0-5 °C then 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine hydrochloride (308 mg, 1.61 mmol, 1.1 eq.) was added. The reaction mixture was risen to room temperature, stirred overnight and concentrated to dryness. The residue was taken up in a small volume of MeOH and EtOAc and washed with an aqueous solution of citric acid, saturated NaHCO3, water then brine. The organic phase was dried over MgSO4 before concentration under vacuum. The crude product was purified by silica gel flash column chromatography (CH2Cl2/MeOH from 100/0 to 90/10). Tert-butyl N-[(5S)-6-[[(2S)- 3-[3-[(allyloxycarbonylamino)methyl]-3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]- 5-(9H-fluoren-9-ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate was obtained (365 mg). Step 8: To a solution of tert-butyl N-[(5S)-6-[[(2S)-3-[3-[(allyloxycarbonylamino)methyl]- 3-hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5-(9H-fluoren-9- ylmethoxycarbonylamino)-6-oxo-hexyl]carbamate (365 mg, 0.51 mmol, 1.0 eq.) in THF was added N-ethylethanamine (2 mL, 19.3 mmol, 37.6 eq.). The mixture was stirred overnight at room temperature then concentrated under vacuum. The crude product was purified by silica gel column chromatography (100% CH2Cl2 then CH2Cl2/MeOH/(NH3 7M in MeOH) 90/9/1). Tert-butyl N-[(5S)-6-[[(2S)-3-[3-[(allyloxycarbonylamino)methyl]-3- hydroxy-azetidin-1-yl]-2-hydroxy-propyl]amino]-5-amino-6-oxohexyl]carbamate (135 mg). Synthesis of compounds of formula (I) Example 4: Loading measurement Before starting the synthesis of peptides, the loading of the first amino acid (or dipeptide) on resin was measured following the steps 1, 2 or 3 (depending on the resin used) and step 4 described below. Step 1: Resin swelling. The resin (5.10-4 mol, 1.0 eq.) was put in the reaction vessel. CH2Cl2 (3.0 mL) was added to immerse all the resin, the mixture was shaken for 30 min. The solvent was removed by filtration under vacuum. Step 2: Esterification on the 2-chlorotrityl resin. The first amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was dissolved in 2.5 mL of CH2Cl2. DIPEA (2.0 mmol, 4.0 eq.) was added and the solution was mixed thoroughly by pipetting. This solution was immediately added to the swollen 2-chlorotrityl resin (5.10-4 mol, 1.0 eq.). The reaction vessel was closed with a cap and the reaction mixture was shaken for 4 h then filtered. DMF (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered. This washing was repeated 3 times. A solution of CH2Cl2/MeOH/DIPEA 80/15/5 (2 mL) was added to the resin. The reaction vessel was closed with a cap and the system was shaken for 10 min. The mixture was filtered under vacuum and this capping procedure was repeated once. DMF (2.5 mL) was added to the resin and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times. Step 3: For the 1,3-diaminopropane trityl resin, 1,5-diaminopentane trityl resin and Rink amide resin, the anchoring of the first amino acid or dipeptide was performed with a standard coupling procedure. DMF (2.5 mL) was added to the swollen resin (5.10-4 mol, 1.0 eq.). The mixture was shaken for 20 seconds, and the solvent was removed under vacuum. The protected amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was added followed by 4.0 mL of DMF. The mixture was shaken for 20 seconds then DIPEA (2.0 mmol, 4.0 eq.) was added and the mixture was shaken until dissolution of the amino-acid or dipeptide. HBTU (1.45 mmol, 2.9 eq.) was added then the reaction vessel was closed with a cap and shaken for 2 h. The mixture was filtered under vacuum. DMF (2.5 mL) was added to the resin and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times. This coupling step was repeated once. MeOH (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered under vacuum. CH2Cl2 (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered under vacuum. The washing with CH2Cl2 was repeated 5 times. Step 4: Loading measurement. Preparation of the sample solution. The resin obtained in step 2 or 3 was dried for 2 h under vacuum then air-dried overnight. About 10 mg of the dry resin was introduced in an Eppendorf tube. 200 µL of a solution of DMF/piperidine 80/20 were added. The mixture was shaken for 10 min then the supernatant was collected and introduce in a 25 mL volumetric flask. This Fmoc-cleavage was repeated once, and the supernatants were combined. The volume was completed to 25 mL with ethanol. Preparation of a standard solution. To 5 mg of the corresponding Fmoc-amino acid or Fmoc-dipeptide was added 400 µL of a solution DMF/piperidine 80/20. This mixture was transferred into a 25 mL volumetric flask and the volume was completed to 25 mL with ethanol. A blank solution was prepared according to the procedure described by Al Musaimi & al, ACS Combinatorial Science 2019, 21, 717-721. The loading was measured following the calculation method described by Al Musaimi & al, ACS Combinatorial Science 2019, 21, 717-721. Example 5: Synthesis of peptide with -NH(CH2)3NH(CH2)3NH2 with or without C- and/or N-substitution, -NH(CH2)3NH(CH2)4NH2 with or without C- and/or N-substitution, - NH(CH2)2NH(CH2)2NH2 with or without C- and/or N-substitution, -NH(CH2)3NH2 with C- and/or N-substitution, -NH(CH2)2NH2 with C- and/or N-substitution and NH CH2CH=CHCH2NH2 scaffolds at the C-terminal position Step 1: The 2-chlorotrityl resin was swollen following step 1 of example 4. Step 2: Esterification on the 2-chlorotrityl resin. The first amino-acid or dipeptide was esterified on the 2-chlorotrityl resin following Step 2 from example 1. At the end of the esterification step, a solution of DMF/piperidine 80:20 (4.0 mL) was added to the resin. The mixture was shaken for 20 minutes then filtered under vacuum to remove the solvent. This step was repeated once. DMF was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times. MeOH was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered. CH2Cl2 was added (2.5 mL) and the mixture was shaken for 20 seconds then filtered. The resin was dried 15 minutes under vacuum. Step 3: Standard coupling procedure and Fmoc removal. DMF (2.5 mL) was added to the swollen resin (5.10-4 mol, 1.0 eq.). The mixture was shaken for 20 seconds, and the solvent was removed under vacuum. The protected amino-acid or dipeptide (1.5 mmol, 3.0 eq.) was added followed by 4.0 mL of DMF. The mixture was shaken for 20 seconds then DIPEA (2.0 mmol, 4.0 eq.) was added and the mixture was shaken until dissolution of the amino-acid or dipeptide. HBTU (1.45 mmol, 2.9 eq.) was added then the reaction vessel was closed with a cap and shaken for 2 h. The mixture was filtered under vacuum. DMF (2.5 mL) was added to the resin and the mixture was shaken for 20 seconds then filtered. This washing was repeated five times. This coupling step was repeated once. MeOH (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered under vacuum. CH2Cl2 (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered under vacuum. The washing with CH2Cl2 was repeated 5 times. DMF (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered under vacuum. This washing was repeated twice. To the resin obtained was added a solution of DMF/piperidine 80:20 (4.0 mL). The mixture was shaken for 20 minutes, then filtered under vacuum. This step was repeated once. DMF (2.5 mL) was added and the mixture was shaken for 20 seconds then filtered. This washing was repeated 5 times. The reaction vessel was filled with MeOH (2.5 mL), shaken for 20 seconds and filtered under vacuum. The reaction vessel was filled with CH2Cl2 (2.5 mL), shaken for 20 seconds and filtered under vacuum. The washing with CH2Cl2 was repeated 5 times. The resin was dried 15 minutes under vacuum then air-dried overnight. This cycle of standard coupling followed by a Fmoc removal was repeated for each amino-acid or dipeptide of the sequence except for the last amino-acid for which Fmoc removal step was not necessary as the N terminal amine was protected by a Boc. Step 4: Cleavage of the peptide from the resin without affecting protecting groups. After the coupling of the last amino-acid, a solution of HFIP/CH2Cl220/80 (2 mL) was added to the swollen resin (4.7.10-5 mol). The mixture was shaken for 1 h at room temperature then filtered under vacuum. CH2Cl2 (0.5 mL) was added to the resin and the mixture was shaken for 20 seconds then filtered. This washing step was repeated twice. The filtrates were combined and washed with 3 mL of water (repeated 3 times) and brine (3 mL). The organic layer was dried over MgSO4 and filtered. This cleavage procedure was repeated twice but for the second, the reaction mixture was shaken for 40 min. The two resulting organic solutions were combined then concentrated down. Step 5: Coupling in solution of the C-terminal building-block. The fully-protected peptide (2.25.10-4 mol, 1.0 eq.) obtained at the end of step 4 was dissolved in CH2Cl2 (4 mL). DIPEA (1.8 mmol, 8.0 eq.), HOBt (1.3 mmol, 5.8 eq.) and EDC.HCl (1.3 mmol, 5.8 eq.) were added, followed by a solution of the amine for C-terminal derivatization (1.35 mmol, 6.0 eq.) in a minimum volume of CH2Cl2. The reaction mixture was stirred for 18 h at room temperature. The mixture was quenched with water and the phases were separated. The organic phase was washed with water (3*6 mL), 1 % aqueous solution of KHSO4 (6 mL) and brine (6 mL). The resulting organic layer was dried over MgSO4, filtered and concentrated down. Step 6: Cleavage of alloc then acid sensitive protecting groups. The peptide from Step 5 (0.25 mmol) was dissolved under argon in anhydrous CH2Cl2 (20 mL). Phenylsilane (1.25 mmol or 2.5 mmol, 5.0 eq. and 10 eq.) was added. The mixture was stirred under argon bubbling for 20 min then palladium-tetrakis(triphenylphosphine) (0.025 mmol, 0.1 eq) was added. The reaction mixture was stirred between 3 h and 4.5 h at room temperature then concentrated under reduced pressure. The completion of the reaction was controlled. If the reaction was not complete, additional phenylsilane (5.0 eq.) and palladium-tetrakis(triphenylphosphine) (0.1 eq.) were added. A solution TFA/triisopropylsilane/water (85/7.5/7.5) was directly added to the residue. The resulting mixture was stirred for 3 to 4 h, concentrated under vacuum (sometimes co-evaporation with water then a mixture of acetonitrile and toluene). The residue obtained was triturated with TBME, diethyl ether or EtOAc to give the crude product. Step 7: Cleavage of acid sensitive protecting groups (Boc and silylated groups): the peptide from Step 5 (2.25.10-4 mol), was dissolved in 4 mL of a solution TFA/triisopropylsilane/water (85/7.5/7.5). The resulting mixture was stirred for 3 h at room temperature, except when a silyl protecting group was present (in that case the mixture was stirred 4 h), then poured dropwise to 30 mL of cold (0 °C) TBME. The mixture was stirred 30 min at 0 °C. The precipitate obtained was isolated by centrifugation (2.200 g for 5 min), washed with 10 mL of cold (0 °C) TBME and centrifuged again (2.200 g for 5 min). The residue obtained was dissolved in water (4 mL). This solution was frozen and freeze-dried to give the crude product. Step 8: Purification. The crude product was dissolved in milliQ water (~250 mg/mL) and was purified by preparative HPLC using TFA as additive. Tubes containing pure product were combined and the solution was frozen to -80 °C and freeze-dried to TFA salt of the peptide. This product was analyzed by HPLC-MS applying the HPLC-MS analytical method for final purity check described above. When the peptide obtained was not pure enough, the product was dissolved in milliQ water (~150 mg/mL) and was purified by preparative HPLC using HFBA as additive. Tubes containing pure product were combined and the solution was frozen to -80 °C and freeze-dried to give the HFBA salt of the peptide. This product was analyzed by HPLC-MS applying the HPLC-MS analytical method for final purity check. Step 9: Dowex 1,4 chloride resin (938 mg, 5.0 eq., resin loading at 1.2 meq/g) was introduced in a reaction vessel. A solution of purified peptide from step 8 (2.25.10-4 mol, 1.0 eq.) in water (15 mL) was added. The mixture was shaken for 2 h then filtered. Water (15 mL) was added to the resin and the mixture was shaken then filtered. This washing was repeated twice. All the filtrates were combined, frozen at -80 °C then freeze-dried. The hydrochloride salt of the peptide was analyzed by HPLC-MS applying the HPLC-MS analytical method for final purity check described above. Example 6: Synthesis of peptide with CO2H at the C-terminal position Peptides were synthesized following step 1 of example 4 then steps 2 and 3 of example 5. Step 4: The cleavage of the peptide from the resin and the deprotection of all the lateral chains were performed in one step as follows. A solution of TFA/triisopropylsilane/water 85/7.5/7.5 (4 mL) was added to the resin (2.5.10-4 mol). The mixture was shaken for 2 h then filtered. The filtrate was poured dropwise to 30 mL of cold (0 °C) TBME. Additional 4 mL of a solution TFA/ triisopropylsilane /water 85/7.5/7.5 were added to the resin. The mixture was shaken for 20 min, filtered and the filtrate was poured dropwise to the previous 30 mL of cold (0 °C) TBME containing the first filtrate. The mixture was stirred 30 min at 0 °C. The precipitate obtained was isolated by centrifugation (2.200 g for 5 min), washed with 10 mL of cold (0 °C) TBME and centrifuged again (2.200 g for 5 min). The residue obtained was dissolved in water (4 mL). This solution was frozen and freeze dried to give the crude product. Step 5: The final purification was performed following the step 8 described in example 5. Step 6: The salt exchange was performed following the step 9 described in example 5. Example 7: Synthesis of peptide with NH(CH2)3NH2 or NH(CH2)5NH2 at the C-terminal position Peptides were synthesized from 1,3-diaminopropane or 1,5-diaminopentane trityl resin. The resin was swollen following the step 1 described in example 4. The coupling of amino acid or dipeptide was achieved following the step 3 described in example 5 and the peptide was cleaved from the resin following the step 4 described in example 6. Final purification and salt exchange were performed following the steps 8 and 9 described in example 5. Example 8: Synthesis of peptides with CONH2 at the C-terminal position Peptides with CONH2 at the C-terminal position were synthesized from Rink amide resin. The resin was swollen following step 1 described in example 4. Then the Fmoc protecting group was removed from the resin following the procedure described in step 3, example 5. The peptide chain was grown by introduction of amino acids or dipeptide following the procedure described in step 3 in example 5. Then the peptide was cleaved from the resin following the procedure described in step 4 in example 6. The final purification and salt exchange were performed following the procedures described in steps 8 and 9 in example 5. Example 9: Synthesis of compound 33 The peptide was synthesized by using a Rink amide resin. The resin was swollen following the step 1 described in example 4. Then the Fmoc protecting group was removed from the resin following the Fmoc removal step described in the step 3, example 5. Fmoc-Asp(Alloc)-OH then the corresponding amino acids or dipeptide were introduced following the step 3 described in example 5. Step 4: Alloc cleavage. Under inert atmosphere, phenylsilane (370 µL, 3 mmol, 10 eq.) and palladium tetrakis-(triphenylphosphine) (35 mg, 0.03 mmol, 0.1 eq.) were dissolved in anhydrous CH2Cl2 (10 mL). This solution was added to the dried resin (0.3 mmol). The reaction vessel was protected from light by an aluminum foil and the mixture was shaken for 15 min. The solvent was removed by filtration and the procedure was repeated once. CH2Cl2 (5 mL) was added and the mixture was shaken for 20 seconds then filtered. This washing was repeated twice. DMF (5 mL) was added and the mixture was shaken for 20 seconds then filtered. This washing was repeated twice. The resin was dried 15 minutes under vacuum then air-dried overnight. Step 5: The N-Boc-1,3-diaminopropane hydrochloride was introduced following the standard coupling procedure described in the step 2, example 4. Step 6: The peptide was cleaved from the resin and protecting groups were removed following the step 4 described in example 6. Step 7: The final purification was performed following the step 8 described in example 5. Step 8: The salt exchange was made following the step 9 described in example 5. The following compounds of formula (I) were prepared in accordance with the methods described herein (table 1). Structures of compounds 1 to 80 are as shown herein before.
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Table 1: Compounds of formula (I) II – In vitro pharmacological properties Minimum inhibitory concentration (MIC): MIC values were determined using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methodology, colony direct suspension, as described in CLSI document M07-A10 (Clinical and Laboratory Standards Institute. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—10th ed. CLSI document M07-A10 Clinical and Laboratory Standards Institute, Wayne, PA.) Determination of mRNA expression levels by RT-qPCR. Bacterial cultures of P. aeruginosa strains (PAO1, ATCC 27853, ATCC 47085, and pae β02) were conducted in Mueller Hinton II Broth (Becton Dickinson, ref: 212322) until OD600nm of 0.5-1.0. Depending on the experiments, 128 µg/mL of NOSO-502, NOSO-95C, or compound 1 according to the invention can be added to cultures before incubation with vigorous shaking at 37°C for 45 min (induction phase). Total mRNA extraction was achieved with the RNeasy Protect Bacteria 50 preps kit (Qiagen ref. 74524) according to the manufacturer’s instructions and was performed on 3 independent biological replicates. RNA Integrity Number (RIN) were determined, and reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen ref. 18064-022) and random hexamer from Applied Biosystems ref. N8080127. RT-qPCR to follow mexY gene expression was carried out using a LightCycler 480 (Roche) with Sensi-Fast SYBR no rox commercialized by Bioline (BIO-98050) and with primers MexY1a (5′-CTA CAA CAT CCC CTA TGA CAC CTC-3′) and MexY1b (5′- ATGGTCAGCACGTTGATCGAGAA-3′). Experiment was performed in triplicates on each cDNA sample. As control, a blank sample (distilled water) and a no reverse transcriptase control were included to exclude DNA contamination. The rpsL gene was used as the reference housekeeping gene. The data for each sample are expressed relative to the level of rpsL, using REST software 2009 and the Pfaffl equation (Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36). In table 2, the levels of expression of mexY in each culture of P. aeruginosa strains studied were reported as fold change relative to that in the P. aeruginosa PAO1 strain. In table 2, the levels of expression of mexY in each culture of P. aeruginosa strains treated with compounds (128 µg/mL for 45 min) were reported as fold change relative to that in their corresponding untreated P. aeruginosa strains. RESULTS
Figure imgf000139_0001
Table 2: antibacterial activity of NOSO-502, NOSO-95C, and compound 1 against a panel of P. aeruginosa strains with different levels of constitutive or inducible MexXY expression. a Pseudomonas strains used in this study: (11B) PAO1:mexX::Tn501 insertion, defective MexXY-OprM efflux pump strain; (PAO1T) PAO1 with deletion of oprM, defective MexXY-OprM efflux pump strain; (ATCC 27853) reference strain; (PAO1) reference strain; (Pae β02) clinical OprD deficient strain. bRT-qPCR comparative analysis of mexY gene expression (as a measure of mexXY) in cultures of different P. aeruginosa isolates (45 min) relative to P. aeruginosa PAO1 culture. Results were expressed in fold change compared to mexY expression in Pa PAO1 culture (arbitrarily equal to 1). (-) not tested because the strains are MexXY-OprM deficient. cRT-qPCR comparative analysis of mexY gene (as a measure of mexXY) differentially expressed in P. aeruginosa culture treated with NOSO-502 (A), NOSO-95C (B) or compound 1 (128 µg/mL, 45 min) relative to untreated culture. Results were expressed in fold change compared to mexY expression in P. aeruginosa untreated culture. NOSO-502, NOSO-95C, and compound 1 exhibit very potent and close antibacterial activity against strains with deficient MexXY-OprM efflux pump (11B, PAO1T). NOSO-502 is inactive against P. aeruginosa strains expressing the MexXY-OprM efflux pump. Indeed, NOSO-502 strongly enhances the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates and is susceptible to the efflux activity of this pump, resulting in a high NOSO-502 resistance level of these strains. NOSO-95C does not induce the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates. However, its antibacterial activity against P. aeruginosa strains varies widely depending on their constitutive MexXY expression level, clearly showing that this compound remains susceptible to the MexXY-OprM efflux activity. Compound 1 according to the invention does not induce the expression of the mexXY multidrug efflux operon in P. aeruginosa isolates. Its antibacterial activity against P. aeruginosa strains remains constant, independently from the MexXY expression level, clearly showing that this compound is not susceptible to the MexXY-OprM efflux activity. These results clearly demonstrate that compound 1 overcomes this resistance mechanism.
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
144 6 7 2 5 i 2 n i n 2 r a 8 a r t 2 n t s i na s r t sl a m r t s ia i u s s ce c ed a e i a m r f a s f no u se c uc mdp c c uee oc o n S o r c o p e r . t S n e t E n E ++ + +++ +++ +++ ++ ++ + ++ ++ ++ ++ + ++ ++ ++ + ++ ++ ++ ++ +++ ++ +++ ++ + +++ ++ ++ ++ +++ +++ +++
Figure imgf000145_0001
145 6 7 2 25 i n i na 8 2 ar r 2 n t t i na s r t s s s l a i m r t as c i u s d e c a e er f a i anm s fs oe u u md cc c up o co ee c c np S o r o e r . t e S n t E n E + + ++ + + ++ + + +++ +++ ++ +++ +++ ++ ++ +++ + ++ ++ + ++ ++ + ++ + + ++ + + ++ + + ++ + + ++ + + ++
Figure imgf000146_0001
146 6 7 2 25 i n i na 8 2 ar r 2 n t t i na s r t s s s l a i m r t as c i u s d e c a e er f a i anm s fs oe u u md cc c up o co ee c c np S o r o e r . t e S n t E n E + + ++ ++ + +++ + + +++ + + ++ + + +++ + + ++ ++ + ++ ++ + +++ + + ++ + + +++ + + +++ +++ ++ + ++ + ++
Figure imgf000147_0001
147 6 7 2 5 i 2 n i n 2 r a 8 a r t 2 n t s i na s r t s s l a i m r t as c i u s d e c e a e f a f i anm r s s oe uc u md c cc upe oc o en o c p S r o e r . t e t S n E n E ++ + ++ ++ ++ +++ ++ ++ ++ ++ + + ++ + + + + ++ ++ + ++ ++ + ++ + + ++ + + +++ + + ++ ++ + +++ ++ + +++
Figure imgf000148_0001
148 6 7 2 5 i 2 n i n 2 r a 8 a r t 2 n t s i na s r t sl a m r t s ia i u s s ce c ed a e i a m r f a s f noe u sud c c mup c c ee oc o n S o r c o p e r . t S n e t E n E + + +++ ++ ++ ++ ++ ++ +++ ++ ++ + + ++ + ++ ++++ + ++ ++ + ++ ++ ++ ++ ++ + ++ ++ + +++ ++ ++ ++ + +++ + + ++
Figure imgf000149_0001
149 6 7 2 25 i n i na 8 2 ar r 2 n t t i na s s r t s s l a i m ac i r u t ss e ce ed a a i a m r f f n s oe u sd c uc mup c c ee oc o n S o r c o e r p. t S n e t E n E + + ++ + ++ ++ + ++ ++ ++ + ++ + + ++ + ++ ++ ++ + ++ + + ++ ++ ++ ++ + + + + + ++ ++ ++ + ++ + +
Figure imgf000150_0001
150 6 7 2 25 i n i na 8 2 ar t r t 2 n i na s s r t sl a m r t s iac i u s s d e c a e em r f a i an s fs oe u u md cp c cc uee oc oc n S o r o e r p. t S n e t E n E + + ++ ++ ++ ++ + + ++ ++ + +++
Figure imgf000151_0001
III- In vivo efficacy in a murine lung infection model Mouse lung infection models. Compounds of the present invention were tested against P. aeruginosa ATCC 27853 in a 6-h immunocompromised mice respiratory tract infection model. Female CD-1 mice (18-22g) were allowed to acclimatize for 7 days, then rendered neutropenic by IP injection of cyclophosphamide (150 mg/kg on day 4 and 100 mg/kg on day 1 before infection). Mice were infected by intranasal route (5 x 105 c.f.u./mouse) under parenteral anaesthesia. The test compounds were formulated in phosphate-buffered saline (PBS, pH 7.4). Treatment was initiated 2 h post infection and 6 doses of each compound (from 0.5 mg/kg to 64 mg/kg depending on the compounds) were administered once by subcutaneous injection in a single dose volume of 4 mL/kg (2 mice per dose). A non- treated group (vehicle-only) was included to serve as a negative treatment control. At 2 h post infection, one infected group was humanely euthanized, and lungs processed for pre-treatment quantitative culture to determine P. aeruginosa burdens. At 8 h post infection, all remaining mice were humanely euthanized, and lungs were aseptically removed. The number of viable bacteria in lung was determined by plating serial tenfold dilutions of homogenates onto LB agar for 24 h at 37 °C. Results are presented in table 5.
Figure imgf000152_0001
Table 5: Efficacy of compounds of the present invention in an immunocompromised mice respiratory tract infection model with P. aeruginosa ATCC 27853. Mice (2 mice/group) were infected by intranasal instillation at t0. Antibacterial administration was performed by subcutaneous route at t0+2h. Bacterial counts in lungs were done at t0+2h for the control group (stasis) and at t0+8h for the treated and vehicle groups. Results were expressed as the doses (in mg/kg) required to achieve bacteriostatic effect and 1-log10 CFU in lungs reduction compared to stasis. (-) dose >120 mg/kg, (+) 120 mg/kg ≥ dose > 90mg/kg, (++) 90 mg/kg ≥ dose > 60mg/kg, (+++) 60 mg/kg ≥ dose > 30mg/kg, (++++) 30 mg/kg ≥ dose > 1 mg/kg. Compound 1 was also challenged in P. aeruginosa ATCC 27853 and A. baumannii ATCC BAA-1710 respiratory tract infection models in neutropenic mice, with minor changes compared to the protocol previously described. Mice were infected by intranasal route with 8 x 105 CFU/mouse of P. aeruginosa ATCC 27853 or with 3 x 106 CFU/mouse of A. baumannii ATCC BAA-1710. Treatment was initiated 2 h post infection and Compound 1 (1, 2, 4, 8, 16, and 32 mg/kg) was administered every 6 h in different groups (2 mice per dose) by subcutaneous injection (0.1 mL).26 h post infection, mice were humanely killed, and bacterial load in lungs was determined. A sigmoid maximum effect (Emax) model was fitted to the total daily dose versus Δlog10 CFU data by non-linear regression, and 1 log kill effect was determined using GraphPad Prism 8.0 (GraphPad Inc, San Diego, CA, USA). Results are presented in table 6.
Figure imgf000153_0001
Table 6: Efficacy of Compound 1 in an immunocompromised mice respiratory tract infection model with P. aeruginosa ATCC 27853 or A. baumannii. Mice (2 mice/group) were infected by intranasal instillation at t0. Antibacterial administration was performed by subcutaneous route at t0+2h. Bacterial counts in lungs were done at t0+2h for the control group (stasis) and at t0+24h for the treated and vehicle groups. Results were expressed as the doses (in mg/kg) required to achieve bacteriostatic effect and 1-log10 CFU in lungs reduction compared to stasis. (-) dose >120 mg/kg, (+) 120 mg/kg ≥ dose > 90mg/kg, (++) 90 mg/kg ≥ dose > 60mg/kg, (+++) 60 mg/kg ≥ dose > 30mg/kg, (++++) 30 mg/kg ≥ dose > 1 mg/kg. All tested compounds exhibit potent in vivo efficacy in a murine lung infection model with P. aeruginosa.

Claims

CLAIMS 1. A compound of formula (I): Ra-Xaa1-Xaa2-Xaa3- Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-(Xaa10)y-Rb (I) wherein: Ra is H or -(C1-C3)-alkyl; y is 0 or 1; Xaa1 is wherein one or more carbon atoms of the chain
Figure imgf000154_0001
bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)-alkyl and carboxyl, wherein: R1, R11 and R’11 are independently H or -(C1-C3)-alkyl; n1 is an integer from 1-4; Xaa2 is
Figure imgf000154_0002
wherein: X2 is NH, N(Me) or O; R2 and R’2 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl or -C(O)-(C1- C3)-haloalkyl; R22 is OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -O-C(O) -(C1-C6)-alkyl or –O- C(O)-(C1-C6)- haloalkyl; n2 is an integer from 1-3; , ,
Figure imgf000155_0001
X3 is N(R33) or O; R3 is halogen, NH2, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C3-C8)-cycloalkyl, -(C2- C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)-alkyl-OR33, -(C1-C6)-alkyl-NR33R’33, -(C1-C6)-alkyl- C(O)NR33R’33, -(C1-C6)-alkyl-C(O)OR33 or -(C1-C6)-alkyl-heteroaryl wherein said alkyl in the -(C1-C6)-alkyl-heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of –OH, –O-C(O)-(C1-C6)-alkyl and –O-C(O)-(C1-C6)- haloalkyl and wherein said heteroaryl is optionally substituted by one or more substituents selected from the group consisting of -(C1-C3)-alkyl and (C1-C6)-alkyl-aryl wherein said aryl is optionally substituted by one or more substituents selected from the group consisting of –OH, -(C1-C3)-alkyl, -(C1-C3)-alkoxy and halogen; R33, R’33 and R’’333 are independently H, -C(=NH)-NH2, -(C1-C3)-alkyl, -C(O)-(C1- C3)-alkyl, -C(O)-(C1-C3)-haloalkyl or -(C1-C6)-alkyl-phenyl, wherein said phenyl may be substituted by one or more substituents selected from the group consisting of –OH, - NH2, -COOH, -CONH2, -CN, -CF3, -(C1-C6)-alkyl, -(C1-C6)-alkyl-COOH, -(C1-C6) hydroxyalkyl, -(C1-C6) aminoalkyl, -(C1-C6) alkyl-NHCONH2, -(C1-C3)-alkoxy, -(C1-C3)- alkoxy-COOH and halogen; R333, R’333 , R’’3333 and R’’’333 are independently H, OH, halogen, -(C1-C3)-alkyl, - (C1-C3)-alkoxy, –O-CO-(C1-C3)-alkyl, –O-CO-(C1-C3)-haloalkyl or -NR’R” wherein R’ and R’’ are independently hydrogen or C1-C6-alkyl; n3 is an integer from 1-3; n4 is an integer from 0-3; Xaa4 is
Figure imgf000156_0001
wherein: X4 is NH, N(Me) or O; R4 and R’4 are independently H, -(C1-C3)-alkyl or -(C1-C3)-haloalkyl; Xaa5 is
Figure imgf000156_0002
wherein: X5 is NH, N(Me) or O; R5, R’5 and R’’5 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, or -C(O)- (C1-C3)-haloalkyl; R555, R’555, R’’555 and R’’’555 are independently H, OH, halogen, -(C1-C3)-alkyl, or - (C1-C3)-alkoxy; n5 is an integer from 1-3; Xaa6 is
Figure imgf000156_0003
, , , , ,
Figure imgf000156_0004
wherein: X6 is N(R66) or O; R6 is H, halogen, NH2, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C3-C8)-cycloalkyl, -(C2- C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)-alkyl-OR66, -(C1-C6)-alkyl-SR66, -(C1-C6)-alkyl- NR66R’66, -(C1-C6)-alkyl-C(O)NR66R’66, -(C1-C6)-alkyl-C(O)OR66 or -(C1-C6)-alkyl- heteroaryl wherein the alkyl in the -(C1-C6)-alkyl-heteroaryl is optionally substituted by one or more substituents selected from the group consisting of –OH, –O-C(O)-(C1-C6)- alkyl and –O-C(O)-(C1-C6)-haloalkyl and the heteroaryl is optionally substituted by one or more substituents selected from the group consisting of -(C1-C3)-alkyl, and (C1-C6)- alkyl-aryl wherein said aryl is optionally substituted by one or more –OH, -(C1-C3)-alkyl, -(C1-C3)-alkoxy or halogen; R66 and R’66 are independently H, OH, halogen, -C(=NH)-NH2, -(C1-C3)-alkyl, -O- (C1-C3)-alkyl, -(C1-C3)-haloalkyl, -(C1-C6)-alkyl-COOH, -C(O)-(C1-C3)-alkyl, -C(O)-(C1- C3)-haloalkyl, -NH2, -NH(C1-C3)-alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl]; n6 is an integer from 0-3; Xaa7 is
Figure imgf000157_0001
wherein: X7 is N(R77) or O; R7 is H, -(C1-C6)-alkyl, -(C1-C6)-haloalkyl, -(C2-C6)-alkenyl, -(C2-C6)-alkynyl, -(C1-C6)- alkyl-OR77, -(C1-C6)-alkyl-SR77, -(C1-C6)-alkyl-S(O)-R77, -(C1-C6)-alkyl-S(O)2-R77, -(C1-C6)- alkyl-NR77R’77, -(C1-C6)-alkyl-C(O)OR77, -(C1-C6)-alkyl-C(O)NR77R’77, -(C1-C6)-alkyl- heteroaryl, -(C1-C6)-alkyl-aryl or -(C1-C6)-alkyl-aryl-heteroaryl, wherein said aryl or heteroaryl in the -(C1-C6)-alkyl-heteroaryl, -(C1-C6)-alkyl-aryl or -(C1-C6)-alkyl-aryl- heteroaryl groups is optionally mono- or poly- substituted with –OH, -NH2, -COOH, - CONH2, -CN, -CF3, -(C1-C6)-alkyl, -(C1-C6)-alkyl-COOH, -(C1-C6)-hydroxyalkyl, -(C1-C6)- aminoalkyl, -(C1-C6)-alkyl-NHCONH2, -(C1-C3)-alkoxy, -(C1-C3)-alkoxy-COOH, or halogen; R77 and R’77 are independently H, OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-haloalkyl, - C(O)-NH2, -C(=NH)-NH2, -C(O)-(C1-C3)-alkyl, -C(O)-(C1-C3)-haloalkyl, -NH2, -NH(C1-C3)- alkyl, or -N[(C1-C3)-alkyl][(C1-C3)-alkyl]; n7 is an integer from 0-3;
Xaa8 is
Figure imgf000158_0001
wherein: X8 is NH, N(Me) or O; R8 and R’8 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl, -C(=NH)-NH2, or -C(O)-(C1-C3)-haloalkyl; R88, R’88 , R’’88 and R’’’88 are independently H, OH, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, or halogen; n8 is an integer from 1-4; Xaa9 is
Figure imgf000158_0002
Figure imgf000158_0003
wherein: X9 is NH, N(Me) or O; R9 and R’9 are independently H, -C(=NH)-NH2, -C(O)NH2, -(C1-C6)-alkyl, -(C1-C6)- haloalkyl, -C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R99 is H or -(C1-C3)-alkyl; n9 is an integer from 1-4; Xaa10 is
Figure imgf000159_0001
or
Figure imgf000159_0002
; wherein: X10 is NH or O; R10 and R’10 are independently H, -C(=NH)-NH2, -(C1-C3)-alkyl, -(C1-C6)-alkyl- NH2,-C(O)-(C1-C3)-alkyl, or -C(O)-(C1-C3)-haloalkyl; R100 is H, OH, -NH2, -(C1-C3)-alkyl, -(C1-C3)-alkoxy or halogen; n10 is independently an integer from 0-5; Rb is -NR5aR6a, wherein R5a is: -A1-NRa-A2-NHRb, -A3-CO-NRc-A4-NHRd, or -A5-NHRe wherein: A1 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; A2 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; Ra is a hydrogen atom, a –(C1-C5)-hydroxyalkyl, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyaminoalkyl or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A1 or A2 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Ra; Rb is a hydrogen atom or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A2 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rb; A3 is an unsubstituted C1-C4-alkanediyl or a C1-C4-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5-aminoalkyl, a –(C1-C5-hydroxyalkyl, a carboxyl and a carbamoyl; A4 is an unsubstituted C1-C5-alkanediyl or a C1-C5-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, an amine, a –(C1-C5)-aminoalkyl, a –(C1-C5)-hydroxyalkyl, a carboxyl and a carbamoyl; Rc is a hydrogen atom, a –(C1-C5)-hydroxyalkyl, a –(C1-C5)-aminoalkyl, a –(C1-C6)-hydroxyaminoalkyl or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A4 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rc; Rd is a hydrogen atom or a C1-C6-alkanediyl that forms a cycle with a carbon atom of A4 in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing Rd; A5 is an unsubstituted C2-C6-alkenediyl group, an unsubstituted C1-C6- alkanediyl or a C1-C6-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl, an amine and a –(C1-C6)- aminoalkyl; Re is a hydrogen atom, a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl, provided that Re is a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl when A5 is an unsubstituted C1-C6-alkanediyl; R6a is a hydrogen atom, a –(C1-C5)-hydroxyalkyl or a C1-C6-alkanediyl group that forms a cycle with a carbon atom of A1, A3 or A5 of R5a in position alpha, beta, gamma, delta or epsilon relative to the nitrogen atom bearing R6a; or hydrates, solvates, or salts thereof.
2. The compound of formula (I) according to claim 1 wherein Xaa1 is
Figure imgf000160_0001
wherein one or more carbon atoms of the chain bearing the NR11R’11 group may be substituted by one or more substituents selected from the group consisting of –(C1-C3)- alkyl and carboxyl, and R11 and R’11 are as recited in claim 1.
3. The compound of formula (I) according to claim 1 or 2 wherein Xaa2 is
Figure imgf000161_0001
wherein: X2 is NH or N(Me), preferably NH; R2 and R’2 are independently H, -(C1-C3)-alkyl, -C(O)-(C1-C3)-alkyl or -C(O)-(C1- C3)-haloalkyl, preferably H; R22 is OH, halogen, -(C1-C3)-alkyl, -(C1-C3)-alkoxy, -O-C(O) -(C1-C6)-alkyl or –O- C(O)-(C1-C6)-haloalkyl, preferably OH.
4. The compound of formula (I) according to any one of claim 1 to 3 wherein Xaa3 is
Figure imgf000161_0002
wherein: X3 is N(R33); R3 is -(C1-C4)-alkyl-NR33R’33, preferably -CH2-CH2-NR33R’33; R33 and R’33 are H.
5. The compound of formula (I) according to any one of claim 1 to 4 wherein Xaa4 is
Figure imgf000161_0003
wherein: X4 is NH or N(Me), preferably NH; R4 and R’4 are independently H.
6. The compound of formula (I) according to any one of claim 1 to 5 wherein Xaa5 is
Figure imgf000162_0001
wherein: X5 is NH, N(Me), preferably NH; R5 and R’5 are independently H or -(C1-C3)-alkyl, preferably H; n5 is an integer from 1-3, preferably 2.
7. The compound of formula (I) according to any one of claim 1 to 6 wherein Xaa9 is
Figure imgf000162_0002
wherein: X9 is NH or N(Me), preferably NH; R9 and R’9 are independently H and -C(=NH)-NH2; n9 is an integer from 1-4, preferably 2.
8. The compound of formula (I) according to any one of claim 1 to 7 wherein wherein: A5 is an unsubstituted C2-C6-alkenediyl group, an unsubstituted C1-C6-alkanediyl or a C1- C6-alkanediyl substituted by one or more substituents selected from the group consisting of a hydroxyl, a –(C1-C3)-hydroxyalkyl, a carboxyl, a halogen atom, a carbamoyl, an amine and a –(C1-C6)-aminoalkyl; and Re is a hydrogen atom, a -(C1-C3)-alkyl or a -(C1-C6)-hydroxyalkyl, provided that Re is a -(C1-C6)-hydroxyalkyl when A5 is an unsubstituted C1-C6-alkanediyl.
9. The compound of formula (I) according to any one of claim 1 to 8 of following formula (Ia):
Figure imgf000163_0001
(Ia) wherein: R1a and R2a are, independently of each other, a hydrogen atom or a -(C1-C3)-alkyl; R3a is H or NH2; R4a is
Figure imgf000163_0002
y is equal to 0 or 1; R5a and R6a are as recited in claim 1.
10. The compound according to claim 9, wherein R1a and R2a are a hydrogen atom.
11. The compound according to claim 9 or 10, wherein R4a is
Figure imgf000163_0003
12. The compound according to any one of claims 9 to 11, wherein R5a is one the following groups:
Figure imgf000163_0004
Figure imgf000164_0001
Figure imgf000164_0002
Figure imgf000165_0001
13. The compound according to any one of claims 1 to 12, wherein the compound is selected from the group consisting of the following compounds 1 to 80:
Figure imgf000165_0002
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
14. A composition comprising a compound of formula (I) according to any one of claims 1 to 13 and a pharmaceutically or veterinary excipient. 15. A compound of formula (I) according to any one of claims 1 to 13 or a composition according to claim 14 for use in medicine. 16. A compound of formula (I) according to any one of claims 1 to 13 or a composition according to claim 14 for use in the treatment or prevention of bacterial infections, preferably caused or suspected to be caused by A.baumannii, E. cloacae, E. coli, Klebsiella spp., M. morganii, P. mirabilis, S. marcescens, Staphylococcus spp., Enterococcus spp., S. pneumoniae, more preferably for use in the treatment or prevention of multi-drug resistant bacterial infections, even more preferably for use in the treatment or prevention of multi-drug resistant bacterial infections caused or suspected to be caused by P. aeruginosa.
PCT/EP2024/058803 2023-03-31 2024-03-29 New odilorhabdins analogues as antibiotics against multi-resistant bacteria WO2024200838A1 (en)

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