JP6223508B2 - Proteasome inhibitor - Google Patents

Description

  The present invention relates to boronic acid and boronic ester compounds that are useful as proteasome inhibitors. The invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of using the compositions for the treatment of various diseases.

  Boronic acid and boronate ester compounds exhibit a variety of pharmaceutically useful biological activities. Shenvi et al. Patent Document 1 (1985) discloses that peptide boronic acid is an inhibitor of a specific proteolytic enzyme. Patent Document 2 (1993), Patent Document 3 (1993), and Patent Document 4 (1993) of Kettner and Shenvi describe a group of peptide boronic acids that inhibit trypsin-like proteases. Kleeman et al. U.S. Pat. No. 6,057,099 (1992) discloses an N-terminal modified peptide boronic acid that inhibits the action of renin. Kinder et al. U.S. Patent No. 6,057,099 (1992) discloses certain boronic acid compounds that inhibit the growth of cancer cells. Bachovin et al. U.S. Patent No. 6,053,077 discloses peptide boronic acid compounds that inhibit fibroblast activation protein.

  Boronic acid and boronic ester compounds are particularly promising as inhibitors of the proteasome, a multi-catalytic protease that is responsible for the majority of intracellular protein turnover. Adams et al. U.S. Patent No. 5,983,098 (1998) describes peptide boronic esters and acid compounds that are useful as proteasome inhibitors. This reference also reduces the rate of muscle protein degradation, reduces the activity of intracellular NF-κB, reduces the rate of intracellular p53 protein degradation, inhibits intracellular cyclin degradation, Of the use of boronic esters and acid compounds to inhibit the growth of NF-κB and to inhibit NF-κB-dependent cell adhesion. Furet et al. Patent Document 9; Patent Document 10 and Bemadini et al. U.S. Patent Nos. 6,099,036 and 5,037,831 disclose further boronic esters and boronic acid compounds that have been reported to have proteasome inhibitory activity.

  Non-Patent Document 1 discloses that the proteasome is a proteolytic component of the ubiquitin-proteasome pathway, where proteins are targeted for degradation by binding to multiple ubiquitin molecules. Ciechanover also discloses that the ubiquitin-proteasome pathway plays an important role in a variety of important physiological processes. Non-Patent Document 2 discloses that the proteasome exhibits trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase activity. It is the 20S proteasome that constitutes the catalytic core of the 26S proteasome. Non-Patent Document 3 describes Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC, and Z-Leu-Leu-Glu-2NA (Suc is N-succinyl, AMC is 7-amino Various peptide substrates, including -4-methylcoumarin, and 2NA is 2-naphthylamine, are taught to be cleaved by the 20S proteasome.

Proteasome inhibition represents an important new strategy for cancer treatment. Non-Patent Document 4 describes the important role that the ubiquitin-proteasome pathway plays in the regulation of cell cycle, tumor growth, and metastasis. The authors teach that a number of key regulatory proteins, including cyclins, and the cyclin-dependent kinases p21 and p27 ^KIP1, are degraded over time during the cell cycle by the ubiquitin-proteasome pathway. Regular degradation of these proteins is necessary for the cell to progress through the cell cycle and to undergo mitosis.

  Furthermore, the ubiquitin-proteasome pathway is required for transcriptional regulation. Non-Patent Document 5 teaches that the activation of the transcription factor NF-κB is regulated by the degradation of the inhibitory protein IκB by the proteasome. NF-κB plays a central role in the regulation of genes involved in immune and inflammatory responses. Non-Patent Document 6 teaches that the ubiquitin-proteasome pathway is required for the expression of cell adhesion molecules such as E-selectin, ICAM-1, and VCAM-1. Non-Patent Document 7 discloses that cell adhesion molecules participate in tumor metastasis and blood vessel formation in vivo by reciprocating the adhesion and overflow of tumor cells between the vasculature in the body and a distant tissue site. Teaches that Furthermore, Non-Patent Document 8 teaches that NF-κB is an anti-apoptotic regulator and that inhibition of NF-κB activation makes cells more sensitive to environmental stress and cytotoxic drugs.

  The proteasome inhibitor VELCADE® (bortezomib, N-2-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid) is the first proteasome inhibitor with regulatory approval. Non-Patent Document 9 investigates clinical studies that have resulted in the approval of bortezomib for the treatment of patients with multiple myeloma who have received at least one prior treatment. Non-Patent Document 10 describes an international multicenter phase II study confirming the activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma. Non-Patent Document 11 and Non-Patent Document 12 discuss a number of molecular mechanisms that can contribute to the antitumor activity of bortezomib.

  As is evident from the above references, the proteasome is an important target for therapeutic intervention. Accordingly, there is a continuing need for new and / or improved proteasome inhibitors.

US Pat. No. 4,499,082 US Pat. No. 5,187,157 US Pat. No. 5,242,904 US Pat. No. 5,250,720 US Pat. No. 5,169,841 US Pat. No. 5,106,948 International Publication No. 07/0005991 Pamphlet US Pat. No. 5,780,454 International Publication No. 02/096933 Pamphlet International Publication No. 05/016859 Pamphlet International Publication No. 05/021558 Pamphlet International Publication No. 06/08660 Pamphlet

Ciechanover, Cell, 79: 13-21 (1994) Rivett et al, Biochem. J. et al. 291: 1 (1993) McCorack et al. Biochemistry 37: 7792 (1998) King et al. . Science 274: 1652-1659 (1996) Palombella et al. Cell, 78: 773 (1994). Read et al. , Immunity 2: 493-506 (1995). Zetter, Seminars in Cancer Biology 4: 219-229 (1993) Beg and Baltimore, Science 274: 782 (1996). Mitsiades et al. , Current Drug Targets, 7: 1341 (2006). Fisher et al. , J. et al. Clin. Oncol. , 30: 4867 Ishii et al. , Anti-Cancer Agents in Medicinal Chemistry, 7: 359 (2007). Roccaro et al. Curr. Pharm. Biotech. , 7: 1341 (2006)

  The present invention provides compounds that are effective inhibitors of the proteasome. These compounds are useful for inhibiting proteasome activity in vitro and in vivo, and are particularly useful for the treatment of various cell proliferative diseases.

  The compounds of the present invention are compounds of general formula (I):

Or a pharmaceutically acceptable salt or boronic anhydride thereof, wherein Z ¹ and Z ² are each independently hydroxy, alkoxy, aryloxy, or aralkoxy, or Z ¹ and Z ² Together form a part derived from a boronic acid complexing agent,
Ring A is

Selected from the group consisting of

The boronic acid compounds of formula (I) (wherein Z ¹ and Z ² are each hydroxy) are referred to by the chemical names below.

For example, the present invention provides the following items.
(Item 1)
Compound of formula (I):


Or a pharmaceutically acceptable salt or boronic acid anhydride thereof,
Wherein Z ¹ and Z ² are each independently hydroxy, alkoxy, aryloxy, or aralkoxy, or Z ¹ and Z ² together form a moiety derived from a boronic acid complexing agent,
Ring A is


Selected from the group consisting of:
Compound.
(Item 2)
[(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(5-chloro-2-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,5-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,5-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-(([(2-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chloro-5-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,4-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1 - ({ [(2, 5- dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,4-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chloro-4-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-(([(2,3-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,4-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-chloro-2-fluorobenzoyl) amino] acetyl) amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,4-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid and [(1R) -1-({[(3,5-dichlorobenzoyl 2. The compound of item 1, selected from the group consisting of:) amino] acetyl} amino) -3-methylbutyl] boronic acid, or a pharmaceutically acceptable salt or boronic anhydride thereof.
(Item 3)
[(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(5-chloro-2-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,5-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,5-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chloro-5-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,4-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,5-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3,4-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(3-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chloro-4-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,3-dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(2,4-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-chloro-2-fluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1-({[(4-chlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid,
[(1R) -1- (| [ (2, 4- dichlorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid, and [(1R) -1 - ({ [(3,5- dichlorobenzoyl ) Amino] acetyl} amino) -3-methylbutyl] boronic acid,
2. The compound of item 1, which is a mannitol ester of a boronic acid compound selected from the group consisting of.
(Item 4)
A pharmaceutical composition comprising the compound according to item 1 and a pharmaceutically acceptable carrier.
(Item 5)
A method for treating cancer comprising administering the pharmaceutical composition according to item 3 to a patient in need of such treatment.

  The term “alkyl” used alone or as part of a larger moiety refers to a straight or branched chain or cyclic aliphatic group having 1 to 12 carbon atoms. The term “alkoxy” refers to an O-alkyl group.

For example, the terms “aryl” and “ar-” used alone or as part of a larger moiety, such as “aralkyl”, “aralkoxy”, or “aryloxyalkyl” include 1-3 rings. Refers to C ₆ -C ₁₄ aromatic hydrocarbons, each of which is optionally substituted. Preferably, the aryl group is a C _6-10 aryl group. Aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. An “aralkyl” or “arylalkyl” group includes an aryl group covalently linked to an alkyl group, both of which are independently optionally substituted. Preferably, aralkyl groups include, but are not limited to, benzyl, phenethyl, and naphthylmethyl, C _6-10 aryl (C _1-6 ) alkyl, C _6-10 aryl (C _1-4 ) alkyl, Or C _6-10 aryl (C _1-3 ) alkyl.

As used herein, the term “substituted” means that the hydrogen moiety at the specified moiety is a specified substituent, provided that the substitution results in a stable or chemically feasible compound. Means it can be replaced with Non-limiting examples of suitable substituents include C _1-6 alkyl, C _3-8 cycloalkyl, C _1-6 alkyl (C _3-8 ) cycloalkyl, C _2-8 alkenyl, C _2-8 alkynyl, Cyano, amino, C _1-6 alkylamino, di (C _1-6 ) alkylamino, benzylamino, dibenzylamino, nitro, carboxy, carbo (C _1-6 ) alkoxy, trifluoromethyl, halogen, C _{1- 6} alkoxy, C _6-10 aryl, C _6-10 aryl (C _1-6 ) alkyl, C _6-10 aryl (C _1-6 ) alkoxy, hydroxy, C _1-6 alkylthio, C _1-6 alkylsulfinyl, C _{1-6 alkylsulfonyl, C 6-10 arylthio, C 6-10 arylsulfinyl, C 6-10} arylsulfonyl, _{6-10 aryl, C 1-6} alkyl _{(C 6-10)} aryl, and halo _{(C 6-10)} mentioned aryl.

  As used herein, the phrase “one or more substituents” is based on the number of available binding sites provided that the above conditions for stability and chemical feasibility are met. Refers to a number of substituents equal to 1 to the maximum possible number of substituents. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and the substituent may be the same or different substituent. As used herein, the term “independently selected” means that the same or different values may be selected for multiple instances of a given variable in a single compound. To do.

  The term “about” is used herein to mean “approximately”, “approximately”, “approximately”, or “around”. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the numerical values recited. In general, the term “about” is used herein to correct a numerical value by a difference of 10% above or below the stated value.

  As used herein, the term “comprises” means “including but not limited to”.

Unless otherwise stated, the structures illustrated herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having this structure are within the scope of the present invention, except for replacement of hydrogen atoms with deuterium or tritium, or replacement of carbon atoms with ¹³ C or ¹⁴ C enriched carbon.

As used herein, the term “boronic acid” refers to a compound containing a —B (OH) ₂ moiety. In some embodiments, the boronic acid compound can form an oligomeric anhydride by dehydration of the boronic acid moiety. For example, Snyder et al. ,
J. et al. Am. Chem. Soc. 80: 3611 (1958) reports oligomeric aryl boronic acids.

  As used herein, the term “boronic anhydride” refers to a compound formed by the combination of two or more molecules of a boronic acid compound with the loss of one or more water molecules. When mixed with water, the boronic acid anhydride compound hydrates and releases the free boronic acid compound. In various embodiments, the boronic acid anhydride can include 2, 3, 4 or more boronic acid units and can have a cyclic or linear structure. Non-limiting examples of oligomeric boronic acid anhydrides of the peptide boronic acid compounds of the present invention are illustrated below.

In formulas (1) and (2), the variable n is an integer from 0 to about 10, preferably 0, 1, 2, 3, or 4. In some embodiments, the boronic acid anhydride compound comprises a cyclic trimer of formula (2) (“boroxine”), where n is 1. The variable W has the formula (3).

Wherein ring A has the values described above with respect to formula (I).

  In some embodiments, at least 80% of the boronic acid present in the boronic acid anhydride compound is present in the form of a single oligomeric anhydride. In some embodiments, at least 85%, about 90%, about 95%, or about 99% of the boronic acid present in the boronic acid anhydride compound is present in the form of a single oligomeric anhydride. In certain preferred embodiments, the boronic acid anhydride compound consists of, or consists essentially of, a boroxine having the formula (3).

  The boronic acid anhydride compound is preferably prepared from the corresponding boronic acid by exposure to dehydration conditions including, but not limited to, recrystallization, lyophilization, exposure to heat, and / or exposure to a desiccant. be able to. Non-limiting examples of suitable recrystallization solvents include ethyl acetate, dichloromethane, hexane, ether, acetonitrile, ethanol, and mixtures thereof.

In some embodiments, Z ¹ and Z ² together form a moiety derived from a boronic acid complexing agent. For the purposes of the present invention, the term “boronic acid complexing agent” refers to any compound having at least two functional groups, each of which is capable of forming a covalent bond with boron. Non-limiting examples of suitable functional groups include amino and hydroxyl. In some embodiments, at least one of the functional groups is a hydroxyl group. The term “moiety derived from a boronic acid complexing agent” refers to a moiety formed by removing a hydrogen atom from two functional groups of a boronic acid complexing agent.

As used herein, the terms “boronate ester” and “boronate ester” are used interchangeably and refer to a compound containing a —B (Z ¹ (Z ² ) moiety, where , Z ¹ or Z ² is alkoxy, aralkoxy, or aryloxy, or both Z ¹ and Z ² are derived from a boronic acid complexing agent having at least one hydroxyl group Forming part.

In some embodiments, Z ¹ and Z ² together form a moiety derived from a compound having at least two hydroxyl groups separated by at least two linking atoms in the chain or ring, The ring includes carbon atoms and, optionally, heteroatom (s) that may be N, S, or O, where the atom bonded to the boron in each case is an oxygen atom.

  As used herein, the term “compound having at least two hydroxy groups” refers to any compound having two or more hydroxyl groups. For the purposes of the present invention, the two hydroxyl groups are preferably separated by at least 2 bonded atoms, preferably about 2-5 bonded atoms, more preferably 2 or 3 bonded atoms. For convenience, the term “dihydroxy compound” may be used to refer to a compound having at least two hydroxyl groups as defined above. Thus, the term “dihydroxy compound” as used herein is not intended to be limited to compounds having only two hydroxyl groups. A moiety derived from a compound having at least two hydroxyl groups may be bound to boron by any two oxygen atoms of the hydroxyl group. Preferably, a boron atom, an oxygen atom bonded to boron, and an atom that bonds two oxygen atoms together form a 5- or 6-membered ring.

For the purposes of the present invention, the boronic acid complexing agent is preferably pharmaceutically acceptable, ie suitable for administration to humans. In some preferred embodiments, the boronic acid complexing agent is a sugar. The term “sugar” includes any polyhydroxy carbohydrate moiety, including monosaccharides, disaccharides, polysaccharides, sugar alcohols, and amino sugars. In some embodiments, the sugar is a monosaccharide, disaccharide, sugar alcohol, or amino sugar. Non-limiting examples of suitable sugars include glucose, sucrose, fructose, trehalose, mannitol, sorbitol, glucosamine, and N-methylglucosamine. In certain embodiments, the sugar is mannitol or sorbitol. Thus, in embodiments where the sugar is mannitol or sorbitol, Z ¹ and Z ² together form a moiety of the formula: C ₆ H ₁₂ O ₆ , where the oxygen atoms of the two deprotonated hydroxyl groups are In order to form a boronate ester compound, a covalent bond is formed with boron. In certain embodiments, Z ¹ and Z ² together form a moiety derived from D-mannitol.

  In some embodiments, the compound of Formula (I) is obtained from Plamondon et al. In WO 02/059131, formulated as a lyophilized powder, which is incorporated herein by reference in its entirety. In some embodiments, the lyophilized powder also includes a free dihydroxy compound. Preferably, the free dihydroxy compound and the compound of formula (I) are present in the mixture in a molar ratio ranging from about 0.5: 1 to about 100: 1, more preferably from about 5: 1 to about 100: 1. . In various embodiments where the dihydroxy compound is mannitol, the lyophilized powder is in the range of about 10: 1 to about 100: 1, about 20: 1 to about 100: 1, or about 40: 1 to about 100: 1. In a molar ratio, it contains free mannitol and mannitol boronate ester.

  In some embodiments, the lyophilized powder comprises mannitol and a compound of formula (I) that is substantially free of other ingredients. However, the composition may include one or more other pharmaceutically acceptable excipients, carriers, diluents, fillers, salts, buffers, stabilizers, solubilizers, and others known in the art. The material may further be included. Preparation of pharmaceutically acceptable dosage forms containing these materials is described, for example, in Remington: The Science and Practice of Pharmacy, 20th Ed. , Ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 or the latest edition.

  The lyophilized powder containing the compound of (I) is preferably obtained from Plamondon et al. Prepared in accordance with the procedure described in WO 02/059131. Thus, in some embodiments, a method for preparing a lyophilized powder comprises: (a) preparing an aqueous mixture comprising a peptide boronic acid and a dihydroxy compound; and (b) lyophilizing the mixture. including.

General Synthetic Methodology Compounds of formula (I) can be prepared by methods known to those skilled in the art. For example, Adams et. al, U.S. Pat. No. 5,780,454, Pickersgill et al. International Patent Publication No. 2005/097809. An exemplary synthetic route is described in Scheme 1 below.

  Scheme 1:

Coupling of compound i with N-protected glycine (ii) followed by N-terminal deprotection yields compound iii. Examples of suitable protecting groups (PG) include, for example, acyl protecting groups such as formyl, acetyl (Ac), succinyl (Suc), and methoxysuccinyl, and tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) And urethane protecting groups such as, but not limited to, fluorenylmethoxycarbonyl (Fmoc). The peptide coupling reaction can be carried out by pre-conversion of compound ii to an activated ester of a carboxylic acid moiety such as O- (N-hydroxysuccinimide) ester followed by treatment with compound i. Alternatively, activated esters can be generated in situ by contacting the carboxylic acid with a peptide coupling reagent. Examples of suitable peptide coupling reagents include, for example, carbodiimide reagents such as dicyclohexylcarbodiimide (DCC) or l- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), such as benzotriazol-1-yloxy Phosphonium reagents such as tris (dimethylamino) phosphonium hexafluorophosphate (BOP) and, for example, O- (lH-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium tetrafluoro Examples include, but are not limited to, uronium reagents such as borate (TBTU).

Compound iii is then coupled with substituted benzoic acid (ArCO ₂ H) to give compound iv. The peptide coupling conditions described above for the coupling of compounds i and ii are also suitable for the coupling of compounds with ArCO ₂ H. Deprotection of the boronic acid moiety then gives compound v. The deprotection step is preferably carried out by transesterification in a biphasic mixture comprising a boronic ester compound iv, an organic boronic acid acceptor, a low alkanol, a C _5-8 hydrocarbon solvent, and a hydrous mineral acid.

  Scheme 2:

Alternatively, the order of the coupling reaction may be reversed as shown in Scheme 2. Thus, O-protected glycine (vi) is first coupled with substituted benzoic acid (ArCO ₂ H) and then ester hydrolyzed to form compound vii. Coupling with compound i and boronic acid deprotection are then performed as described above with respect to Scheme 1 to give compound v.

Use, Formulation, and Administration The present invention provides compounds that are potent inhibitors of the proteasome. The compounds can be analyzed in vitro or in vivo for their ability to inhibit peptide hydrolysis or proteolysis by the proteasome.

  Accordingly, in another aspect, the invention provides a step of contacting a cell in which proteasome inhibition is desired with a compound described herein, or a pharmaceutically acceptable salt, boronic ester, or boronic anhydride thereof. A method for inhibiting one or more peptidase activities of a proteasome in a cell is provided.

  The invention also provides a method for inhibiting cell proliferation comprising the step of contacting a cell in which such inhibition is desirable with a compound described herein. The phrase “inhibits cell proliferation” is used to indicate the ability of a compound of the invention to inhibit cell number or cell growth in a contacted cell as compared to a cell not contacted with an inhibitor. Assessment of cell proliferation can be done by counting cell numbers using a cell counter or by a cell viability assay such as, for example, an MTT or WST assay. When cells are in solid growth (eg, solid tumors or organs), assessment of such cell proliferation is performed, for example, by measuring growth with a caliper and comparing the size of the growth of contacted cells with non-contacted cells. be able to.

  Preferably, the growth of cells contacted with the inhibitor is delayed by at least about 50% compared to the growth of non-contacting cells. In various embodiments, cell proliferation of contacted cells is inhibited by at least about 75%, at least about 90%, or at least about 95% compared to non-contacted cells. In some embodiments, the phrase “inhibits cell proliferation” includes a reduction in the number of contacted cells as compared to non-contacted cells. Thus, proteasome inhibitors that inhibit cell proliferation in contacted cells can cause growth delay, growth arrest, programmed cell death (ie, apoptosis), or necrotic cell death of contacted cells.

  In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or boronic anhydride thereof, and a pharmaceutically acceptable carrier.

  When pharmaceutically acceptable salts of the compounds of this invention are used in these compositions, the salts are preferably derived from inorganic or organic acids or bases. To identify suitable salts, see, eg, Berge et al, J. MoI. Pharm. Sci. 66: 1-19 (1977), and Remington: The Science and Practice of Pharmacy, 20th Ed. , Ed. A. See Gennaro, Lippincott Williams & Wilkins, 2000.

  Non-limiting examples of suitable acid addition salts include: Acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, di Gluconate, dodecyl sulfate, ethane sulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodic acid Salt, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectate, persulfate, 3 Phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

Suitable base addition salts include ammonium salts, alkali metal salts such as lithium, sodium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, and other polyvalent metal salts such as zinc And salts having an organic base, such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylenediamine, ethanolamine, and choline, and salts having an amino acid, such as arginine. It is not limited to. In some embodiments, the pharmaceutically acceptable salt is a base addition salt of a boronic acid compound of formula (I), wherein Z ¹ and Z ² are both hydroxy.

  The term “pharmaceutically acceptable carrier” is compatible with the recipient, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to a target site without terminating the activity of the agent. As used herein to refer to material. Where there are toxic and adverse effects associated with the carrier, it corresponds to a reasonable risk-to-effect ratio for the intended use of the active agent.

  The terms “carrier”, “adjuvant”, or “vehicle” are used interchangeably herein, and any solvent, diluent, and other liquid vehicle, dispersion or as appropriate for the particular dosage form desired. Contains suspending aids, surfactants, pH adjusters, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like. Remington: The Science and Practice of Pharmacy, 20th Ed. , Ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for their preparation. Any conventional carrier medium may, for example, cause any undesirable biological effect or otherwise interact in a deleterious manner with any other component of the pharmaceutically acceptable composition. Thus, its use is considered to be within the scope of the present invention, unless it is incompatible with the compounds of the present invention. Some examples of materials that can serve as a pharmaceutically acceptable carrier include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as , Phosphates, carbonates, magnesium hydroxide, and aluminum hydroxide, glycine, sorbic acid or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, pyrogen-free water, salts, or electrolytes, For example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts such as colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylate, wax, polyethylene-polyoxypropylene block Polymer, wool fat, sugar, eg Starch, such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients, such as fructose, glucose, sucrose, and mannitol For example cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol and polyethylene glycol, esters such as olein Ethyl acetate and ethyl laurate, agar, alginic acid, isotonic saline, Ringer's solution, alcohols such as ethanol, isopropyl alcohol, hexade Such as alcohols, glycerol, cyclodextrins such as hydroxypropyl β-cyclodextrin, and sulfobutyl ether β-cyclodextrins, lubricants such as sodium lauryl sulfate and magnesium stearate, petroleum hydrocarbons such as mineral oil and Although petrolatum etc. are mentioned, it is not limited to this. Coloring agents, release agents, coating agents, sweeteners, flavoring and flavoring agents, preservatives, and antioxidants can also be present in the composition according to the judgment of the formulator.

  The pharmaceutical composition of the present invention can be produced by known methods such as conventional granulation, mixing, dissolution, encapsulation, lyophilization, or emulsification process. Compositions include granules, precipitates or particulates, freeze-dried powders, powders containing spin-dried powders or spray-dried powders, amorphous powders, tablets, capsules, syrups, suppositories, infusions, emulsions, elixirs It may be produced in a variety of forms, including suspensions or solutions.

  According to a preferred embodiment, the composition of the invention is formulated for drug administration to a mammal, preferably a human. Such pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implantable reservoir. As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection. Or an injection technique is included. Preferably the composition is administered orally, intravenously, subcutaneously. The formulations of the invention may be designed to be short acting, immediate release, or long acting. Furthermore, the compounds can be administered by local rather than systemic means, such as administration at the tumor site (eg, by injection).

  Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsion solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms can contain, for example, water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid, propylene glycol, 1, 3-butylene glycol, cyclodextrin, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid ester, As well as inert diluents commonly used in the art, such as mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
A sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Also good. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.A. S. P. And isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. Can do. Compositions formulated for parenteral administration may be injected by bolus injection or by timed infusion, or may be administered by continuous infusion.

  Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms the active compound contains at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) a filler or bulking agent, such as Starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia c) humectants such as glycerol, d A) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarders such as paraffin, f) absorption enhancers such as quaternary Ammonium compounds, g) wetting agents such as Alcohols and glycerol monostearate, h) absorbents such as kaolin and bentonite clays, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof Mixed with. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents such as phosphates or carbonates.

  Similar types of solid compositions may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings known in the pharmaceutical formulating art. They may optionally contain opacifiers, which are solid dosage forms of compositions that release the active ingredient alone or selectively in certain parts of the intestinal tract, optionally in a delayed manner. May be. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols.

  The active compound can also be in the form of a microencapsulation with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also contain additional materials other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and macrocrystalline cellulose, as a common practice. In the case of capsules, tablets, and pills, the dosage form may also include a buffer. They may optionally contain opacifiers, which are solid dosage forms of compositions that release the active ingredient alone or selectively in certain parts of the intestinal tract, optionally in a delayed manner. May be. Examples of embedding compositions that can be used include polymeric substances and waxes.

  Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalations, or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and eye drops are also considered to be within the scope of this invention. Furthermore, the present invention contemplates the use of a transdermal patch, which has the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

In some embodiments, the compound of formula (I) is administered intravenously. In such embodiments, the compound of formula (I) (wherein Z ¹ and Z ² together form a moiety derived from a boronic acid complexing agent) is prepared in the form of a lyophilized powder as described above. be able to. The lyophilized powder is preferably reconstituted by adding an aqueous solvent suitable for drug administration. Examples of suitable reconstitution solvents include, but are not limited to, water, saline, phosphate buffered saline (PBS). Preferably, the lyophilized powder is reconstituted with physiological (0.9%) saline. Reconstitution establishes an equilibrium between the boronate ester compound and the corresponding free boronic acid compound. In some embodiments, equilibration is achieved rapidly after addition of the aqueous medium, for example, within 10-15 minutes. The relative concentrations of boronate ester and boronic acid present in the equilibrium are, for example, the pH of the solution, the temperature, the nature of the boronic acid complexing agent, and the boronate complexing agent versus boronate ester compound present in the lyophilized powder. Depends on parameters such as ratio.

  The pharmaceutical composition of the present invention is preferably formulated for administration to a patient having a proteasome-mediated disease or at risk for its onset or recurrence. As used herein, the term “patient” means an animal, preferably a mammal, more preferably a human. Preferred pharmaceutical compositions of the invention are those that are formulated for oral, intravenous, or subcutaneous administration. However, any of the above dosage forms containing a therapeutically effective amount of a compound of the invention are well within the scope of routine experimentation and are thus well within the scope of the invention. In some embodiments, the pharmaceutical composition of the present invention may further comprise another therapeutic agent. In some embodiments, such other therapeutic agent is a therapeutic agent that is normally administered to a patient having the disease or condition being treated.

  “Therapeutically effective amount” means an amount sufficient to result in a detectable reduction in proteasome activity or the severity of a proteasome-mediated disease. The amount of proteasome inhibitor required will depend on the effectiveness of the inhibitor for a given cell type and the length of time required to treat the disease. In addition, specific dosages and treatment plans for any particular patient will include the activity of the particular compound employed, the patient's age, weight, general health, sex, and dietary habits, administration time, excretion rate, concomitant medications, It should be understood that this will depend on various factors, including the judgment of the treating physician and the severity of the particular disease being treated. The amount of additional therapeutic agent present in the composition of the invention is typically below the amount normally administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent ranges from about 50% to about 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

  In another aspect, the present invention provides a method for treating a patient having a proteasome-mediated disease or at risk of developing or relapse thereof. As used herein, the term “proteasome-mediated disease” refers to any disease, condition caused by or characterized by increased proteasome expression or activity or requiring proteasome activity. Or including state. The term “proteasome-mediated disease” also includes any disease, illness, or condition in which inhibition of proteasome activity is beneficial.

For example, compounds and pharmaceutical compositions of the present invention, the protein is regulated by proteasome activity _{^{(e.g., NF K B, p27 Kip,}} p21 WAF / CIP1, p53) are useful in the treatment of diseases mediated by. Related diseases include inflammatory diseases (eg, rheumatoid arthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, dermatitis (eg, atopic dermatitis, psoriasis)), proliferation Vascular disease (eg, atherosclerosis, restenosis), proliferative eye disease (eg, diabetic retinopathy), benign proliferative disease (eg, hemangioma), autoimmune disease (eg, multiple sclerosis) Tissue and organ rejection), and inflammation associated with infection (eg, immune response), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, motor neuron disease, neuropathic pain, triplet repeat disease, astrocytoma, and Neurodegeneration as a result of alcoholic liver disease), ischemic injury (eg, stroke), and cachexia (eg, various physiological and pathological conditions (eg, , Nerve injury, fasting, fever, acidosis, HIV infection, eye disorders, and accelerated muscle proteolysis with specific endocrine disorders)) can be mentioned.

  The compounds and pharmaceutical compositions of the invention are particularly useful for the treatment of cancer. As used herein, the term “cancer” establishes uncontrolled or unregulated cell proliferation, decreased cell differentiation, inappropriate ability to invade surrounding tissues, and / or new growth at ectopic sites. Refers to cell damage characterized by ability. The term “cancer” includes, but is not limited to, solid tumors and blood-borne tumors. The term “cancer” encompasses skin, tissue, organ, bone, cartilage, blood, and vascular diseases. The term “cancer” further encompasses primary and metastatic cancers.

  Non-limiting examples of solid tumors that can be treated with the disclosed proteasome inhibitors include pancreatic cancer, bladder cancer, colorectal cancer, breast cancer including metastatic breast cancer, androgen-dependent and androgen-independent prostate cancer Prostate cancer, including, for example, renal cancer, including metastatic renal cell carcinoma, hepatocellular carcinoma, such as non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and lung cancer, including lung adenocarcinoma, such as Ovarian cancer including advanced epithelial cancer or primary peritoneal cancer, cervical cancer, gastric cancer, esophageal cancer, eg head and neck cancer including head and neck squamous cell carcinoma, melanoma, neuroendocrine cancer including metastatic neuroendocrine tumor Examples include brain tumors including glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma, osteosarcoma, and soft tissue sarcoma.

  Non-limiting examples of hematological malignancies that can be treated with the disclosed proteasome inhibitors include acute myeloid leukemia (AML), chronic CML and accelerated CML and CML blast crisis (CML-BP) ( CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's disease (HD), non-Hodgkin lymphoma (NHL) including follicular lymphoma and mantle cell lymphoma, B cell lymphoma, T cell lymphoma, Multiple myeloma (MM), Waldenstrom macroglobulinemia, refractory anemia (RA), refractory anemia with cyclic iron blasts (RARS), (refractory anemia with excess blasts (RAEB), And myelodysplastic syndrome (MDS), including RAEB in transformation (RAEB-T), and myeloproliferative syndrome

  In some embodiments, the compounds or compositions of the invention are used to treat a patient having, or at risk of developing or recurring, a cancer selected from the group consisting of multiple myeloma and mantle cell lymphoma Is done.

  In some embodiments, the proteasome inhibitors of the present invention are administered in conjunction with another therapeutic agent. Other therapeutic agents can also inhibit the proteasome or act by different mechanisms. In some embodiments, the other therapeutic agent is a therapeutic agent normally administered to a patient having the disease or condition being treated. The proteasome inhibitors of the present invention may be administered with other therapeutic agents in a single dosage form or in different dosage forms. When administered as a separate dosage form, the other therapeutic agent may be administered before, simultaneously with, or after administration of the proteasome inhibitor of the present invention.

  In some embodiments, the proteasome inhibitor of formula (I) is administered in conjunction with an anticancer agent. As used herein, the term “anticancer agent” refers to any agent that is administered to a subject with cancer for the purpose of treating the cancer.

  Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (eg, irinotecan, topotecan, camptothecin, and analogs or metabolites thereof, and doxorubicin), topoisomerase II inhibitors (eg, etoposide, teniposide, and daunorubicin) ), Alkylating agents (eg, melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide), DNA intercalating agents (eg, cisplatin) Oxaliplatin, and carboplatin), DNA intercalators and free radical generators, such as bleomycin, and nucleoside mimetics (e.g. Orourashiru, Kapeshichibin, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea) and the like.

Chemotherapeutic agents that block cell replication include paclitaxel, docetaxel, and related analogs, vincristine, vinblastine, and related analogs, thalidomide, lenalidomide, and related analogs (eg, CC-5013 and CC-4047), proteins tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib), proteasome inhibitors (e.g., bortezomib), NF-? K _B inhibitors, including inhibitors of I K _B kinase, combined with over-expressed protein in cancer, it Antibodies that down-regulate cell replication by (eg, trastuzumab, rituximab, cetuximab, and bevacizumab) and other inhibitors of proteins or enzymes that are known to be upregulated, overexpressed, or activated in cancer Harms down-regulate cell replication).

  In order to more fully understand the present invention, the following preparation and test examples are described. These examples illustrate how to make or test certain compounds and are not to be construed as limiting the scope of the invention in any way.

Analytical LC-MS Method Spectra were run on a Symmetry C18-3.5 μm-4.6 × 50 mm column using the following gradient:
Solvent A: 2% isopropyl alcohol, 98% water, 10 mM NH4OAc
Solvent B: 75% acetonitrile, 25% methanol, 10 mM NH4OAc.

Example 1: [(1R) -l - ({[(2, 3- difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid. Synthesis of 20D-mannitol (I-1)

Step 1: To a solution of 2,3-difluorobenzoic acid (0.190 g, 1.2 mmol) in methyl [(2,3-difluorobenzoyl) amino] acetate tetrahydrofuran (5 mL) was added glycine methyl ester hydrochloride (0 .150 g, 1.2 mmol), HOBt (0.162 g, 1.2 mmol), DIEA (0.209 mL, 1.2 mmol), and EDCI (0.252 g, 1.3 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was quenched with a saturated solution of sodium bicarbonate and the product was separated in DCM. The organic layer was separated and the solvent removed to give methyl [(2,3-difluorobenzoyl) amino] acetate that was used in the next step without purification.

Step 2: [(2,3-Difluorobenzoyl) amino] acetic acid To a solution of methyl [(2,3-difluorobenzoyl) amino] acetate (0.250 g, 1.1 mmol) in methanol (7 mL) was hydroxylated. Lithium (0.053 g, 2.2 mmol) and water (3 mL) were added. The reaction mixture was stirred overnight. The mixture was diluted with water (20 mL) and acidified with 1N HCl (5 mL). The product was separated into DCM / methanol (4: 1). The organic layer was dried over sodium sulfate and the solvent removed to give [(2,3-difluorobenzoyl) amino] acetic acid, which was used in the next step without purification.

Step 3: 2,3-difluoro-N- [2-({(1R) -3-methyl-1-[(3aR, 4R, 6R, 7aS) -3a, 5,5-trimethylhexahydro-4,6 [(2,3-Difluorobenzoyl) amino] acetic acid (0 in methano-1,3,2-benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] benzamidodimethylformamide (10 mL) .205 g, 0.95 mmol) in a solution of TBTU (0.337 g, 1.0 mmol) and (1R) -3-methyl-1-[(3aS, 4S, 6S) as its trifluoroacetate salt. , 7aR) -3a, 5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl] butan-1-amine (0.362 g, 0.95 mm) Le) was added. The mixture was cooled to 0 ° C. and DIEA (0.498 mL, 2.9 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched with water (100 mL) and the product was separated in DCM. The organic layer was dried over sodium sulfate, the solvent was removed, and 2,3-difluoro-N- [2-({(1R) -3-methyl-1-[(3aR, 4R _, 6R, 7aS) -3a, 5,5-Trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] benzamide was obtained.

Step 4: [(1R) -l-({[(2,3-difluorobenzoyl) amino] acetyl] amino) -3-methylbutyl] boronic acid in methanol / 1N HCl (1: 1) (1.5 mL) 2,3-difluoro-N- [2-({(1R) -3-methyl-1-[(3aR, 4R, 6R, 7aS) -3a, 5,5-trimethylhexahydro-4,6-methano- To a solution of 1,3,2-benzodioxaborol-2-yl] butyl} amino) 2-oxoethyl] benzamide (0.536 g, 1.2 mmol) was added heptanol (1 mL) and isobutyl boronate (0. 207 g, 2.0 mmol) was added. The reaction mixture was stirred overnight. The heptanol layer was separated and the methanol / HCl layer was concentrated. The crude product was purified by reverse phase HPLC to give [(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid.

Step 5: [(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid. 20D-mannitol] (I-1)
[(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid (0.085 g, in t-butyl alcohol (2 mL) and water (5 mL) D-mannitol (0.943 g, 5.2 mmol) was added to the 0.26 mmol) solution. The solution was warmed and stirred until everything was dissolved. The solution is then frozen and the solvent removed by lyophilization to give [(1R) -1-({[(2,3-difluorobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid. 20D-mannitol (I-1) (0.98 g, 97%) was obtained.

Example 2: [(1R) -1-({[(2-Bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid. Synthesis of 20D-mannitol (I-5)

Step 1: tert-butyl [2-({(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methano-1, 3,2-Benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] carbamate (1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR as trifluoroacetate salt ) -3a, 5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl] butan-1-amine (4.9 g, 10.8 mmol) To the mixture was added N -.- (tert-butoxycarbonyl) glycine (1.98 g, 11.3 mmol) and TBTU (3.81 g, 11.9 mmol) in DCM (100 mL) was added to DCM (25 m ) In DIEA (5.64 mL, it was added dropwise over 15 min 32.4 mmol). The reaction mixture was stirred overnight and concentrated. The crude product was purified by column chromatography and tert-butyl [2-({(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4 , 6-Methano-1,3,2-benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] carbamate (2.5 g, 55%) was obtained.

Step 2: 2-amino-N-{(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methano-1,3 , 2-Benzodioxaborol-2-yl] butyl} acetamide in tert-butyl [2-({(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) ] in DCM (15 mL) -3a, 5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] carbamate (2.5 g, 5. To a solution of 9 mmol) 4M HCl in dioxane (5.9 mL) was added. The reaction mixture was stirred for 2 hours, concentrated and 2-amino-N-{(1R) -3-methyl-1-[(3aS, 4S _, 6S _, 7aR) -3a, 5,5-trimethylhexahydro-4. _{, 6-methano-1,3,} to give the 2-benzodioxole Sabo roll 2-yl] butyl} acetamide, which was used in the next step without purification.

Step 3: 2-Bromo-N- [2-({(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methano 2-Bromobenzoic acid (0.124 g, 0.62 mmol) in -1,3,2 -benzodioxaborol-2-yl] butyl} amino) -2- oxoethylbenzamide DCM (2.25 mL) Solution of EDCI (0.119 g, 0.62 mmol), HOBt (0.084 g, 0.62 mmol), N-methylmorpholine (0.185 mL, 1.68 mmol), and 2-amino-N- {(1R) -3-Methyl-1-[(3aS, 4S, 6S _, 7aR) -3a _, 5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol- 2-yl] butyl } Acetamide (0.2 g, 0.56 mmol) was added. The reaction mixture was stirred for 2 hours and concentrated. The residue was diluted with water and extracted with EtOAc. The organic solutions were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated. The crude product was purified by column chromatography and 2-bromo-N- [2-({(1R) -3-methyl-1-[(3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexa _{hydro-4,6-methano-1,3,} to give 2-benzodioxole Sabo roll 2-yl] butyl} amino) -2-oxoethyl] benzamide (0.22 g, 78%).

Step 4: 2- (bromo in [(1R) -1-({[(2-bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid methanol / hexane (1: 1) (2.2 mL) -N- [2 - ({(1R ) -3- methyl -1 - [(3aS, 4S, 6S, 7aR) -3a, 5,5- tri methylhexahydrophthalic _{4,6-methano-1,3,} 2 To a solution of -benzodioxaborol-2-yl] butyl} amino) -2-oxoethyl] benzamide (0.220 g, 0.44 mmol) (1 mL, 1.0 mmol) and isobutyl boronate (0. 078 g, 0.76 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was concentrated and purified by reverse phase HPLC to obtain [(1R) -1-({[(2-bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid (0.119 g, 73%). Got.

Step 5: [(1R) -1-({[(2-Bromobenzoyl) amino] acetyl} amino) 3-methylbutyl] boronic acid. 20D-mannitol (I-5)
[(1R) -1-({[(2-Bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid (0.103 g, 0. 1) in tert-butyl alcohol (9 mL) and water (15 mL). D-mannitol (1.01 g, 5.5 mmol) was added to a solution of 28 mmol). The solution was warmed and stirred until everything was dissolved. The solution is then frozen and the solvent removed by lyophilization to give [(1R) -1-({[(2-bromobenzoyl) amino] acetyl} amino) -3-methylbutyl] boronic acid. 20D-mannitol (I-5) (0.92 g, 84%) was obtained.

  The compounds in the following table were prepared from the appropriate starting materials in a manner analogous to that of Example 1 or 2.

Example 2: 20S Proteasome Assay 1 μL of test compound dissolved in DMSO in a 384 well black microtiter plate was added to human PA28 activator (Boston Biochem, final 12 nM) and Ac-WLA-AMC (β5 selective substrate) ( 25 μL assay buffer at 37 ° C. (final 15 μM) was added followed by 25 μL assay buffer at 37 ° C. containing human 20S proteasome (Boston Biochem, final 0.25 nM). The assay buffer consists of 20 mM HEPES, 0.5 mM EDTA, and 0.01% BSA, pH 7.4. Subsequently, the reaction was subjected to a BMG Galaxy plate reader (37 ° C., excitation 380 nm, emission 460 nm, gain 20). The percent inhibition was calculated relative to the 0% inhibition (DMSO) and 100% inhibition (10 μM bortezomib) controls.

When tested in this assay, all of compounds I-1 to I-21 exhibited IC ₅₀ values of less than 50 nM.

Example 3: Anti-proliferation assay HCT-116 (1000) or other tumor in 100 μL of appropriate cell culture medium (McCoy's 5A for HCT-116, Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) Cells are seeded in the wells of a 96 well cell culture plate and the plate is incubated at 37 ° C. for 96 hours. MTT or WST reagent (10 μL, Roche) is added to each well and incubated for 4 hours at 37 ° C. as described by the manufacturer. For MTT, solubilize metabolic dye overnight according to manufacturer's instructions (Roche). Read the optical density of each well using a spectrophotometer (Molecular Devices) at 595 nm (main) and 690 nm (standard) for MTT and 450 nm for WST. For MTT, the standard optical density value is subtracted from the dominant wavelength value. The percentage of inhibition is calculated using the value from the DMSO control set at 100%.

Example 4: In vivo tumor efficacy model The right dorsal side of female CD-1 nude mice (5-8 weeks old, Charles River) using a 1 mL 26 3/8 gauge needle (Becton Dickinson Ref # 309625) The subcutaneous space in the abdomen is aseptically injected with HCT-116 (2-5 × 10 ⁶ ) or other tumor cells immediately after dissociation in 100 μL of RPMI-1640 medium (Sigma-Aldrich). Alternatively, some xenograft models require serial passage of tumor fragments. In these cases, anesthetized (3-5% isoflorane / oxygen mixture) was applied to C.I. A small piece of tumor tissue (approximately 1 mm ³ ) is implanted subcutaneously into the right dorsal flank of a B-17 / SCID mouse (5-8 weeks old, Charles River) via a 13 gauge trocar (Popper & Sons 7927). From 7 days after inoculation, tumors are measured twice a week using a vernier caliper. Calculate tumor volume using standard procedures (0.5 × (length × width ² )). When the tumor reaches a volume of about 200 mm ³ , mice are randomly assigned to treatment groups and begin to receive drug treatment. The dosing and schedule for each experiment will be determined based on previous results obtained from pharmacokinetic / pharmacodynamic and maximum tolerated studies. The control group receives vehicle without any drug. Typically, test compound (100-200 μL) is administered via intravenous (27 gauge needle), oral (20 gauge feeding needle), or subcutaneous (27 gauge needle) route at various doses and schedules. . Tumor size and body weight are measured twice a week and the study is terminated when the control tumor reaches approximately 2000 mm ³ .

  Although the foregoing invention has been described in some detail for purposes of clarity and understanding, these specific embodiments are illustrative and are not to be considered limiting. Those skilled in the art can read this disclosure and make various changes in form and detail without departing from the true scope of the invention as defined by the appended claims rather than by the specific embodiments. You will understand.

  The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The issued patents, applications, and references cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. Incorporated into. In case of conflict, the present disclosure, including definitions, will prevail.

Claims (18)

Compound of formula (v):

Is the process of generating
Wherein ring A is

Selected from the group consisting of
The process is:
(1) reacting a compound of formula (i) with a compound of formula (ii) to form a compound of formula (iii):


Wherein PG is a protecting group;
(2) Deprotecting the compound of formula (iii) to form the compound of formula (iii):


(3) reacting a compound of formula (iii) with a compound of formula (viii) to form a compound of formula (iv):


And (4) deprotecting the compound of formula (iv) to form a compound of formula (v),
Where Z ¹ and Z ² together with the boron atom to which they are attached


Forming;
X ₁ ^- is _CF 3 CO ₂ ^- and is, then, _{X 2} ^- is Cl ^- is, process.   The process according to claim 1, wherein the reaction of steps (1) and (3) is performed in the presence of a peptide coupling reagent. The process according to claim 2, wherein the peptide coupling reagent is selected from carbodiimide reagents, phosphonium reagents, and uronium reagents.   The peptide coupling reagent is dicyclohexylcarbodiimide (DCC), l- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP). ), O- (lH-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBt), The process of claim 3.   The process according to claim 1, wherein the reaction in steps (1) and (3) is carried out in the presence of a solvent.   The process of claim 5, wherein the solvent is selected from dichloromethane, tetrahydrofuran, and dimethylformamide.   The process of claim 1 wherein PG is a protecting group selected from acyl protecting groups and urethane protecting groups.   8. The process of claim 7, wherein the protecting group is selected from formyl, acetyl, succinyl, methoxysuccinyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). . Ring A is


The process of claim 1, wherein Compound of formula (v):


Is the process of generating
Wherein ring A is


Selected from the group consisting of
The process is:
(1a) reacting a compound of formula (viii) with a compound of formula (vi) to form a compound of formula (via):


Wherein PG is a protecting group;
(2a) deprotecting the compound of formula (via) to form the compound of formula (vii):


(3a) reacting a compound of formula (vii) with a compound of formula (i) to form a compound of formula (iv):


And (4a) deprotecting the compound of formula (iv) to form a compound of formula (v),
Where Z ¹ and Z ² together with the boron atom to which they are attached,


Forming;
X ₁ ^- is _CF 3 CO ₂ ^- and is, then, _{X 2} ^- is Cl ^- is, process.   The process according to claim 10, wherein the reaction of steps (1a) and (3a) is performed in the presence of a peptide coupling reagent. 12. The process of claim 11, wherein the peptide coupling reagent is selected from carbodiimide reagents, phosphonium reagents, and uronium reagents.   The peptide coupling reagent is dicyclohexylcarbodiimide (DCC), l- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP). ), O- (lH-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBt), The process according to claim 12.   The process according to claim 10, wherein the reaction of steps (1a) and (3a) is carried out in the presence of a solvent.   15. The process according to claim 14, wherein the solvent is selected from dichloromethane, tetrahydrofuran, and dimethylformamide.   The process according to claim 10, wherein PG is a protecting group selected from an acyl protecting group and a urethane protecting group.   17. The process of claim 16, wherein the protecting group is selected from formyl, acetyl, succinyl, methoxysuccinyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). . Ring A is


The process of claim 10, wherein