Proteasome inhibitor

Abstract

To provide a novel compound useful as a proteasome inhibitor.SOLUTION: The invention also provides a pharmaceutical composition comprising the compound, and a method of using the composition for treatment of various diseases. The compound is a compound of the general formula (I) or a pharmaceutical acceptable salt or boronic acid anhydride thereof. In the formula, Z1 and Z2 are each independently hydroxy, alkoxy, aryloxy or aralkoxy, or Z1 and Z2 together form a moiety derived from a boronic acid complexing agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to boronic acid and boronic ester compounds that are useful as proteasome inhibitors. The present invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various diseases. [Background technology]

[0002] Boronic acid and boronic ester compounds exhibit a variety of pharma- ceutical useful biological activities. Shenvi et al., U.S. Patent No. 5,399,431 (1985), discloses that peptide boronic acids are inhibitors of certain proteolytic enzymes. Kettner and Shenvi, U.S. Patent No. 5,499,492 (1993), U.S. Patent No. 5,499,433 (1993), and U.S. Patent No. 5,499,434 (1993), describe a class of peptide boronic acids that inhibit trypsin-like proteases. Kleeman et al., U.S. Patent No. 5,499,432 (1992), discloses N-terminally modified peptide boronic acids that inhibit the action of renin. Kinder et al., U.S. Patent No. 5,499,432 (1992), discloses certain boronic acid compounds that inhibit the growth of cancer cells. Bachovchin et al., U.S. Patent No. 5,499,432 (1992), discloses peptide boronic acid compounds that inhibit fibroblast activation protein.

[0003] Boronic acid and boronic acid ester compounds are particularly promising as inhibitors of the proteasome, a multicatalytic protease responsible for most of the intracellular protein turnover. Adams et al., US Pat. No. 6,299,433 (1998), describes peptide boronic acid ester and acid compounds that are useful as proteasome inhibitors. This reference also describes the use of boronic acid ester and acid compounds to reduce the rate of muscle protein degradation, reduce the activity of NF-κB in cells, reduce the rate of degradation of p53 protein in cells, inhibit cyclin degradation in cells, inhibit cancer cell growth, and inhibit NF-κB-dependent cell adhesion. Furet et al., US Pat. No. 6,299,433, Chatterjee et al., US Pat. No. 6,299,433, and Bemadini et al., US Pat. No. 6,371,633, disclose additional boronic acid ester and acid compounds that are reported to have proteasome inhibitory activity.

[0004] Non-Patent Document 1 discloses that the proteasome is a proteolytic component of the ubiquitin-proteasome pathway, in which proteins are targeted for degradation by conjugation with 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 peptidyl glutamyl peptidase activities. The 20S proteasome constitutes the catalytic core of the 26S proteasome. Non-patent literature 3 teaches that various peptide substrates, including 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-4-methylcoumarin, and 2NA is 2-naphthylamine), are cleaved by the 20S proteasome.

[0005] Proteasome inhibition represents an important new strategy for cancer therapy. Non-Patent Document 4 describes the important role that the ubiquitin-proteasome pathway plays in regulating the cell cycle, tumor growth, and metastasis. The authors report that the ubiquitin-proteasome pathway inhibits a number of key regulatory proteins, including cyclins, as well as the cyclin-dependent kinases p21 and p27. KIP1 are degraded sequentially during the cell cycle by the ubiquitin-proteasome pathway. The ordered degradation of these proteins is necessary for cells to progress through the cell cycle and undergo mitosis.

[0006] Furthermore, the ubiquitin-proteasome pathway is necessary for transcriptional regulation. Non-Patent Document 5 teaches that activation of the transcription factor NF-κB is regulated by proteasomal degradation of the inhibitor protein IκB. NF-κB plays a central role in regulating genes involved in immune and inflammatory responses. Non-Patent Document 6 teaches that the ubiquitin-proteasome pathway is necessary for the expression of cell adhesion molecules such as E-selectin, ICAM-1, and VCAM-1. Non-Patent Document 7 teaches that cell adhesion molecules are involved in tumor metastasis and angiogenesis in vivo by shuttling tumor cell adhesion and extravasation between the vasculature in the body and distant tissue sites. Furthermore, Non-Patent Document 8 teaches that NF-κB is an anti-apoptotic regulator, and inhibition of NF-κB activation further sensitizes cells to environmental stress and cytotoxic drugs.

[0007] The proteasome inhibitor VELCADE® (bortezomib, N-2-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid) is the first proteasome inhibitor to receive regulatory approval. Non-Patent Document 9 reviews the clinical studies that led to the approval of bortezomib for the treatment of patients with multiple myeloma who have received at least one prior therapy. 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 may contribute to the antitumor activity of bortezomib.

[0008] As is evident from the above references, the proteasome represents an important target for therapeutic intervention. Thus, there is a continuing need for new and / or improved proteasome inhibitors. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Pat. No. 4,499,082 [Patent Document 2] U.S. Pat. No. 5,187,157 [Patent Document 3] U.S. Pat. No. 5,242,904 [Patent Document 4] U.S. Patent No. 5,250,720 [Patent Document 5] U.S. Pat. No. 5,169,841 [Patent Document 6] U.S. Pat. No. 5,106,948 [Patent Document 7] International Publication No. 07 / 0005991 Brochure [Patent Document 8] U.S. Patent No. 5,780,454 [Patent Document 9] International Publication No. 02 / 096933 Brochure

Patent document 10

Patent document 11

Patent document 12

Non-licensed literature

[0010]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0011] The present invention provides compounds that are effective inhibitors of the proteasome, which are useful for inhibiting proteasome activity in vitro and in vivo, and are particularly useful in the treatment of a variety of cell proliferative disorders.

[0012] The compounds of the present invention are compounds of the general formula (I):

[0013] [ka] or a pharma- ceutically 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

[0014] [ka] is selected from the group consisting of:

[0015] A boronic acid compound of formula (I), 1 and Z 2 are hydroxyl groups) are referred to by the following chemical names:

[0016] [Table 1-1]

[0017] [Table 1-2] For example, the present invention provides the following: (Item 1) Compounds of formula (I): [ka] or a pharma- ceutically acceptable salt or boronic acid anhydride thereof, In the formula, 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 [ka] 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 Item 1. The compound according to item 1, selected from the group consisting of [(1R)-1-({[(3,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid, or a pharma- ceutically acceptable salt or boronic acid 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)-l-({[(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)-l-({[(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, The compound according to 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 pharma- ceutically 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The term "alkyl" used alone or as part of a larger moiety refers to a straight or branched chain or cyclic aliphatic group having from 1 to 12 carbon atoms. The term "alkoxy" refers to an O-alkyl group.

[0019] For example, the terms "aryl" and "ar-" used alone or as part of a larger moiety, such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refer to an alkyl group having 1 to 3 rings and a C6-C 14 Aryl groups refer to aromatic hydrocarbons, each of which is optionally substituted. Preferably, an aryl group is C 6-10 Aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. An "aralkyl" or "arylalkyl" group comprises an aryl group covalently bonded to an alkyl group, either of which is independently optionally substituted. Preferably, aralkyl groups include, but are not limited to, C aryl groups, such as benzyl, phenethyl, and naphthylmethyl. 6-10 Aryl (C 1-6 ) Alkyl, C 6-10 Aryl (C 1-4 ) alkyl, or C 6-10 Aryl (C 1-3 ) alkyl.

[0020] As used herein, the term "substituted" means that a hydrogen radical of the specified moiety is replaced with a specified substituent, provided that the substitution results in a stable or chemically feasible compound. Non-limiting examples of suitable substituents include, but are not limited to, 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, C1-6 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, C 6-10 Aryl, C 1-6 Alkyl(C 6-10 ) aryl, and halo(C 6-10 ) aryl.

[0021] As used herein, the phrase "one or more substituents" refers to a number of substituents equal to one to the maximum number of possible substituents based on the number of available binding sites, provided that the above conditions of stability and chemical feasibility are met. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and the substituents may be either the same or different substituents. 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.

[0022] The term "about" is used herein to mean approximately, roughly, approximately, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the limits above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value above and below the set forth value by a variance of 10%.

[0023] As used herein, the term "comprises" means "including but not limited to."

[0024] Unless otherwise stated, structures depicted herein are meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of a hydrogen atom by deuterium or tritium, or 13 C or 14 Compounds having the present structure except for the replacement of a carbon atom with a C-enriched carbon are within the scope of this invention.

[0025] As used herein, the term "boronic acid" refers to a compound containing a -B(OH)2 moiety. In some embodiments, boronic acid compounds can form oligomeric anhydrides by dehydration of the boronic acid moiety. See, e.g., Snyder et al., J. Am. Chem. Soc. 80:3611 (1958) reports oligomeric arylboronic acids.

[0026] As used herein, the term "boronic acid 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 contain two, three, four 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 invention are illustrated below.

[0027] [ka] 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 anhydride compound comprises a cyclic trimer ("boroxine") of formula (2), where n is 1. The variable W has the formula (3).

[0028] [ka] wherein ring A has the values ​​described above for formula (I).

[0029] In some embodiments, at least 80% of the boronic acid present in the boronic 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 anhydride compound is present in the form of a single oligomeric anhydride. In some preferred embodiments, the boronic anhydride compound consists of, or consists essentially of, a boroxine having the formula (3).

[0030] Boronic acid anhydride compounds can be prepared from the corresponding boronic acids, preferably by exposure to dehydrating conditions, including but not limited to recrystallization, lyophilization, exposure to heat, and / or exposure to a drying agent. Non-limiting examples of suitable recrystallization solvents include ethyl acetate, dichloromethane, hexane, ether, acetonitrile, ethanol, and mixtures thereof.

[0031] In some embodiments, Z 1 and Z 2 together form a moiety derived from a boronic acid complexing agent. For purposes of this 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 "a moiety derived from a boronic acid complexing agent" refers to a moiety formed by removing hydrogen atoms from two functional groups of a boronic acid complexing agent.

[0032] As used herein, the terms “boronate ester” and “boronic acid ester” are used interchangeably and refer to —B(Z 1 (Z 2 ) moiety, where Z 1 or Z2 at least one of Z is alkoxy, aralkoxy, or aryloxy; 1 and Z 2 together form a moiety derived from a boronic acid complexing agent having at least one hydroxyl group.

[0033] 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 bonded atoms in a chain or ring containing carbon atoms and, optionally, heteroatom(s) which may be N, S, or O, where the atom bonded to boron in each case is an oxygen atom.

[0034] As used herein, the term "compound having at least two hydroxyl groups" refers to any compound having two or more hydroxyl groups. For purposes of the present invention, the two hydroxyl groups are preferably separated by at least two bond atoms, preferably about 2-5 bond atoms, more preferably 2 or 3 bond 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 bonded to boron by any two oxygen atoms of its hydroxyl groups. Preferably, the boron atom, the oxygen atom bonded to boron, and the atom bonding the two oxygen atoms together form a five- or six-membered ring.

[0035] For purposes of the present invention, the boronic acid complexing agent is preferably pharma- ceutically acceptable, i.e., 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 Both have the formula: CH 12 O6 moiety, where the oxygen atoms of the two deprotonated hydroxyl groups form covalent bonds with boron to form a boronate ester compound. 1 and Z 2 together form the moiety derived from D-mannitol.

[0036] In some embodiments, the compound of formula (I) is formulated as a lyophilized powder as described in WO 02 / 059131 to Plamondon et al., 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 includes free mannitol and mannitol boronate ester in a molar ratio ranging from about 10:1 to about 100:1, from about 20:1 to about 100:1, or from about 40:1 to about 100:1.

[0037] In some embodiments, lyophilized powder comprises mannitol and the compound of formula (I) substantially free of other components.However, the composition can further comprise one or more other pharma- ceutically acceptable excipients, carriers, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art.The preparation of pharma-ceutically 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 latest edition.

[0038] The lyophilized powder comprising the compound of formula (I) is preferably prepared according to the procedures described in WO 02 / 059131 to Plamondon et al. Thus, in some embodiments, a method for preparing the lyophilized powder comprises (a) preparing an aqueous mixture comprising a peptide boronic acid and a dihydroxy compound, and (b) lyophilizing the mixture.

[0039] General synthesis methodology Compounds of formula (I) can be prepared by methods known to those skilled in the art. For example, see U.S. Patent No. 5,780,454 to Adams et al., International Patent Publication No. 2005 / 097809 to Pickersgill et al. An exemplary synthetic route is depicted in Scheme 1 below.

[0040] Scheme 1:

[0041] [ka] Coupling of compound i with N-protected glycine (ii) and subsequent N-terminal deprotection results in compound iii. Examples of suitable protecting groups (PG) include, but are not limited to, acyl protecting groups, such as formyl, acetyl (Ac), succinyl (Suc), and methoxysuccinyl, and urethane protecting groups, such as tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). The peptide coupling reaction can be carried out by pre-conversion of the carboxylic acid moiety of compound ii to an activated ester, such as, for example, O-(N-hydroxysuccinimide) ester, followed by treatment with compound i. Alternatively, the activated ester can be generated in situ by contacting the carboxylic acid with a peptide coupling reagent. Examples of suitable peptide coupling reagents include, but are not limited to, carbodiimide reagents such as, for example, dicyclohexylcarbodiimide (DCC) or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phosphonium reagents such as, for example, benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), and uronium reagents such as, for example, O-(lH-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0042] Compound iii is then coupled with a substituted benzoic acid (ArCOH) to give compound iv. The peptide coupling conditions described above for the coupling of compounds i and ii are also suitable for coupling of compounds with ArCOH. Deprotection of the boronic acid moiety then gives compound v. The deprotection step is preferably carried out by reacting a boronic ester compound iv, an organoboronic acid acceptor, a lower alkanol, C 5-8 This is carried out by transesterification in a two-phase mixture containing a hydrocarbon solvent and an aqueous mineral acid.

[0043] Scheme 2:

[0044] [ka] Alternatively, the order of coupling reactions may be reversed as shown in Scheme 2. Thus, O-protected glycine (vi) is first coupled with a substituted benzoic acid (ArCOH) followed by ester hydrolysis to form compound vii. Coupling with compound i and boronic acid deprotection are then carried out as described above for Scheme 1 to give compound v.

[0045] Use, Prescription, and Administration The present invention provides compounds that are potent inhibitors of the proteasome, which can be assayed in vitro or in vivo for their ability to inhibit peptide hydrolysis or protein degradation by the proteasome.

[0046] Thus, in another aspect, the present invention provides a method for inhibiting one or more peptidase activities of the proteasome in a cell, comprising contacting a cell in which proteasome inhibition is desired with a compound described herein, or a pharma- ceutically acceptable salt, boronic ester, or boronic anhydride thereof.

[0047] The present invention also provides a method for inhibiting cell proliferation, comprising contacting a cell for which such inhibition is desired with a compound as described herein. The phrase "inhibit cell proliferation" is used to indicate the ability of the compound of the present invention to inhibit cell number or cell growth in contacted cells, compared to cells not contacted with the inhibitor. Cell proliferation can be assessed by counting cell number using a cell counter or by cell viability assays, such as MTT or WST assays. If the cell is in solid proliferation (e.g., solid tumor or organ), such cell proliferation can be assessed by, for example, measuring growth with a caliper and comparing the size of growth of contacted cells with non-contacted cells.

[0048] Preferably, the growth of the cells contacted with the inhibitor is retarded by at least about 50% compared to the growth of non-contacted cells. In various embodiments, the cell growth of the 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 "inhibit cell growth" includes a reduction in the number of contacted cells compared to non-contacted cells. Thus, proteasome inhibitors that inhibit cell growth in contacted cells can cause growth retardation, growth arrest, programmed cell death (i.e., apoptosis), or necrotic cell death of the contacted cells.

[0049] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharma- ceutically acceptable salt or boronic acid anhydride thereof, and a pharma- ceutically acceptable carrier.

[0050] When pharma- ceutically acceptable salts of the compounds of the present invention are used in these compositions, the salts are preferably obtained from inorganic or organic acids or bases.For identification of suitable salts, see, for example, Berge et al, J. Pharm. Sci. 66:1-19 (1977), and Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000.

[0051] Non-limiting examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glycoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

[0052] Suitable base addition salts include, but are not limited to, ammonium salts, alkali metal salts such as lithium, sodium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, other polyvalent metal salts such as zinc salts, salts with organic bases such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylenediamine, ethanolamine, and choline, and salts with amino acids such as arginine, etc. In some embodiments, the pharma- ceutically acceptable salt is a base addition salt of a boronic acid compound of formula (I), where Z 1 and Z 2 are both hydroxy.

[0053] The term "pharmaceutically acceptable carrier" is used herein to refer to a material that is compatible with a recipient subject, 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. The toxicity and adverse effects, if any, associated with the carrier are commensurate with a reasonable risk-benefit ratio for the intended use of the active agent.

[0054] The terms "carrier", "adjuvant" or "vehicle" are used interchangeably herein and include any solvents, diluents and other liquid vehicles, dispersion or suspension aids, surfactants, pH adjusters, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000, discloses various carriers used in formulating pharma-ceutically acceptable compositions, and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, for example, by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharma-ceutically acceptable composition, its use is considered to be within the scope of the present invention.Some examples of materials which can serve as pharma- ceutically acceptable carriers 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 such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose, sucrose, and mannitol, starches such as corn starch and potato starch, cellulose and its derivatives such as calcium carbonate, calcium phosphate ... Examples of suitable surfactants include, but are not limited to, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth, malt, gelatin, talc; excipients such as cocoa butter and suppository wax; 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 ethyl oleate and ethyl laurate; agar, alginic acid, isotonic saline, Ringer's solution, alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, and glycerol; cyclodextrins such as hydroxypropyl β-cyclodextrin, and sulfobutyl ether β-cyclodextrin; lubricants such as sodium lauryl sulfate and magnesium stearate; and petroleum hydrocarbons such as mineral oil and petrolatum. Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0055] The pharmaceutical compositions of the present invention can be manufactured by known methods such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. The compositions may be produced in a variety of forms, including granules, precipitates, or particulates, powders, including freeze-dried, rotary dried, or spray-dried powders, amorphous powders, tablets, capsules, syrups, suppositories, infusions, emulsions, elixirs, suspensions, or solutions.

[0056] According to a preferred embodiment, the compositions of the present invention are formulated for pharmaceutical administration to mammals, preferably humans. Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intravenously, or subcutaneously. The formulations of the present invention may be designed to be short-acting, immediate-release, or long-acting. Additionally, the compounds may be administered by local rather than systemic means, such as administration (e.g., by injection) at the site of a tumor.

[0057] Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, suspensions, syrups, and elixirs.In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as 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 esters, and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and perfumes.

[0058] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any sterile fixed oil may 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 prior to use in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media. Compositions formulated for parenteral administration may be injected by bolus injection or by timed infusion, or may be administered by continuous infusion.

[0059] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dibasic calcium phosphate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, and the like. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents such as phosphates or carbonates.

[0060] Similar types of solid compositions may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like. 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 formulation art. They may optionally contain opacifying agents, and may be solid dosage forms of compositions that release the active ingredient singly or selectively in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may 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 milk sugar, and high molecular weight polyethylene glycols, and the like.

[0061] The active compound can also be in the form of 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, release-controlling coatings, and other coatings known in the pharmaceutical formulation art. In such solid dosage forms, the active compound can be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms can also contain, as is customary, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and macrocrystalline cellulose. In addition, in the case of capsules, tablets, and pills, the dosage forms can contain buffering agents. They can optionally contain opacifying agents, and they can be solid dosage forms of compositions that release the active ingredient alone or selectively, optionally in a delayed manner, in a certain part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0062] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any necessary preservatives or buffers, as appropriate. Ophthalmic preparations, ear drops, and eye drops are also considered to be within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable 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 dispersing the compound in a polymer matrix or gel.

[0063] In some embodiments, the compound of formula (I) is administered intravenously.1 and Z 2 (both of which form a moiety derived from the boronic acid complexing agent) may be prepared in the form of a lyophilized powder as described above. 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, and phosphate buffered saline (PBS). Preferably, the lyophilized powder is reconstituted with physiological (0.9%) saline. Upon reconstitution, an equilibrium is established between the boronate ester compound and the corresponding free boronic acid compound. In some embodiments, equilibrium is achieved quickly, e.g., within 10-15 minutes, after addition of the aqueous medium. The relative concentrations of boronate ester and boronic acid present at equilibrium depend on parameters such as, for example, the pH of the solution, temperature, the nature of the boronic acid complexing agent, and the ratio of boronic acid complexing agent to boronate ester compound present in the lyophilized powder.

[0064] The pharmaceutical compositions of the present invention are preferably formulated for administration to patients with or at risk of developing or recurring a proteasome-mediated disease. As used herein, the term "patient" refers to an animal, preferably a mammal, more preferably a human. A preferred pharmaceutical composition of the present invention is a pharmaceutical composition formulated for oral, intravenous, or subcutaneous administration. However, any of the above dosage forms containing a therapeutically effective amount of a compound of the present invention are well within the bounds of routine experimentation and therefore well within the scope of the present 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 patients with the disease or condition being treated.

[0065] By "therapeutically effective amount" is meant an amount sufficient to result in a detectable decrease 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. It should also be understood that the specific dosage and treatment regime for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, the patient's age, weight, general health, sex, and dietary habits, time of administration, rate of excretion, co-medications, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of additional therapeutic agent present in the compositions of the invention is typically equal to or less than 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.

[0066] In another aspect, the present invention provides a method for treating a patient having or at risk of developing or recurring a proteasome-mediated disease. As used herein, the term "proteasome-mediated disease" includes any disease, disorder, or condition caused by or characterized by increased proteasome expression or activity, or that requires proteasome activity. The term "proteasome-mediated disease" also includes any disease, disorder, or condition in which inhibition of proteasome activity is beneficial.

[0067] For example, the compounds and pharmaceutical compositions of the present invention can be used to inhibit proteins regulated by proteasome activity (e.g., NF-. K B, p27 Kip , p21 WAF / CIP1, p53). Associated diseases include inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, dermatitis (e.g., atopic dermatitis, psoriasis)), proliferative vascular diseases (e.g., atherosclerosis, restenosis), proliferative eye diseases (e.g., diabetic retinopathy), benign proliferative diseases (e.g., hemangiomas), autoimmune diseases (e.g., multiple sclerosis, tissue and organ rejection), and inflammation (e.g., immune response) associated with infection, neurodegenerative diseases (e.g., 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 (e.g., stroke), and cachexia (e.g., accelerated muscle protein breakdown that accompanies various physiological and pathological conditions (e.g., nerve damage, hunger, fever, acidosis, HIV infection, eye disorders, and certain endocrine disorders).

[0068] The compounds and pharmaceutical compositions of the present invention are particularly useful for the treatment of cancer. As used herein, the term "cancer" refers to a cellular disorder characterized by uncontrolled or unregulated cell proliferation, reduced cell differentiation, inappropriate ability to invade surrounding tissues, and / or ability to establish new growth in ectopic locations. The term "cancer" includes, but is not limited to, solid tumors and blood-borne tumors. The term "cancer" encompasses diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" further encompasses primary and metastatic cancers.

[0069] 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, prostate cancer, including androgen-dependent and androgen-independent prostate cancer, kidney cancer, including metastatic renal cell carcinoma, hepatocellular carcinoma, lung cancer, including non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and lung adenocarcinoma, ovarian cancer, including advanced epithelial carcinoma or primary peritoneal carcinoma, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, including head and neck squamous cell carcinoma, melanoma, neuroendocrine cancer, including metastatic neuroendocrine tumors, such as brain tumors, including glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma, osteosarcoma, and soft tissue sarcoma.

[0070] Non-limiting examples of hematological malignancies that can be treated with the disclosed proteasome inhibitors include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), including accelerated CML and CML blast crisis phase (CML-BP), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), including follicular lymphoma and mantle cell lymphoma, B cell lymphoma, T cell lymphoma, multiple myeloma (MM), Waldenstrom's macroglobulinemia, myelodysplastic syndromes (MDS), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), and RAEB in transformation (RAEB-T), and myeloproliferative syndromes.

[0071] In some embodiments, the compounds or compositions of the invention are used to treat patients having, or at risk of developing or recurring with, a cancer selected from the group consisting of multiple myeloma and mantle cell lymphoma.

[0072] In some embodiments, the proteasome inhibitor of the present invention is administered in conjunction with another therapeutic agent. The other therapeutic agent may also inhibit the proteasome or may act by a different mechanism. In some embodiments, the other therapeutic agent is a therapeutic agent that is normally administered to patients with the disease or condition being treated. The proteasome inhibitor of the present invention may be administered with the other therapeutic agent in a single dosage form or in different dosage forms. When administered as separate dosage forms, the other therapeutic agent may be administered before, simultaneously with, or after administration of the proteasome inhibitor of the present invention.

[0073] In some embodiments, the proteasome inhibitor of formula (I) is administered in conjunction with an anti-cancer agent. As used herein, the term "anti-cancer agent" refers to any agent administered to a subject with cancer for the purpose of treating the cancer.

[0074] Non-limiting examples of DNA damaging chemotherapy agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin, and its analogs or metabolites, and doxorubicin), topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin), alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, , decarbazine, methotrexate, mitomycin C, and cyclophosphamide), DNA intercalating agents (e.g., cisplatin, oxaliplatin, and carboplatin), DNA intercalating agents and free radical generators such as bleomycin, and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0075] Chemotherapeutic agents that block cell replication include paclitaxel, docetaxel, and related analogues, vincristine, vinblastine, and related analogues, thalidomide, lenalidomide, and related analogues (e.g., CC-5013 and CC-4047), protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib), proteasome inhibitors (e.g., bortezomib), I K NF-Kinase B inhibitors K These include B inhibitors, antibodies (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab) that bind to proteins overexpressed in cancer, thereby downregulating cell replication, as well as other inhibitors of proteins or enzymes known to be upregulated, overexpressed, or activated in cancer, the inhibition of which downregulates cell replication.

[0076] In order that this invention may be more fully understood, the following preparative and testing examples are set forth. These examples illustrate how to make or test particular compounds and are not to be construed as limiting the scope of the invention in any way. EXAMPLES

[0077]

number

[0078] [Table 2] Example 1: [(1R)-l-({[(2 , 3-Difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid . 20. Synthesis of D-mannitol (I-1)

[0079] [ka] Step 1: Methyl [(2,3-difluorobenzoyl)amino]acetate To a solution of 2,3-difluorobenzoic acid (0.190 g, 1.2 mmol) in 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). The reaction mixture was stirred overnight. The reaction mixture was quenched with a saturated solution of sodium bicarbonate and the product was partitioned into DCM. The organic layer was separated and the solvent was removed to give methyl [(2,3-difluorobenzoyl)amino]acetate, which was used in the next step without purification.

[0080] 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 added lithium hydroxide (0.053 g, 2.2 mmol) and water (3 mL). The reaction mixture was stirred overnight. The mixture was diluted with water (20 mL) and acidified with 1N HCl (5 mL). The product was partitioned in DCM / methanol (4:1). The organic layer was dried over sodium sulfate and the solvent was removed to give [(2,3-difluorobenzoyl)amino]acetic acid, which was used in the next step without purification.

[0081] Step 3: 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 To a solution of [(2,3-difluorobenzoyl)amino]acetic acid (0.205 g, 0.95 mmol) in dimethylformamide (10 mL) was added TBTU (0.337 g, 1.0 mmol) and (1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butan-1-amine (0.362 g, 0.95 mmol) as its trifluoroacetate salt. The mixture was cooled to 0° C. and DIEA (0.498 mL, 2.9 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with water (100 mL) and the product was partitioned into 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.

[0082] Step 4: [(1R)-l-({[(2,3-difluorobenzoyl)amino]acetyl]amino)-3-methylbutyl]boronic acid To a solution of 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 (0.536 g, 1.2 mmol) in methanol / 1N HCl (1:1) (1.5 mL) was added heptanol (1 mL) and isobutyl boronate (0.207 g, 2.0 mmol). 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.

[0083] Step 5: [(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid.20D-mannitol] (I-1) To a solution of [(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid (0.085 g, 0.26 mmol) in t-butyl alcohol (2 mL) and water (5 mL) was added D-mannitol (0.943 g, 5.2 mmol). The solution was warmed and stirred until everything was dissolved. The solution was then frozen and the solvent removed by lyophilization to give [(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid.20 D-mannitol (I-1) (0.98 g, 97%).

[0084] Example 2: Synthesis of [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid.20D-mannitol (I-5)

[0085] [ka] 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 To a mixture of (1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butan-1-amine (4.9 g, 10.8 mmol) as the trifluoroacetate salt, N-·-(tert-butoxycarbonyl)glycine (1.98 g, 11.3 mmol) was added and a solution of TBTU (3.81 g, 11.9 mmol) in DCM (100 mL) was added dropwise over 15 min to a solution of DIEA (5.64 mL, 32.4 mmol) in DCM (25 mL). The reaction mixture was stirred overnight and concentrated. The crude product was purified by column chromatography to give 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%).

[0086] 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 To a solution of 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, 5.9 mmol) in DCM (15 mL) was added 4 M HCl in dioxane (5.9 mL). The reaction mixture was stirred for 2 h, concentrated 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 was obtained, which was used in the next step without purification.

[0087] Step 3: 2-Bromo-N-[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-oxoethylbenzamide To a solution of 2-bromobenzoic acid (0.124 g, 0.62 mmol) in DCM (2.25 mL) was added 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 h and concentrated. The residue was diluted with water and extracted with EtOAc. The organic solutions were combined, washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography to give 2-bromo-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3 , To obtain 2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide (0.22 g, 78%).

[0088] Step 4: [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid 2-Bromo-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3 , To a solution of [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid (0.119 g, 73%) was added 1-((1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid (0.119 g, 73%).

[0089] Step 5: [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)3-methylbutyl]boronic acid.20D-mannitol (I-5) To a solution of [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid (0.103 g, 0.28 mmol) in tert-butyl alcohol (9 mL) and water (15 mL) was added D-mannitol (1.01 g, 5.5 mmol). The solution was warmed and stirred until everything was dissolved. The solution was 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%).

[0090] The compounds in the following table were prepared from the appropriate starting materials in a manner analogous to that of Examples 1 or 2.

[0091] [Table 3] Example 2: 20S proteasome assay To 1 μL of test compound dissolved in DMSO in a 384-well black microtiter plate, 25 μL of assay buffer at 37 °C containing human PA28 activator (Boston Biochem, 12 nM final) and Ac-WLA-AMC (β5 selective substrate) (15 μM final) was added, followed by 25 μL of assay buffer at 37 °C containing human 20S proteasome (Boston Biochem, 0.25 nM final). Assay buffer consisted of 20 mM HEPES, 0.5 mM EDTA, and 0.01% BSA, pH 7.4. Reactions were then run on a BMG Galaxy plate reader (37 °C, excitation 380 nm, emission 460 nm, gain 20). Percentage of inhibition was calculated relative to controls of 0% inhibition (DMSO) and 100% inhibition (10 μM bortezomib).

[0092] When tested in this assay, compounds I-1 to I-21 all had IC 50 The values ​​were shown.

[0093] Example 3: Antiproliferative Assay HCT-116 (1000) or other tumor cells in 100 μL of the appropriate cell culture medium (McCoy's 5A for HCT-116, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) are seeded in wells of a 96-well cell culture plate and the plate is incubated at 37 °C for 96 h. MTT or WST reagent (10 μL, Roche) is added to each well and incubated at 37 °C for 4 h as described by the manufacturer. For MTT, the metabolic dye is solubilized overnight according to the manufacturer's instructions (Roche). The optical density of each well is read using a spectrophotometer (Molecular Devices) at 595 nm (primary) and 690 nm (standard) for MTT and at 450 nm for WST. For MTT, the standard optical density value is subtracted from the value at the primary wavelength. The value from the DMSO control set at 100% is used to calculate the percentage of inhibition.

[0094] Example 4: In vivo tumor efficacy model Freshly dissociated HCT-116 (2-5 × 10 cells) in 100 μL of RPMI-1640 medium (Sigma-Aldrich) were injected into the subcutaneous space in the right dorsal flank 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). 6 ) or other tumor cells are aseptically injected. Alternatively, some xenograft models require serial passaging of tumor fragments. In these cases, a small fragment of tumor tissue (approximately 1 mm 2 ) is injected into the right dorsal flank of anesthetized (3-5% isoflorane / oxygen mixture) CB-17 / SCID mice (5-8 weeks old, Charles River) via a 13-gauge trocar (Popper & Sons 7927). 3 ) are implanted subcutaneously. Starting 7 days after inoculation, tumors are measured twice a week using a Vernier caliper. Tumor volume is calculated using standard procedures (0.5 x (length x width)). 2 The tumor was about 200 mm 3 Once tumor volume reaches 100 mm, mice are randomized into treatment groups and begin receiving drug treatment. Dosing and schedule for each experiment are determined based on previous results from pharmacokinetic / pharmacodynamic and maximum tolerated dose studies. Control groups receive vehicle without any drug. Test compounds (100-200 μL) are typically administered via intravenous (27-gauge needle), oral (20-gauge feeding needle), or subcutaneous (27-gauge needle) routes at various doses and schedules. Tumor size and body weight are measured twice weekly, and control tumors reach approximately 2000 mm. 3 When this is reached, the study ends.

[0095] 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 will appreciate, upon reading this disclosure, that various changes in form and detail may be made without departing from the true scope of the invention, which is intended to be defined by the appended claims rather than the specific embodiments.

[0096] The patents and scientific literature referred to in this specification establish knowledge available to those skilled in the art.Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs.The issued patents, applications, and references cited in this specification are incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.In case of conflict, the present disclosure, including definitions, will control.

Claims

[Claim 1] Boronic acid and boronic ester compounds that are useful as proteasome inhibitors.

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