Novel USP7 inhibitors for treating multiple myelomas

Abstract

To provide novel USP7 inhibitors for treating multiple myelomas.SOLUTION: The present invention relates to inhibitors of R1 useful in the treatment of cancer and other USP7 mediated diseases, having the formula: USP7, R2, R3, R4, R5, R6 and n as described herein. In certain embodiments, the present invention provides a pharmaceutical composition suitable for use in a subject in the treatment or prevention of a disorder associated with modulation of the USP7, comprising an effective amount of any of the compounds described herein (e.g., a compound of the present invention, such as a compound of Formula (I)) and one or more pharmaceutically acceptable excipients.SELECTED DRAWING: Figure 3C

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 563,375, filed September 26, 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] Rights Statement This invention was made with government support under Grant RO1 CA211681 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Over the past five to ten years, deubiquitinating enzymes (DUBs) have attracted considerable attention as potential drug targets. Because DUB family members stabilize oncogenic proteins, DUB inhibitors effectively promote the degradation of oncogenic proteins, particularly proteins targeted directly. Therefore, there has been considerable research into well-optimized and well-characterized DUB inhibitors. However, many reported DUB inhibitors are polypharmacological agents with weak (micromolar) potency against their primary targets, limiting their usefulness in target validation and mechanistic studies. Due to the lack of high-resolution DUB-small molecule ligand complex structures, no structurally based optimization efforts have been reported for mammalian DUBs.

[0004] The DUB enzyme USP7 has been shown to be involved in the regulation of numerous cellular processes, including epigenetics, cell cycle, DNA repair, immunity, viral infection, and tumorigenesis. USP7, also known as herpesvirus-associated ubiquitin-specific protease (HAUSP), was first discovered as a protein that plays a role in viral lytic growth (Everett et al., Novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J, 16, 1997, 566-77). USP7 has attracted attention because it is involved in regulating the degradation of the tumor suppressor p53 by stabilizing the major E3 ligase for p53, MDM2 (Li et al., Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature, 416, 2002, 648-53; Cummins et al., Tumor suppression: disruption of HAUSP gene stabilizes p53. Nature, 428, 2004, 1p following 486; Li et al., A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol Cell, 13, 2004, 879-86). Consistent with its regulation of diverse substrates and biological processes, USP7 has emerged as a drug target in a wide range of malignancies, including multiple myeloma, breast cancer, neuroblastoma, glioma, and ovarian cancer (Chauhan et al., A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma). cells and overcomes bortezomib resistance. Cancer Cell, 22, 2012, 345-58; Wang et al., J Clin Invest, 126, 2016, 2205-20; Tavana et al., Nat Med, 22, 2016, 1180-1186; Cheng et al., Expression of HAUSP in gliomas correlates with disease progression and survival of patients. Oncol Rep, 29, 2013, 1730-6; Zhang et al., Expression of USP7 and MARCH7 Is Correlated with Poor Prognosis in Epithelial Ovarian Cancer. Tohoku J Exp Med, 239, 2016, 165-75). However, known USP7 inhibitors have been shown to exhibit moderate potency against USP7 and low selectivity against DUBs. Reported drawbacks of known USP7 inhibitor compounds include low solubility and general toxicity, along with moderate potency and selectivity (Chen et al., Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors. Bioorg Med Chem Lett, 27, 2017, 845-849). Therefore, there is a need for the development of more potent, selective, soluble USP7 inhibitors with reduced toxicity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Everett et al.,Novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein.EMBO J,16,1997,566-77 [Non-patent document 2] Li et al.,Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization.Nature,416,2002,648-53 [Non-patent document 3] Cummins et al.,Tumour suppression:disruption of HAUSP gene stabilizes p53.Nature,428,2004,1p following 486 [Non-patent document 4] Li et al.,A dynamic role of HAUSP in the p53-Mdm2 pathway.Mol Cell,13,2004,879-86 [Non-Patent Document 5] Chauhan et al.,A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance.Cancer Cell,22,2012,345-58 [Non-patent document 6] Wang et al.,J Clin Invest,126,2016,2205-20 [Non-Patent Document 7] Tavana et al.,Nat Med,22,2016,1180-1186 [Non-patent document 8] Cheng et al.,Expression of HAUSP in gliomas correlates with disease progression and survival of patients.Oncol Rep,29,2013,1730-6 [Non-Patent Document 9] Zhang et al.,Expression of USP7 and MARCH7 Is Correlated with Poor Prognosis in Epithelial Ovarian Cancer.Tohoku J Exp Med,239,2016,165-75 [Non-Patent Document 10] Chen et al.,Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors.Bioorg Med Chem Lett,27,2017,845-849 Summary of the Invention

[0006] Formula (I) [ka] (In the formula, R1 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R2 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R3 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R4 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; provided that R1, R2, R3, and R4 are not simultaneously H; R5 is H, halogen, -CN, -OR7 or -NR7R8; R6 is alkyl, -C(=O)R 10 , -C(=S)R 10 , —C(O)NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 11 and the alkyl is optionally substituted with one or more R 12 is replaced by each R7 and R8 is independently H, alkenyl, or alkyl; Each R9, independently at each occurrence, is -NR7R8, alkoxy, -(OCH2CH2) m alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkoxy, haloalkoxy, halogen, and -OH, and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, -N3, and -OH; R 10is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, heterocycloalkyl, aryl, amino, heteroalkyl, alkylamino, aminoalkyl, or heteroaryl, and each of the alkyl, alkenyl, and alkynyl groups independently represents one or more R 13 wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently substituted with one or more R 12 and each R 11 is independently at each occurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO, or —OH; Each R 12 is independently at each occurrence aryl or heteroaryl, wherein said aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, and —OH; Each R 13 is independently at each occurrence -OH, alkoxy, heteroalkyl, aryloxy, -NH2, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -O-aryl, -O-heteroaryl, -NR7aryl, -NR7heteroaryl, or -NR7C(=O)R 14 wherein the cycloalkyl, heterocycloalkyl, aryl, heteroalkyl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO2, and —OH; R 14 is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, each of which independently represents one or more R 15 wherein the alkyl, alkenyl, and alkynyl are each independently optionally substituted with one or more substituents selected from halogen and —OH; Each R 15is independently at each occurrence halogen, alkyl, CN, —C(═O)alkyl, or —C(═O)alkenyl, wherein said alkyl and alkenyl are each independently substituted with one or more substituents selected from halogen and —OH; m is 1, 2, or 3; n is 0 or 1, however, (i) When R2 is -NO2, -NHC(O)Me or -NH2 and R1, R2 and R4 are each H, or when R1 is Me and R2, R3 and R4 are each H, R6 is -C(O)R 10 Instead, in the formula, R 10 is -(CH2)-(CHMe)-phenyl, (ii) R2 is Cl, R1, R3 and R4 are each H, and R6 is -C(=O)R 10 and R 10 is one R 13 When R is (C-C) alkyl substituted with 13 is not an unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted 2-thiophenyl group, (iii) When R2 is Cl, and R1, R3, and R4 are each H, R6 is —C(═O)R 10 and R 10 is not 1-ethylpropyl), or a pharmaceutically acceptable salt thereof, are disclosed herein.

[0007] In certain embodiments, the present invention provides pharmaceutical compositions suitable for use in a subject in the treatment or prevention of a disorder associated with the modulation of USP7, comprising an effective amount of any of the compounds described herein (e.g., a compound of the invention, such as a compound of Formula (I)) and one or more pharmaceutically acceptable excipients. In certain embodiments, pharmaceuticals may be used in the treatment or prevention of conditions or diseases as described herein.

[0008] Disclosed herein are methods of inhibiting USP7, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0009] Disclosed herein are methods for treating diseases and conditions that benefit from modulation of USP7, comprising administering to a subject a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the diseases and conditions benefit from inhibition of USP7. The diseases and conditions include, but are not limited to, cancer and cancer metastasis, neurodegenerative diseases, immune disorders, diabetes, bone and joint diseases, osteoporosis, arthritic disorders, cardiovascular diseases, ischemic diseases, viral infections and diseases, and viral and / or latent bacterial infections and diseases.

[0010] Disclosed herein is a method for treating cancer, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is multiple myeloma.

[0011] Disclosed herein are compounds of formula (I) or pharmaceutically acceptable salts thereof for use in the manufacture of a medicament for treating a disease or condition associated with the inhibition of USP7.

[0012] Disclosed herein is the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the treatment of a disease or condition associated with the inhibition of USP7. In certain embodiments, for example, the following are provided: (Item 1) Formula I [ka] wherein R1 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R2 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R3 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R4 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; However, R1, R2, R3 and R4 are not H at the same time, R5 is H, halogen, -CN, -OR7 or -NR7R8; R6 is alkyl, -C(=O)R 10 , -C(=S)R 10 , —C(O)NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; The cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 11 optionally replaced by The alkyl may be one or more R 12 is replaced by each R7 and R8 is independently H, alkenyl, or alkyl; Each R9, independently at each occurrence, is -NR7R8, alkoxy, -(OCH2CH2) m alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkoxy, haloalkoxy, halogen, and —OH; wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, -N3, and -OH; R 10is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, heterocycloalkyl, aryl, amino, heteroalkyl, alkylamino, aminoalkyl, or heteroaryl; The alkyl, alkenyl, and alkynyl each independently represent one or more R 13 optionally replaced by The cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 12 optionally replaced by Each R 11 is independently at each occurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO, or —OH; Each R 12 is independently at each occurrence aryl or heteroaryl; wherein the aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, and —OH; Each R 13 is independently at each occurrence -OH, alkoxy, heteroalkyl, aryloxy, -NH2, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -O-aryl, -O-heteroaryl, -NR7aryl, -NR7heteroaryl, or -NR7C(=O)R 14 and wherein the cycloalkyl, heterocycloalkyl, aryl, heteroalkyl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO2, and —OH; R 14 is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; The aryl and heteroaryl each independently represent one or more R 15 optionally replaced by wherein the alkyl, alkenyl, and alkynyl are each independently optionally substituted with one or more substituents selected from halogen and —OH; Each R 15 is independently at each occurrence halogen, alkyl, —CN—C(═O)alkyl, or —C(═O)alkenyl; the alkyl and alkenyl are each independently substituted with one or more substituents selected from halogen and —OH; m is 1, 2, or 3; n is 0 or 1, provided that (i) when R2 is -NO2, -NHC(O)Me or -NH2 and R1, R2 and R4 are each H, or when R1 is Me and R2, R3 and R4 are each H, R6 is -C(O)R 10 Instead, in the formula, R 10 is -(CH2)-(CHMe)-phenyl, (ii) R2 is Cl, R1, R3 and R4 are each H, and R6 is -C(=O)R 10 and R 10 is one R 13 When R is (C-C) alkyl substituted with 13 is not unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted 2-thiophenyl, (iii) When R2 is Cl, and R1, R3, and R4 are each H, R6 is —C(═O)R 10 and R 10 is not 1-ethylpropyl) or a pharmaceutically acceptable salt thereof. (Item 2) The compound according to item 1, wherein R1 is H, -NR7C(=O)alkyl or -NR7R8. (Item 3) The compound according to item 1 or 2, wherein R1 is H. (Item 4) The compound according to any one of items 1 to 3, wherein R3 is H, -NO2 or -NR7R8. (Item 5) 5. The compound according to any one of items 1 to 4, wherein R3 is H. (Item 6) 6. The compound according to any one of items 1 to 5, wherein R4 is H. (Item 7) Each R9, independently at each occurrence, is -NR7R8, alkoxy, -(OCH2CH2) m alkyl, heterocycloalkyl, or heteroaryl; 7. The compound according to any one of items 1 to 6, wherein said heterocycloalkyl or heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, alkoxy and -N3. (Item 8) 8. The compound according to any one of items 1 to 7, wherein n is 0. (Item 9) 8. The compound according to any one of items 1 to 7, wherein n is 1. (Item 10) The compound is represented by formula (Ia) [ka] Item 1, wherein the compound has the structure: or a pharmaceutically acceptable salt thereof. (Item 11) 11. The compound according to any one of items 1 to 10, wherein R2 is selected from halogen, —NH2, —NO2, CN, —NR7C(═O) and —C(═O)NR7 alkyl, wherein each alkyl is independently optionally substituted with one or more R9. (Item 12) 12. The compound according to any one of items 1 to 11, wherein R2 is nitro or -NHalkyl. (Item 13) 12. The compound according to any one of items 1 to 11, wherein R2 is halo. (Item 14) 12. The compound according to any one of items 1 to 11, wherein R2 is chloro. (Item 15) 12. The compound according to any one of items 1 to 11, wherein R2 is -NR7C(=O)alkyl or -C(=O)NR7alkyl, said alkyl group being substituted with one R9. (Item 16) The compound according to item 15, wherein R9 is heterocycloalkyl or heteroaryl. (Item 17) 17. The compound according to item 16, wherein R9 is N-methylpiperazinyl, piperidinyl or morpholinyl. (Item 18) Item 17. The compound according to item 16, wherein R9 is imidazolyl. (Item 19) Item 16. The compound according to item 15, wherein R9 is azide. (Item 20) Item 16. The compound according to item 15, wherein R9 is -NR7H. (Item 21) Item 16. The compound according to item 15, wherein R7 is acyl, alkylacyl or alkenylacyl. (Item 22) R7 [ka] 22. The compound according to item 21, wherein (Item 23) The compound is represented by formula (Ib) [ka] 18. The compound according to any one of items 1 to 17, having the structure: or a pharmaceutically acceptable salt thereof. (Item 24) 24. The compound according to any one of items 1 to 23, wherein R5 is H, CN, —OH or —NR7R8. (Item 25) 24. The compound according to any one of items 1 to 23, wherein R5 is -OH or -NR7R8. (Item 26) R5 is -OH, -NH2, -N(H)CH3 or -N(CH3)2; The compound according to any one of items 1 to 23. (Item 27) 27. The compound according to any one of items 1 to 26, wherein R5 is —OH. (Item 28) R6 is alkyl, -C(=O)R 10 , -C(=S)R10 , aryl or heteroaryl. (Item 29) R6 is -C(=O)R 10 29. The compound according to any one of items 1 to 28, wherein (Item 30) R 10 30. The compound according to item 28 or 29, wherein is alkyl, alkenyl, amino, alkylamino, alkynyl, cycloalkyl, cycloalkyl, alkylamino, heteroaryl or aminoalkyl. (Item 31) R 10 31. The compound according to item 30, wherein is heteroalkyl. (Item 32) 32. The compound according to item 31, wherein said aryl or heteroaryl is further substituted with alkyl, halo, alkyloxy or nitro. (Item 33) 32. The compound according to item 31, wherein said aryl or heteroaryl is further substituted with halo, alkyloxy or nitro. (Item 34) 32. The compound according to item 31, wherein said acylamino is substituted with halo, alkenyl, heteroaryl or heterocycloalkyl. (Item 35) 35. The compound according to item 34, wherein said heteroaryl or heterocycloalkyl is substituted with alkyl acyl, alkenylacyl or hydroxyl. (Item 36) R 10 Each has at least one R 13 30. The compound according to any one of items 1 to 29, wherein R is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl or heterocycloalkyl optionally substituted with (Item 37) Each R 13 is independently at each occurrence -OH, alkoxy, aryloxy, -NH, arylalkyl, cycloalkyl, aryl, heteroaryl, or -NRC(=O)R 1437. The compound according to item 36, wherein (Item 38) Each R 14 38. The compound according to item 37, wherein, at each occurrence, is independently alkyl, haloalkyl, arylalkyl, alkenyl, heterocyclyl, or heteroaryl. (Item 39) R2 is Cl, —NO2, —NH2, or —NR7C(═O)alkyl; said alkyl optionally substituted with one or more R9; R6 is -C(=O)R 10 and R 10 is alkyl, alkenyl, cycloalkyl, and heterocycloalkyl, wherein the alkyl, alkenyl, and alkynyl each independently represent one or more R 13 optionally replaced by The cycloalkyl and heterocycloalkyl each may be one or more R 12 11. The compound according to item 10, optionally substituted with (Item 40) Formula (Ib) [ka] wherein R1 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R2 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -R7R8, wherein each alkyl is independently optionally substituted with one or more R9; R3 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; R4 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, wherein each alkyl is independently optionally substituted with one or more R9; However, R1, R2, R3 and R4 are not H at the same time, R5 is H, halogen -CN, -OR7 or -NR7R8; R6 is alkyl, -C(=O)R 10 , -C(=S)R 10 , —C(O)NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; The cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 11 optionally replaced by The alkyl group may be one or more R 12 is replaced by each R7 and R8 is independently H or alkyl; Each R9, independently at each occurrence, is -NR7R8, alkoxy, -(OCH2CH2) m alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkoxy, haloalkoxy, halogen, and —OH; wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, -N3, and -OH; R 10 is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; The alkyl, alkenyl, and alkynyl each independently represent one or more R 13 optionally replaced by The cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 12 optionally replaced by Each R 11 is independently at each occurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO, or —OH; Each R 12 is independently at each occurrence aryl or heteroaryl; wherein the aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, and —OH; Each R 13 is independently at each occurrence -OH, alkoxy, aryloxy, -NH2, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -O-aryl, -O-heteroaryl, -NR7aryl, -NR7heteroaryl, or -NR7C(=O)R 14 and wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, —NO2, and —OH; R 14 is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl; The aryl and heteroaryl each independently represent one or more R 15 optionally replaced by wherein the alkyl, alkenyl, and alkynyl are each independently optionally substituted with one or more substituents selected from halogen and —OH; Each R 15 is independently at each occurrence halogen, alkyl, —CN—C(═O)alkyl, or —C(═O)alkenyl; the alkyl and alkenyl are each independently substituted with one or more substituents selected from halogen and —OH; m is 1, 2, or 3; n is 0 or 1, provided that (i) when R2 is -NO2, -NHC(O)Me or -NH2 and R1, R2 and R4 are each H, or when R1 is Me and R2, R3 and R4 are each H, R6 is -C(O)R 10 Instead, in the formula, R 10 is -(CH2)-(CHMe)-phenyl, (ii) R2 is Cl, R1, R3 and R4 are each H, and R6 is -C(=O)R 10 and R 10 is one R 13 When R is (C-C) alkyl substituted with 13 is not unsubstituted cyclopentyl, unsubstituted phenyl or unsubstituted 2-thiophenyl, (iii) When R2 is Cl, and R1, R3, and R4 are each H, R6 is —C(═O)R 10 and R 10 is not 1-ethylpropyl) or a pharmaceutically acceptable salt thereof. (Item 41) A compound selected from the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. (Item 42) A compound selected from the following compounds: [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. (Item 43) 43. A pharmaceutical composition comprising the compound according to any one of items 1 to 42 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. (Item 44) 43. A method for treating a disease or disorder modulated by USP7, comprising administering to a subject in need thereof a compound according to any one of items 1 to 42. (Item 45) 43. A method of inhibiting USP7, comprising administering to a subject in need thereof a compound according to any one of items 1 to 42. (Item 46) 46. ​​The method according to item 45, wherein the disease or disorder associated with the inhibition of USP7 is cancer and cancer metastasis, neurodegenerative diseases, immune disorders, diabetes, bone and joint diseases, osteoporosis, arthritic disorders, cardiovascular diseases, ischemic diseases, viral infections and diseases, viral infectious and / or latent and bacterial infections and diseases. (Item 47) 43. A method for treating cancer, comprising administering to a subject in need thereof a compound according to any one of items 1 to 42. (Item 48) 48. The method of item 47, wherein the cancer is liposarcoma, neuroblastoma, glioblastoma, breast cancer, bladder cancer, glioma, neuroblastoma, adrenocortical carcinoma, multiple myeloma, colorectal cancer, non-small cell lung cancer, human papillomavirus-associated cervical, oropharyngeal, penile, ovarian, anal, thyroid or vaginal cancer, Epstein-Barr virus-associated nasopharyngeal carcinoma, gastric cancer, rectal cancer, thyroid cancer, Hodgkin's lymphoma or diffuse large B-cell lymphoma. (Item 49) 48. The method of item 47, wherein the cancer is multiple myeloma, breast cancer, neuroblastoma, glioma, colon cancer, prostate cancer or ovarian cancer. (Item 50) Item 48. The method of item 47, wherein the cancer is multiple myeloma. (Item 51) Use of a compound according to any one of items 1 to 42 for the manufacture of a medicament for treating a disease modulated by USP7. (Item 52) 43. A compound according to any one of items 1 to 42 for use in treating a disease modulated by USP7. [Brief explanation of the drawings]

[0013] [Figure 1A] The known compound P22077 and its close analogue P5091. [Figure 1B] Known USP7 inhibitors. [Figure 2A] Optimization based on the structural information of compound A led to compounds 10 and 11. Compound 11, the enantiomer of compound 10, is 80 times less active. [Figure 2B] Dose-dependent inhibition of the catalytic domain of USP7 (amino acids 208–560) and full-length USP7 (amino acids 1–1102) by compounds 10 and 11 using Ub-AMC as a substrate. [Figure 2C] Evaluation of compound 10 binding to USP7 using isothermal calorimetry. [Figure 2D] Inhibitory activity of compound 10 across a panel of 41 purified DUBs using ubiquitin-rhodamine (Ub-Rho) as a substrate. [Figure 3A] Characterization of compound 10 binding to USP7 using ribbon diagrams of compound 10 and USP7. [Figure 3B] Stereoview of USP7 (light blue) bound to compound 10 (yellow). Hydrogen bonds are indicated by dashed lines. [Figure 3C] Molecular surface representation of the USP7-compound 10 co-structure. Highlighted areas indicate regions of altered HDX in the presence of compound 10. Dark areas correspond to significant changes, while light areas correspond to areas with minor changes. [Figure 4A] Analysis of USP7 mutant proteins. Detailed ligand interaction diagram of compound 10 with USP7. Residues containing amino acids that belong to the same class as more than 80% of other UPSs are circled in red. [Figure 4B] Summary of activity against Ub-AMC and inhibition by Compound A against USP7 mutant catalytic domain proteins. [Figure 4C] Dose-responsive inhibition of full-length USP7Q351 (amino acids 1–1102) by compound A. [Figure 4D] Dose-responsive inhibition of full-length USP7Q351 (amino acids 1–1102) by compound 10. [Figure 5A] USP7 inhibitory activity and mouse liver microsome (MLM) stability of the disclosed compounds. [Figure 5B] Structures, USP7 inhibitory activity and mouse liver microsome (MLM) stability of the disclosed compounds. [Figure 5C]Analysis of the ability of compounds 10 and 11 to bind native USP7 at multiple doses in HEK293T lysates using competitive activity-based protein profiling. [Figure 6A] Compound 10 promotes HDM2 loss and accumulation of p53 and p21. Analysis of HDM2, p53, and p21 protein levels in MCF7 cells treated with compounds 10 or 11 at the indicated concentrations for 16 hours. [Figure 6B] Analysis of HDM2, p53, and p21 protein levels in MCF7 cells treated with compounds 10 or 11 at the indicated concentrations for 16 hours, with cycloheximide added for the final 2 hours. [Figure 6C] Analysis of HDM2, p53, and p21 protein levels in MM.1S cells treated with compounds 10 or 11 at the indicated concentrations for 6 hours. [Figure 6D] Analysis of HDM2, p53, and p21 protein levels in MM.1S cells treated with compounds 10 or 11 at the indicated concentrations for 6 hours, with cycloheximide added for the final 2 hours. DETAILED DESCRIPTION OF THE INVENTION

[0014] Formula (I) [ka] wherein R1 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, where each alkyl is independently optionally substituted with one or more R9; R2 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -R7R8, where each alkyl is independently optionally substituted with one or more R9; and R3 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy. R is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO, -CN, -NRC(=O)alkyl, -C(=O)NRalkyl, or -NRR, wherein each alkyl is independently optionally substituted with one or more R; R is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO, -NH, CN, -NRC(=O)alkyl, -C(=O)NRalkyl, or -NRR, wherein each alkyl is independently optionally substituted with one or more R; provided that R, R, R, and R are not simultaneously H; R is H, halogen, -CN, -OR, or -NRR; R is alkyl, -C(=O)R 10 , -C(=S)R 10 , —C(O)NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 11 and the alkyl group is optionally substituted with one or more R 12 each R and R is independently H, alkenyl, or alkyl; and each R is independently at each occurrence -NR, R, alkoxy, -(OCH, CH), malkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkoxy, haloalkoxy, halogen, and —OH; the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N, and —OH; R 10 is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, heterocycloalkyl, aryl, amino, heteroalkyl, alkylamino, aminoalkyl, or heteroaryl, and each of the alkyl, alkenyl, and alkynyl groups independently represents one or more R 13 wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently substituted with one or more R 12 and each R 11 is independently at each occurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, -NO2, or -OH; and each R 12 is independently at each occurrence aryl or heteroaryl, wherein said aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, and —OH; and each R 13 is independently at each occurrence -OH, alkoxy, heteroalkyl, aryloxy, -NH2, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -O-aryl, -O-heteroaryl, -NR7aryl, -NR7heteroaryl, or -NR7C(=O)R 14 wherein the cycloalkyl, heterocycloalkyl, aryl, heteroalkyl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —NO, and —OH; and R 14is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, each of which independently represents one or more R 15 wherein the alkyl, alkenyl, and alkynyl are each independently optionally substituted with one or more substituents selected from halogen and —OH; and each R 15 is independently at each occurrence halogen, alkyl, CN-C(=O)alkyl, or -C(=O)alkenyl, wherein said alkyl and alkenyl are each independently substituted with one or more substituents selected from halogen and -OH; m is 1, 2, or 3; and n is 0 or 1, with the proviso that (i) when R2 is -NO2, -NHC(O)Me, or -NH2, and R1, R2, and R4 are each H, or when R1 is Me, and R2, R3, and R4 are each H, then R6 is -C(O)R 10 Instead, in the formula, R 10 is —(CH)—(CHMe)-phenyl, (ii) R is Cl, R, R, and R are each H, and R is —C(═O)R 10 and R 10 is one R 13 When R is (C-C) alkyl substituted with 13 is not unsubstituted cyclopentyl, unsubstituted phenyl, or unsubstituted 2-thiophenyl; (iii) when R2 is Cl, and R1, R3, and R4 are each H, R6 is —C(═O)R 10 and R 10 is not 1-ethylpropyl), or a pharmaceutically acceptable salt thereof, are disclosed herein.

[0015] In some embodiments, the compound has formula (Id): [ka] wherein R1 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO2, -NH2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -NR7R8, where each alkyl is independently optionally substituted with one or more R9; R2 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NH2, -NO2, CN, -NR7C(=O)alkyl, -C(=O)NR7alkyl, or -R7R8, where each alkyl is independently optionally substituted with one or more R9; and R3 is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy. R is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO, -CN, -NRC(=O)alkyl, -C(=O)NRalkyl, or -NRR, wherein each alkyl is independently optionally substituted with one or more R; R is H, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, -OH, -NO, -NH, CN, -NRC(=O)alkyl, -C(=O)NRalkyl, or -NRR, wherein each alkyl is independently optionally substituted with one or more R; provided that R, R, R, and R are not simultaneously H; R is H, halogen, -CN, -OR, or -NRR; R is alkyl, -C(=O)R 10 , -C(=S)R 10 , —C(O)NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl each independently represent one or more R 11 and the alkyl is optionally substituted with one or more R 12 each R and R is independently H or alkyl; and each R is independently at each occurrence -NR, R, alkoxy, -(OCH, CH) malkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkoxy, haloalkoxy, halogen, and —OH; the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, —N, and —OH; R 10 is alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, and each of the alkyl, alkenyl, and alkynyl groups independently is selected from one or more R 13 wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently substituted with one or more R 12 and each R 11 is independently at each occurrence alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, -NO2, or -OH; and each R 12 is independently at each occurrence aryl or heteroaryl, wherein said aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, halogen, and —OH; and each R 13 is independently at each occurrence -OH, alkoxy, aryloxy, -NH2, arylalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -O-aryl, -O-heteroaryl, -NR7aryl, -NR7heteroaryl, or -NR7C(=O)R 14 wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from alkyl, haloalkyl, alkoxy, haloalkoxy, —NO, and —OH; and R 14 is alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl, each of which independently represents one or more R15 wherein the alkyl, alkenyl, and alkynyl are each independently optionally substituted with one or more substituents selected from halogen and —OH; and each R 15 is independently at each occurrence halogen, alkyl, CN-C(=O)alkyl, or -C(=O)alkenyl, wherein said alkyl and alkenyl are each independently substituted with one or more substituents selected from halogen and -OH; m is 1, 2, or 3; and n is 0 or 1, with the proviso that (i) when R2 is -NO2, -NHC(O)Me, or -NH2, and R1, R2, and R4 are each H, or when R1 is Me, and R2, R3, and R4 are each H, then R6 is -C(O)R 10 Instead, in the formula, R 10 is —(CH)—(CHMe)-phenyl, (ii) R is Cl, R, R, and R are each H, and R is —C(═O)R 10 and R 10 is one R 13 When R is (C-C) alkyl substituted with 13 is not unsubstituted cyclopentyl, unsubstituted phenyl, or unsubstituted 2-thiophenyl; (iii) when R2 is Cl, and R1, R3, and R4 are each H, R6 is —C(═O)R 10 and R 10 is not 1-ethylpropyl) or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, R1 is H, -NR7C(=O)alkyl, or -NR7R8. In certain embodiments, R1 is H. In some embodiments, R3 is H, -NO2, or -NR7R8. In certain embodiments, R3 is H. In some embodiments, R4 is H. In some embodiments, each R9, independently at each occurrence, is -NR7R8, alkoxy, -(OCH2CH2) mIn some embodiments, n is 0, while in other embodiments, n is 1. In some embodiments, R9 is heterocycloalkyl or heteroaryl. In some embodiments, R9 is N-methylpiperazinyl, piperidinyl, or morpholinyl. In some embodiments, R9 is imidazolyl. In some embodiments, R9 is azide. In some embodiments, R9 is -NR7H. In some embodiments, R7 is acyl, alkylacyl, or alkenylacyl. In some embodiments, R7 is [ka] is.

[0017] In some embodiments, R2 is selected from halogen, —NH2, —NO2, CN, —NR7C(═O), and —C(═O)NR7 alkyl, where each alkyl is independently substituted with one or more R9. In certain embodiments, R2 is a halo group such as chloro, fluoro, or bromo. In some embodiments, R2 is chloro. In some embodiments, R2 is —NR7C(═O) alkyl or —C(═O)NR7 alkyl, where the alkyl is substituted with one R9. In some embodiments, R2 is nitro or —NHalkyl.

[0018] In some embodiments, R5 is H, CN, -OH, or -NR7R8. In other embodiments, R5 is -OH or -NR7R8. In certain embodiments, R5 is -OH, -HN2, -N(H)CH3, or -N(CH3)2. In some embodiments, R5 is -OH.

[0019] In some embodiments, R6 is alkyl, —C(═O)R 10 , -C(=S)R 10, aryl, or heteroaryl. In some embodiments, R6 is -C(=O)R 10 In certain embodiments, R 10 Each has at least one R 13 In another embodiment, R is an alkyl, alkenyl, alkynyl, -NR7R8, cycloalkyl, or heterocycloalkyl optionally substituted with 13 is independently at each occurrence -OH, alkoxy, aryloxy, -NH, arylalkyl, cycloalkyl, aryl, heteroaryl, or -NRC(=O)R 14 In some embodiments, R 14 is independently at each occurrence alkyl, haloalkyl, arylalkyl, alkenyl, heterocyclyl, or heteroaryl.

[0020] In some embodiments, R 10 is alkyl, alkenyl, amino, alkylamino, alkynyl, cycloalkyl, cycloalkyl, alkylamino, heteroaryl, or aminoalkyl. In some embodiments, the alkyl, amino, alkylamino, or cycloalkyl is substituted with aryl, aralkyl, heteroaryl, heterocyclyl, acylamino, aryloxy, or hydroxyl. In some embodiments, the aryl or heteroaryl is further substituted with alkyl, halo, alkyloxy, or nitro. In some embodiments, the aryl or heteroaryl is further substituted with halo, alkyloxy, or nitro. In some embodiments, the acylamino is substituted with halo, alkenyl, heteroaryl, or heterocycloalkyl. In some embodiments, the heteroaryl or heterocycloalkyl is substituted with alkylacyl, alkenylacyl, or hydroxyl.

[0021] In some embodiments, R2 is Cl, -NO2, -NH2, or -NR7C(=O)alkyl, wherein the alkyl is optionally substituted with one or more R9, and R6 is -C(=O)R 10 and R 10is alkyl, alkenyl, cycloalkyl, and heterocycloalkyl, and the alkyl, alkenyl, and alkynyl each represent one or more R 13 wherein the cycloalkyl and heterocycloalkyl are each optionally substituted with one or more R 12 is optionally replaced by

[0022] Formula (Ib) [ka] Also disclosed is a compound of the formula: or a pharmaceutically acceptable salt thereof, wherein R2, R5, and R6 are as defined above and herein.

[0023] Formula (Ic) [ka] Also disclosed is a compound of the formula: or a pharmaceutically acceptable salt thereof, wherein the variables R5 and R6 are as defined above and herein.

[0024] In some embodiments, the compound of the invention is a compound shown in Table 3 or 4.

[0025] In certain embodiments, the present invention provides a pharmaceutical preparation suitable for use in a human patient, comprising any of the compounds set forth above (e.g., a compound of the invention, such as a compound of Formula (I)) and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical preparation may be used in the treatment or prevention of a condition or disease as described herein. Any of the disclosed compounds may be used in the manufacture of a medicament for the treatment of any disease or condition disclosed herein.

[0026] Details of the disclosure are set forth in the accompanying specification below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, representative methods and materials are described here. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Unless otherwise defined, 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 disclosure belongs. All patents and publications cited herein are incorporated by reference in their entirety.

[0027] definition Unless otherwise defined, all technical scientific terms used herein have the meanings that are commonly understood by those skilled in the art of this disclosure. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms have the meanings that are used herein.

[0028] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have their meaning in U.S. patent law and can mean "includes," "including," etc. Similarly, "consisting essentially of" or "consists essentially of" have their meaning in U.S. patent law and contemplate the existence of more than what is recited, excluding prior art embodiments, so long as the presence of more than what is recited does not alter a basic or novel characteristic of what is recited, and the term does not delimit.

[0029] As used herein, the term "or" is understood to be inclusive unless specifically stated or clear from the context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless specifically stated or clear from the context.

[0030] In this disclosure, the term "and / or" is used to mean "and" or "or," unless otherwise indicated.

[0031] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O).

[0032] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0033] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably C(O)O-.

[0034] The term "alkoxy" refers to an alkyl, preferably a lower alkyl, group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy groups, and the like.

[0035] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0036] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl." The latter refers to an alkenyl moiety having substituents replacing hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons included or not included in one or more double bonds. Furthermore, such substituents include all possible substituents for an alkyl group, except where such substitution is highly stable, as discussed below. For example, substitution of an alkenyl group with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0037] An "alkyl" or "alkane" is a fully saturated, straight- or branched-chain non-aromatic hydrocarbon. Unless otherwise defined, a straight- or branched-chain alkyl group typically has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms. Examples of straight- or branched-chain alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight- or branched-chain alkyl groups are also referred to as "lower alkyl" groups.

[0038] Furthermore, the term "alkyl group" (or "lower alkyl group") as used in the specification, examples, and claims is intended to include both "unsubstituted alkyl groups" and "substituted alkyl groups," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbon atoms of the hydrocarbon backbone. Unless otherwise specified, such substituents can include, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate ester, phosphonate ester, phosphinate ester, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate ester, sulfonate ester, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that moieties substituted on the hydrocarbon chain can themselves be substituted, where appropriate. For example, substituents of substituted alkyls may include substituted and unsubstituted forms of amino, azide, imino, amide, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate) and silyl groups, ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylic acid ester, and ester), -CF3, -CN, and the like. Representative substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxy, alkylthio, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0039] The term "Cx-y" when used with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups containing x to y carbons in the chain. For example, the term "Cx-y alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight- and branched-chain alkyl groups containing x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. C0 alkyl indicates that, if present, the hydrogen bond is at the terminal position of the group. The terms "C2-y alkenyl" and "C2-y alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups containing at least one double or triple bond, respectively, but similar in length and possible substitution to the alkyls described above.

[0040] The term "heteroalkyl," as used herein, means a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent in the chain.

[0041] Furthermore, the term "heteroalkyl" (or "lower heteroalkyl") as used in the specification, examples, and claims is intended to include both "unsubstituted heteroalkyl" and "substituted heteroalkyl," the latter of which refers to heteroalkyl moieties having substituents replacing a hydrogen on one or more carbon or heteroatoms of the backbone. Unless otherwise specified, such substituents can include, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate ester, phosphonate ester, phosphinate ester, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate ester, sulfonate ester, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those of skill in the art will understand that moieties substituted on the heteroalkyl chain can themselves be substituted, where appropriate. For example, substituents of substituted heteroalkyls may include substituted and unsubstituted forms of amino, azide, imino, amide, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate) and silyl groups, ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylic acid ester, and ester), -CF, -CN, and the like.

[0042] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0043] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0044] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl." The latter refers to an alkynyl moiety having substituents replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons included or not included in one or more triple bonds. Furthermore, such substituents include all possible substituents for an alkyl group, except where such substitution is highly stable, as discussed above. For example, substitution of an alkynyl group with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0045] As used herein, the term "amide" refers to [ka] (In the formula, each R 10 each independently represents hydrogen or a hydrocarbyl group, or two R 10 means a group which, together with the N atom to which it is attached, forms a heterocycle having 4 to 8 atoms in the ring structure.

[0046] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, e.g., [ka] (In the formula, each R 10 independently represent hydrogen or a hydrocarbyl group, or two R 10 means a moiety that can be represented by the following formula: (which together with the N atom to which they are attached form a heterocycle having 4 to 8 atoms in the ring structure). As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0047] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0048] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is 5- to 7-membered, and more preferably 6-membered. The term "aryl" also includes polycyclic ring systems having two or more rings in which at least one of two adjacent rings is aromatic and the other ring has two or more carbons in common, e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0049] The term "carbamate" is art-recognized and [ka] wherein R9 and R10 independently represent hydrogen or a hydrocarbyl group such as an alkyl group, or R9 and R10 together with the intervening atom(s) form a heterocycle having 4 to 8 atoms in the ring structure.

[0050] As used herein, the terms "carbocycle" and "carbocyclic" refer to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings in which all carbon atoms are saturated and cycloalkene rings that contain at least one double bond.

[0051] The term "carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring in a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycle includes bicyclic molecules in which one, two, or more atoms are shared between the two rings of the bicyclic molecule. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring in the bicyclic carbocycle shares two adjacent atoms with the other ring. Each ring in a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings, as valences permit, is included in the definition of carbocycle. Representative "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Representative fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0052] A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Unless otherwise defined, monocyclic cycloalkyl groups typically have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings of the bicyclic molecule. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each ring of the bicyclic cycloalkyl shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0053] The term "carbocyclyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0054] The term "carbonate" is art-recognized and refers to the group --OCO.sub.2--R.sub.10, where R.sub.10 represents a hydrocarbyl group.

[0055] As used herein, the term "carboxy" refers to a group of the formula -CO2H.

[0056] As used herein, the term "ester" refers to the group -C(O)OR10, where R10 represents a hydrocarbyl group.

[0057] The term "ether" as used herein refers to a hydrocarbyl group bonded to another hydrocarbyl group via an oxygen atom. Thus, the ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

[0058] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo.

[0059] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a hetaryl group.

[0060] The terms "heteroaryl" and "hetaryl" refer to substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, which ring structures contain at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also refer to polycyclic ring systems having two or more rings in which at least one of two adjacent rings is heteroaromatic and the other ring has two or more carbon atoms in common, e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0061] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0062] The terms "heterocyclyl," "heterocycle," and "heterocyclic" include substituted or unsubstituted non-aromatic monocyclic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, which ring structures contain at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocycle" also include polycyclic ring systems having two or more rings in which at least one of two adjacent rings is a heterocycle, e.g., the other ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl, sharing two or more carbon atoms in common with the two adjacent rings. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0063] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0064] As used herein, the term "hydrocarbyl" refers to a group that does not have =O or =S and that is bonded through a carbon atom that typically has at least one carbon-hydrogen bond and a primary carbon backbone, but may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for purposes of this application, as are substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon). Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0065] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0066] The term "lower" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which the number of non-hydrogen atoms in the substituent is 10 or fewer, preferably 6 or fewer. "Lower alkyl," for example, refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether these groups are designated alone or in combination with other substituents, such as hydroxyalkyl and aralkyl (e.g., atoms in aryl groups are not counted when counting carbon atoms in an alkyl substituent), as specified.

[0067] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) that have two or more atoms in common with two adjacent rings, e.g., the rings are "fused rings." Each ring of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10, preferably 5 to 7, atoms in the ring.

[0068] The term "silyl" means a silicon moiety bonded to three hydrocarbyl moieties.

[0069] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more backbone carbons. "Substituted" or "substituted with" is understood to include the implicit condition that such substituents are consistent with the substituted atom and the allowed valences of the substituent, as well as the implicit condition that the substitution results in a stable compound, e.g., one that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is considered to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Permissible substituents may be one or more and may be the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Substituents may include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate ester, phosphonate ester, phosphinate ester, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate ester, sulfonate ester, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the substituents themselves can be substituted, where appropriate. Unless specifically described as "substituted," reference to a chemical moiety herein is understood to include substituted versions. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted versions.

[0070] The term "sulfate ester" is art-recognized and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0071] The term "sulfonamide" is art-recognized and has the general formula [ka] (wherein R9 and R10 independently represent hydrogen or hydrocarbyl such as alkyl, or R9 and R10 together with the intervening atom(s) represent a group that forms a heterocycle having 4 to 8 atoms in the ring structure).

[0072] The term "sulfoxide" is art-recognized and refers to a group of the formula --S(O)--R.sup.10, where R.sup.10 is hydrocarbyl.

[0073] The term "sulfonate ester" is art-recognized and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0074] The term "sulfone" is art-recognized and refers to a group of the formula -S(O)2-R10, where R10 is hydrocarbyl.

[0075] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0076] As used herein, the term "thioester" refers to a -C(O)SR10 or -SC(O)R10 group, where R10 represents a hydrocarbyl group.

[0077] As used herein, the term "thioether" is equivalent to an ether, where the oxygen is replaced by a nitrogen.

[0078] The term "urea" is a technically recognized and generally [ka] wherein R9 and R10 independently represent hydrogen or hydrocarbyl such as alkyl, or together with R10, the presence of R9 and the intervening atom(s) form a heterocycle having 4 to 8 atoms in the ring structure.

[0079] The term "protecting group" means a group of atoms that, when attached to a reactive functional group of a molecule, masks, reduces, or prevents the reactivity of the functional group. Generally, a protecting group may be selectively removed as desired during the course of synthesis. Examples of protecting groups are described in Greene and Wuts, Protective Groups in Organic Chemistry, 3 rd Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-enanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratyloxycarbonyl ("NVOC"), and the like. Representative hydroxyl protecting groups include, but are not limited to, acylated (esterified) or alkylated hydroxyl groups such as benzyl and trityl ethers, alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0080] The term "prodrug" is intended to encompass compounds that are converted under physiological conditions to the therapeutically active agents of the present invention (e.g., compounds of Formula I). ​​A common method for making a prodrug is to include one or more selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by enzymatic activity of the subject. For example, preferred prodrugs of the present invention are esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids). In certain embodiments, some or all of the compounds of Formula I in the formulations shown above can be replaced with a corresponding suitable prodrug, for example, a prodrug in which a hydroxyl group in the parent compound is present as an ester, or a carbonate or carboxylic acid present in the parent compound is present as an ester.

[0081] The present invention includes all pharmaceutically acceptable isotopically labeled compounds used herein, in which one or more atoms are replaced with atoms having the same atomic number, essentially an atomic mass or mass number different from that normally found. In certain embodiments, the compounds of the present invention are enriched in such isotopically labeled materials (e.g., compounds in which the distribution of isotopes in the compounds in the composition differs from the natural or typical distribution of isotopes).

[0082] Examples of isotopes suitable for inclusion in the compounds of the present invention include: 2 H and 3 Hydrogen such as H 11 C. 13 C and 14 Carbon, such as C, 36 chlorine such as Cl, 18 Fluorine such as F, 123 I and 125 Iodine, such as I 13 N and 15 Nitrogen such as N 15 O. 17 O and 18 Oxygen, such as O 32 Phosphorus, such as P, 35 Examples include isotopes of sulfur such as S.

[0083] Certain isotopically labeled compounds disclosed herein, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H and carbon-14, i.e. 14 C is useful for this purpose in view of its ease of incorporation and rapid means of detection.

[0084] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H, may be preferable in some circumstances as greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements may afford certain therapeutic advantages.

[0085] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be useful in positron emission tomography (PET) studies for examining substrate receptor occupancy.

[0086] The compounds of the present invention may have one or more asymmetric carbon atoms and may exist in the form of optically pure enantiomers, mixtures of enantiomers, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, racemates of diastereoisomers, or mixtures of racemates of diastereoisomers. For example, optically active forms can be obtained by asymmetric synthesis or by resolution of racemates by asymmetric chromatography (chromatography using a chiral adsorbent or eluent). That is, some of the disclosed compounds may exist in various stereoisomeric forms.

[0087] Stereoisomers are compounds that differ only in their configuration. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain asymmetrically substituted carbon atoms that act as chiral centers. "Enantiomer" refers to one of a pair of molecules that are mirror images of each other and are not superimposable. "Diastereomers" are stereoisomers that most commonly contain two or more asymmetrically substituted carbon atoms and are not related as mirror images, reflecting the arrangement of substituents around one or more asymmetric carbon atoms. For example, enantiomers of a compound can be prepared by separating the enantiomer from a racemate using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. Those skilled in the art can readily determine appropriate techniques and / or methods for separating enantiomers of the compounds described herein from a racemic mixture.

[0088] "Geometric isomer" means an isomer that differs in the orientation of substituent atoms in relationship to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. The atoms (other than H) at each end of the carbon-carbon double bond may be in the E (substituents on opposite sides of the carbon-carbon double bond) or Z (substituents oriented on the same side) configuration. "R", "S", "S" * "," "R *"," "E," "Z," "cis," and "trans" indicate configurations relative to a core molecule. Some of the disclosed compounds may exist in the form of atropisomers. Atropisomers are stereoisomers resulting from hindrance of rotation about a single bond where the steric strain hindrance to rotation is high enough to allow isolation of the conformers. The compounds of the invention may be prepared as individual isomers by isomer-specific synthesis or may be reduced from an isomeric mixture. Traditional resolution techniques include forming a salt of the free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming a salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each isomer of an isomeric pair using an optically pure acid, amine, or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or separating isomeric mixtures of starting materials or final products using various well-known chromatographic methods.

[0089] Diastereomeric purity by weight is the ratio of the weight of one diastereomer to the weight of all diastereomers. When the stereochemistry of a disclosed compound is named or depicted from a structure, the named or depicted stereoisomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to the other stereoisomers. When a single enantiomer is named or depicted from a structure, the named or depicted enantiomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% optically pure. When a single diastereomer is named or depicted from a structure, the named or depicted diastereomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. The percentage of optical purity is the ratio of the weight of an enantiomer or the weight of an enantiomer to the weight of its optical isomer.

[0090] A purity percentage based on mole fraction is the ratio of moles of an enantiomer (diastereomer) or moles of an enantiomer (diastereomer) to moles of its optical isomer. When the stereochemistry of a disclosed compound is named or depicted from the structure, the named or depicted stereoisomer is at least about 60 mole%, about 70 mole%, about 80 mole%, about 90 mole%, about 99 mole%, or about 99.9 mole% relative to the other stereoisomer. When a single enantiomer is named or depicted from the structure, the named or depicted enantiomer is at least about 60 mole%, about 70 mole%, about 80 mole%, about 90 mole%, about 99 mole%, or about 99.9 mole% optically pure. When a single diastereomer is named or depicted from the structure, the named or depicted diastereomer is at least about 60 mol%, about 70 mol%, about 80 mol%, about 90 mol%, about 99 mol%, or about 99.9 mol% pure.

[0091] When a disclosed compound is named or depicted from a structure without indicating stereochemistry, and the compound has at least one chiral center, the name or structure should be understood to encompass enantiomers of the compound free of the corresponding optical isomer, a racemic mixture of the compound, or a mixture enriched in one enantiomer relative to the corresponding optical isomer. When a disclosed compound is named or depicted from a structure without indicating stereochemistry, and the compound has two or more chiral centers, the name or structure should be understood to encompass diastereomers free of other diastereomers, many diastereomers free of other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers enriched in one diastereomer relative to the other diastereomer(s), or mixtures of diastereomers enriched in one or more diastereomers relative to the other diastereomers. The present invention encompasses all of these forms.

[0092] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of a compound of Formula (I). For example, any pharmaceutically acceptable salt of a compound described herein includes those that are within the scope of sound medical judgment and suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic response, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during final isolation, purification of the compounds described herein, or separately by reacting the free base with a suitable organic acid.

[0093] The compounds of the present invention may have ionic groups that allow them to be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or, in the case of the compounds of the present invention in their acid form, these salts may be prepared from inorganic or organic bases. The compounds are often prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, as well as methods for preparing suitable salts, are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.

[0094] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, sodium, lithium, potassium, calcium, and magnesium salts, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0095] The term "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or mammals, including commercially relevant mammals such as other primates (e.g., cynomolgus monkeys, monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, cats, and / or dogs, and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys. A preferred subject is a human.

[0096] As used herein, a therapeutic that "prevents" a disease or condition refers to a compound that, for a given sample, reduces or delays the onset of the disease or condition in a treated sample relative to an untreated control sample, or reduces the severity of one or more symptoms of the disease or condition relative to an untreated control sample.

[0097] In treatment, the goal is to prevent or slow (reduce) an undesired physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of disease symptoms, reduction in the extent of the condition, disorder, or disease, stabilization (i.e., not worsening) of the condition, disorder, or disease, delay in the onset or slowing of the progression of the condition, disorder, or disease, detectable or undetectable improvement in the condition, disorder, or disease state or remission (partial or complete), improvement in at least one measurable physical parameter, not necessarily discernible by the patient, or enhancement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if not receiving treatment.

[0098] How to use Ubiquitin is a 76-amino acid protein that is post-translationally attached to substrate proteins via the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the substrate lysine side chain. Linear and branched polyubiquitin chains are constructed via the attachment of ubiquitin to one of the seven lysines of another molecule or to the N-terminal methionine of ubiquitin (Pickart and Fushman, Curr Opin Chem Biol, 8, 610-6, 2004). Ubiquitin is attached to substrate proteins through the coordinated action of ubiquitin-activating (E1), conjugating (E2), and ligating (E3) enzymes and is removed by a family of proteases known as deubiquitinating enzymes (DUBs). The first recognized role of the ubiquitin system was in the regulation of protein turnover (Lecker et al., J Am Soc Nephrol, 17, 1807-19, 2006). Ubiquitin tags are also responsible for signaling a wide range of non-degradative functions (O'Neill, J Biol Chem, 284, 8209, 2009). Ubiquitination can influence protein activity by modulating conformational changes, complexation with other proteins, susceptibility to the addition of other post-translational modifications (PTMs) including phosphorylation and acetylation, and cellular localization. Through complex degradative and non-degradative functions, ubiquitination coordinates a wide range of cellular processes, including protein degradation, DNA repair, chromatin remodeling, receptor signaling, and immunity, among others (Pinto-Fernandez and Kessler, Front Genet, 7, 133, 2016). Not surprisingly, aberrant ubiquitin system activity is associated with disease, most notably cancer, infection, and neurodegeneration (D'Arcy et al., 2016). al.,Pharmacol Ther,147,32-54,2015;Atkin and Paulson, Front Mol Neurosci, 7, 63, 2014; Nanduri et al., Curr Pharm Des, 19, 3234-47, 2013). Deregulation of the ubiquitin-proteasome system is a major risk factor for cancer (Hoeller Ubiquitin has been implicated in the pathogenesis of many human diseases, including neurodegenerative disorders (Rubinsztein, Nature 2006,443(7113),780-786), and viral diseases (Gao & Luo Can J Physiol Pharmacol 2006,84(1),5-14). The relationship between ubiquitin and cancer biology was validated clinically with the FDA approval of the proteasome inhibitor bortezomib for multiple myeloma (Kane et al., Oncologist,8,508-13,2003).

[0099] There are approximately 100 human DUBs belonging to six different families. Five of the families [ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephin, and Mindy] are cysteine ​​proteases, and the sixth [JAB / MPN / MOV34 (JAMM / MPN)] consists of zinc metalloproteases (Komander et al., Nat Rev Mol Cell Biol, 10, 550-63, 2009; Clague et al., Physiol Rev, 93, 1289-315, 2013; Abdul Rehman et al., Mol Cell, 63, 146-55, 2016). Many DUBs have been implicated in physiological and / or pathophysiological functions. Ubiquitin-specific proteases and ubiquitin C-terminal hydrolase (UCH) enzymes are the best-characterized members of the DUB family (Komander et al. 2009; Nijman et al. Cell 2005, 123(5), 773-786). For example, USP1 and USP4 are involved in DNA break repair (Kee and Huang, Mol Cell Biol, 36, 524-44, 2015). USP22 and BAP1 play roles in chromatin function (Atanassov et al., FEBS Lett, 585, 2016-23, 2011), while USP2 and USP8 have been reported to stabilize the oncogenic protein cyclin D1 (Shan et al., 2009) and mutant EGFR (Byun et al., Clin Cancer Res, 19, 3894-904, 2013), respectively. X-ray crystal structures of the catalytic cores of each family reveal that all, except for the Mindy family, adopt a common three-domain fold, in which the Finger domain coordinates the ubiquitin core, and the Sam and Palm domains coordinate the ubiquitin tail in the catalytic triad (Komander et al., 2009). While numerous apo-ubiquitin and ubiquitin-bound structures have been analyzed, very few studies have been performed using non-ubiquitin-based compounds. Notably, there are no reported small molecule DUB complex structures for the mammalian USP family, which contains up to 56 members (Komander et al., 2009), (Davies et al., Bioorg Med Chem Lett, 22, 3900-4, 2012; Ratia et al., Proc Natl Acad Sci USA, 105, 16119-24, 2008; Schlierf et al., Nat Commun, 7, 13166, 2016).

[0100] Although DUBs are generally considered a targetable class for drug development, inhibitor development is still in its infancy. The first DUB inhibitor, the dual USP14 / UCHL5 inhibitor VLX1570, entered clinical trials in 2015 (Wang et al., Sci Rep, 6, 26979, 2016b). The only example of a DUB inhibitor developed that targets the SARs DUB PLPro (Baez-Santos et al., Antiviral Res, 115, 21-38, 2015), has an IC of less than 500 nM. 50s The results of this study have yielded compounds that exhibit high selectivity against mammalian DUBs. In this case, selectivity may be due to significant structural differences between viral and mammalian DUBs. However, no examples of optimization incorporating the structure of mammalian DUBs have been reported. Breakthroughs in X-ray crystallography of small molecule DUB-inhibitor complexes may enable the rapid development of potent and selective inhibitors.

[0101] USP7 (ubiquitin-specific protease 7) / HAUSP (herpesvirus-associated ubiquitin-specific protease) is a 135 kDa protein of the USP family. USP7 has been shown to interact with viral proteins such as ICP0 (Vmw 110), an inhibitor of the herpes simplex virus immediate-early gene stimulating factor of the viral lytic cycle (Everett et al., J Virol 73, 1999, 417-426), and EBNA1 (Epstein-Barr Nuclear Antigen-1) (Holowaty et al., J Biol Chem 2003, 278, 29987-29994 and 47753-47761). The DUB USP7 has been shown to be involved in the regulation of numerous cellular processes, including epigenetics, cell cycle, DNA repair, immunity, viral infection, and tumorigenesis. Interest in the enzyme grew when USP7 was shown to regulate the degradation of the tumor suppressor p53 (Li et al., Nature, 416, 648-53, 2002) by stabilizing the key E3 ligase for p53, MDM2 (Cummins et al., Nature, 428, 1 p following 486, 2004; Li et al., Mol Cell, 13, 879-86, 2004). Consistently, recent reports have also shown that USP7 silencing increases basal p53 levels by promoting MDM2 degradation. Recently, it has been shown that USP7 binding to p53 is regulated by TSPYL5, a protein potentially involved in breast carcinogenesis, through competition with p53 for binding to the same region of USP7 (Epping et al., Nat Cell Biol. 2011, 13(1):102-8). More recently, up- and down-regulation of USP7 has been shown to inhibit colon cancer cell proliferation in vitro and tumor growth in vivo by resulting in constitutively high p53 levels (Becker et al. Cell Cycle 2008, 7(9), 1205-13).

[0102] USP7 mediates Bmi1 / Mel18 stabilization and regulates p16 INK4aIt also alters the levels of tumor suppressors (Maertens et al., Embo J. 2010 29, 2553-2565). Additional proteins involved in genome integrity / regulation, such as DNMT1 DNA methylase and claspin adaptors, are also stabilized by USP7 (Du et al., Science Signaling 2010, 3(146):ra80; Faustrup et al., J. Cell Biol. 2009, 184(1):13-9). Importantly, the abundance of USP7 and DNMT1, a protein involved in maintaining epigenetic methylation required to silence genes involved in progression and cancer, correlates in human colorectal cancer (Du et al., 2010). USP7 has been shown to deubiquitinate the well-known master repressor gene PTEN in human cells, inducing its nuclear export and thus its inactivation (Song et al., Nature 2008, 455(7214), 813-7). More importantly, USP7 overexpression was first reported in prostate cancer, and this overexpression is directly associated with tumor aggressiveness (Song et al., Nature 2008, 455(7214), 813-7).

[0103] Recently, the methyltransferase PHF8 (Wang et al., 2016a) and the demethylase DNMT1 (Du et al., 2010, Felle et al., Nucleic Acids Res, 39, 8355-65, 2011, Qin et al.) have been identified. Various epigenetic regulators, including the acetyltransferase Tip60 (Dar et al., J Cell Biochem, 112, 439-44, 2011) and the acetyltransferase Tip60 (Dar et al., Mol Cell Biol, 33, 3309-20, 2013), as well as H2B itself (van der Knaap et al., Mol Cell, 17, 695-707, 2005), have been identified as direct targets of USP7. Other prominent targets of USP7 include the transcription factor FOXP3, which links this DUB enzyme to immune responses in Treg cells (van Loosdregt et al., Immunity, 39, 259-71, 2013), and N-Myc, which it stabilizes in neuroblastoma cells (Tavana et al., Nat Med, 22, 1180-1186, 2016). Consistent with its regulation of diverse substrates and biological processes, USP7 has emerged as a drug target in a wide range of malignancies, including multiple myeloma (Chauhan et al., Cancer Cell, 22, 345-58, 2012), breast cancer (Wang et al., 2016a), neuroblastoma (Tavana et al., 2016), glioma (Cheng et al., Oncol Rep, 29, 1730-6, 2013), and ovarian cancer (Zhang et al., Tohoku J Exp Med, 239, 165-75, 2016). USP7 has been shown to deubiquitinate FOXO4 in human cells, inducing its nuclear export and subsequent inactivation, resulting in activation of the oncogenic PI3K / PKB signaling pathway (van der Horst et al., Nat Cell Biol. 2006, 8, 1064-1073). Finally, USP7 plays an important role in p53-mediated cellular responses to various stresses, including DNA damage and oxidative stress (Marchenko et al., Embo J. 2007 26, 923-934; Meulmeester et al., Mol Cell 2005, 18, 565-576; van der Horst et al., Nat Cell Biol. 2006, 8, 1064-1073).

[0104] Multiple myeloma (MMz) is an incurable hematological malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow, which interferes with the production of normal blood cells. While the average survival of MM patients has improved in recent years as a result of the introduction of proteasome inhibitor and immunomodulatory treatment regimens, it is still quite low at only 5 years. The proteasome inhibitor bortezomib identifies the ubiquitin proteasome as a therapeutic target for MM drug development. USP7 is a therapeutic target in MM due to its role in the degradation of p53. USP7 is highly expressed in tumor cells from MM patients and in MM cell lines versus normal bone marrow cells. Mutation or deletion of p53 is a late-stage phenomenon in MM, suggesting that increasing p53 through pharmaceutical inhibition of USP7 may be an effective treatment strategy for this malignancy.

[0105] P22077 and its close analog P5091 (structures in Figure 1A) are the most frequently utilized inhibitors to probe USP7 function. P22077 exhibits moderate potency against USP7 (IC50 = 8.0 μM) and equipotent inhibition of two additional DUBs, USP10 and USP47 (Altun et al., 2011, Ritorto et al., 2014). Reported drawbacks of these nitro-thiophene compounds include low solubility and general toxicity, along with moderate potency and selectivity (Chen et al., 2017). Although not as well characterized as P5091 / P22077, additional USP7 inhibitors (shown in Figure 1B) have been identified without significant optimization efforts (Reverdy et al., Chem Biol, 19, 467-77, 2012; Colland et al., Mol Cancer Ther, 8, 2286-95, 2009; Aleo et al., Cancer Res, 66, 9235-44, 2006; Nicholson et al., Protein Sci, 17, 1035-43, 2008; Yamaguchi et al., Bioorg Med Chem Lett, 23, 3884-6, 2013; Tanokashira et al., Tetrahedron, 72, 5530-5540, 2016).

[0106] Disclosed herein are methods for treating and preventing diseases and conditions that benefit from modulation of USP7, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0107] Disclosed herein are methods for treating and preventing diseases and conditions that benefit from the inhibition of USP7, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0108] Disclosed herein is a method of inhibiting USP7, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0109] In certain embodiments, disclosed herein are methods for treating a disease or disorder modulated by USP7, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, disclosed herein are methods for preventing a disease or disorder modulated by USP7, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, modulating USP7 is associated with inhibiting USP7.

[0110] In one embodiment, the disease or disorder is selected from cancer and cancer metastasis, neurodegenerative diseases, immune disorders, diabetes, bone and joint diseases, osteoporosis, arthritic disorders, cardiovascular diseases, ischemic diseases, viral infections and diseases, viral infectious and / or latent and bacterial infections and diseases.

[0111] Disclosed herein is the use of an inhibitor of USP7 for the preparation of a medicament for treating or preventing a disease or condition modulated by USP7, wherein the medicament comprises a compound of Formula (I). In some embodiments, modulating USP7 is associated with inhibiting USP7.

[0112] Disclosed herein are compounds of Formula (I) for use in treating diseases or conditions modulated by USP7. In some embodiments, modulating USP7 is associated with inhibiting USP7.

[0113] Disclosed herein are methods of treating cancer, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0114] In some embodiments, exemplary cancers include, but are not limited to, liposarcoma, neuroblastoma, glioblastoma, breast cancer, bladder cancer, glioma, neuroblastoma, adrenocortical carcinoma, multiple myeloma, colorectal cancer, non-small cell lung cancer, human papillomavirus-associated cervical, oropharyngeal, penile, ovarian, anal, thyroid or vaginal cancer, Epstein-Barr virus-associated nasopharyngeal carcinoma, gastric cancer, rectal cancer, thyroid cancer, Hodgkin's lymphoma or diffuse large B-cell lymphoma.

[0115] In some embodiments, the cancer is selected from multiple myeloma, breast cancer, neuroblastoma, glioma, colon cancer, prostate cancer, neuroblastoma, and ovarian cancer. In some embodiments, the cancer is breast cancer, glioma, neuroblastoma, multiple myeloma, or ovarian cancer. In some embodiments, the cancer is multiple myeloma.

[0116] Disclosed herein is a method of treating neuroblastoma, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0117] In some embodiments, neurodegenerative diseases include, but are not limited to, Alzheimer's disease, multiple sclerosis, Huntington's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, amyotrophic lateral sclerosis, or encephalitis.

[0118] Pharmaceutical Composition The compositions and methods of the present invention can be used to treat a subject in need of treatment. In certain embodiments, the subject is a mammal, such as a human, or a non-human mammal. When administered to a subject, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water, physiologically buffered saline, or other solvents, vehicles such as glycols, oils such as glycerin, olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for human administration, particularly for invasive routes of administration (i.e., routes that avoid transport or diffusion through epithelial barriers, such as injection or insertion), the aqueous solution is pyrogen-free or substantially pyrogen-free. For example, excipients can be selected to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in unit dosage form, such as tablets, capsules (including small capsules and gelatin capsules), granules, lyophilic agents for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be included in a transdermal delivery system, for example, a skin patch.The composition may also be included in a solution suitable for topical administration, such as eye drops.

[0119] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that act, for example, to stabilize, improve the solubility, or increase the absorption of a compound, such as a compound of the present invention. Such physiologically acceptable agents include, for example, sugars such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; small proteins; or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix, which may incorporate the composition, e.g., a compound of the present invention. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0120] The phrase "pharmaceutically acceptable" as used herein means such compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or other problem or difficulty, commensurate with a reasonable benefit / risk ratio.

[0121] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; and (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum oxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0122] For example, the pharmaceutical composition (formulation) can be administered to a subject by any of a number of routes of administration, including orally (e.g., aqueous or non-aqueous solutions or suspensions for application to the tongue, tablets, capsules (including small capsules and gelatin capsules), boluses, powders, granules, drenches such as pastes), absorption through the oral mucosa (e.g., sublingually), anally, rectally, or vaginally (e.g., as a pessary, cream, or foam), parenterally (e.g., intramuscularly, intravenously, subcutaneously, or intrathecally as a sterile solution or suspension), nasally, intraperitoneally, subcutaneously, transdermally (e.g., as a patch applied to the skin), and topically (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compound may also be prepared for inhalation. In certain embodiments, the compound may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for such administration can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, and patents cited therein.

[0123] The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. Generally, the amount of active ingredient that may be combined with a carrier material to produce a single dosage form will be that amount of compound that produces a therapeutic effect. Out of 100%, this amount will usually be about 1% to about 99% of the active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0124] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with a carrier and, optionally, one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0125] Formulations of the present invention suitable for oral administration may be in the form of capsules (including small capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), lyophilized tablets, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, as an oil-in-water or water-in-oil liquid emulsion, as an elixir or syrup, as a lozenge (using an inactive base such as gelatin and glycine or sucrose and gum arabic), and / or as a mouthwash, each containing a predetermined amount of a compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0126] To prepare solid dosage forms for oral administration (such as capsules (including small capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerin; (4) agar, calcium carbonate, potato; The active ingredient is mixed with any of the following: (1) disintegrating agents such as maize or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (2) dissolution retarders such as paraffin; (3) absorption accelerators such as quaternary ammonium compounds; (4) wetting agents such as cetyl alcohol and glycerol monostearate; (5) absorbents such as kaolin and bentonite clay; (6) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (7) complexing agents such as modified and unmodified cyclodextrins; and (8) coloring agents. For capsules (including small capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0127] Tablets may be formed by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Wet formulations may also be formed by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0128] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including small capsules and gelatin capsules), pills, and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide slow or controlled release of the active ingredient therein, using, for example, hydropropylmethylcellulose in varying proportions to provide the desired release characteristics, other polymer matrices, liposomes, and / or microparticles. They may also be sterilized by, for example, filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid component that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a predetermined portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may, where appropriate, be in microencapsulated form with one or more of the above-mentioned excipients.

[0129] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0130] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0131] In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0132] Formulations of pharmaceutical compositions for rectal, vaginal, or intraurethral administration may be presented as suppositories, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers, including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and will melt in the rectum or vaginal cavity, releasing the active compound(s).

[0133] Formulations of the pharmaceutical composition for oral administration may be presented as a mouthwash, mouth spray, or mouth ointment.

[0134] Alternatively or additionally, the compositions may be prepared for delivery via a catheter, stent, wire, or other intraluminal device, which may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestinal tract.

[0135] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0136] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0137] In addition to the active compound, the ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0138] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0139] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present invention to the body.Such dosage forms can be formed by dissolving or dispersing the active compound in a suitable medium.Absorption enhancers can also be used to increase the flux of the compound across the skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0140] Ophthalmic formulations, eye ointments, powders, solutions, and the like, are also contemplated as being within the scope of the present invention. Representative ophthalmic formulations are described in U.S. Patent Application Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. Optionally, the ophthalmic formulation has properties similar to those of tears, aqueous humor, or vitreous humor, or is compatible with such fluids. A preferred route of administration is topical administration (e.g., topical administration, such as eye drops, or administration via an implant).

[0141] As used herein, the phrases "parenteral administration" and "administered parenterally" mean administration other than enteral and local administration, usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0142] Pharmaceutically acceptable compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile, isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders, which can be reconstituted immediately before use into sterile, injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents that render the formulation isotonic with the blood of the intended recipient.

[0143] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0144] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0145] In some cases, it is desirable to slow the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of the drug.This can be achieved by using a suspension of crystalline or amorphous material with poor water solubility.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0146] Injectable depot forms are prepared by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0147] For use in the methods of the present invention, the active compound may be provided by itself or, for example, in combination with a pharmaceutically acceptable carrier, as a pharmaceutical composition containing 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient.

[0148] Methods of introduction may be provided by rechargeable or biodegradable devices. A variety of sustained-release polymeric devices have been developed and recently tested in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including biodegradable and non-degradable polymers, can be used to form implants for the sustained release of compounds at specific target sites.

[0149] The actual dosage level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, provided that it is not toxic to the patient.

[0150] The selected dose level will depend on a variety of factors, including the activity of the particular compound or combination of compounds used or their esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the particular compound(s) being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) being used, the age, sex, weight, symptoms, overall health, and prior medical history of the subject being treated, and other factors well known in the medical arts.

[0151] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start the administration of a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. By "therapeutically effective amount" is meant a concentration of a compound sufficient to elicit the desired therapeutic effect. It is generally understood that an effective amount of a compound will vary according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the subject's symptoms, the disorder being treated, the stability of the compound, and, if necessary, other therapeutic agents being administered with the compound of the present invention. Multiple administrations of the drug may deliver a larger total amount. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th ed., 1814-1882, incorporated herein by reference).

[0152] Generally, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0153] If necessary, the effective daily dose of the active compound may be administered in one, two, three, four, five, six or more subdoses, optionally in unit dosage forms, administered separately at appropriate intervals throughout the day. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In a preferred embodiment, the active compound is administered once daily.

[0154] Effective dosages of the disclosed compounds, when used for the indicated effect, range from about 0.5 mg to about 5000 mg of the disclosed compounds needed to treat the condition. Compositions for in vivo or in vitro use can contain about 0.5, about 5, about 20, about 50, about 75, about 100, about 150, about 250, about 500, about 750, about 1000, about 1250, about 2500, about 3500, or about 5000 mg of the disclosed compounds, or any range from one amount to another listed in the dosage list.

[0155] In certain embodiments, the compounds of the present invention may be used alone or co-administered with other therapeutic agents. As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds, in which a first therapeutic compound is administered while the first is still effective in the body (e.g., the two compounds are simultaneously effective in a subject, and this effect may include a synergistic effect of the two compounds). For example, different therapeutic compounds can be administered simultaneously or sequentially in the same formulation or in separate formulations. In certain embodiments, different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other. Thus, subjects receiving such treatment can benefit from the combined effects of the different therapeutic compounds.

[0156] In certain embodiments, co-administration of a compound of the invention with one or more additional therapeutic agents(s) enhances the effectiveness of either the compound of the invention (e.g., a compound of Formula I or Ia) or the one or more additional therapeutic agents(s) administered separately. In certain embodiments, co-administration results in an additive effect, where additive effect refers to the sum of the respective effects of the compound of the invention and the one or more additional therapeutic agents(s) administered separately.

[0157] The present invention encompasses the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

[0158] Pharmaceutically acceptable acid addition salts can exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. Such solvates can be prepared from, inherent in, or adventitious to the solvent of crystallization.

[0159] Wetting agents, emulsifying agents, lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0160] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. [Example]

[0161] Compounds of formula (I) may be prepared by methods known in the art of organic synthesis, as described in part by the following synthetic scheme: The compounds described herein may be formed from commercially available starting materials or synthesized using known organic, inorganic and / or enzymatic processes.

[0162] In the schemes described below, where necessary according to general principles or chemistry, the use of protecting groups for sensitive or reactive groups is well known. Protecting groups are manipulated according to standard methods of organic synthesis (T.W. Greene and P.G.M. Buts, "Protective Groups"). "Groups in Organic Synthesis", Third edition, Wiley, New York 1999). These groups are removed at a convenient stage of the compound synthesis using methods that will be readily apparent to those skilled in the art. The processes selected, as well as the reaction conditions and order of their execution, should be consistent with the preparation of compounds of formula (I).

[0163] Those skilled in the art will recognize that stereocenters exist in compounds of Formula (I). Accordingly, the present disclosure encompasses both possible stereoisomers (unless specified in the synthesis), including not only the racemate but also the individual enantiomers and / or diastereomers. When a compound is desired as a single enantiomer or diastereomer, it can be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, intermediate, or starting material can be affected by any conventional method known in the art. See, for example, "Stereochemistry of Organic Compounds" by EL Eliel, SH Wilen, and LN Mander (Wiley-Interscience, 1994). Mixtures of enantiomers, diastereomers, and cis / trans isomers resulting from the above processes can be separated into their individual components by chiral salt techniques, chromatography using normal-phase, reverse-phase, or chiral columns, depending on the nature of the separation.

[0164] The present disclosure is further illustrated by the following examples and synthetic schemes, which are not to be construed as limiting the scope or spirit of the disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate specific embodiments and are not intended to limit the scope of the disclosure. It should be further understood that various other embodiments, modifications, and equivalents thereof will be relied upon to suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0165] Analytical methods, materials and measurements Unless otherwise noted, reagents and solvents were used as received from commercial sources. All commercially available starting materials were purchased from Sigma Aldrich, Fisher Scientific, Oakwood Chemical, and Combi Block. All reagents were used as received without further purification. Known compounds were synthesized according to published literature procedures; any variations are noted. Anhydrous solvents, such as tetrahydrofuran (THF), diethyl ether, dichloromethane (DCM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, and toluene (PhMe), were purchased from Fisher Scientific and used as received. Where necessary, air- or moisture-sensitive reactions were performed under an inert atmosphere of nitrogen.

[0166] Solvent removal was performed on a Buchi R-300 rotary evaporator, and further concentration was achieved using a Welch 1400B-01 vacuum line and a Labconco FreeZone 6 plus system. Compounds were purified by normal-phase column chromatography using a Teledyne CombiFlash chromatography system and / or reverse-phase chromatography on a Waters Micromass ZQ preparative system using a SunFire® Prep C18 OBD™ 5 μM column. Purity was analyzed using a Waters Acquity UPLC system. Analytical thin-layer chromatography (TLC) plates were purchased from Fisher Scientific (EMD Millipore TLC Silica Gel 60 F254). Visualization was achieved by irradiation with ultraviolet light (254 nm).

[0167] All H-NMR spectra were recorded on a Bruker ARX 500 (500 MHz) spectrometer at 298 K. C-NMR spectra were recorded on a Bruker ARX 500 (126 MHz) spectrometer. Samples were dissolved in CDCl, DMSO-d, or CD. The spectra were referenced to residual solvent peaks (chloroform-d: 7.26 ppm for H-NMR and 77.16 ppm for C-NMR; DMSO-d: 2.50 ppm for H-NMR and 39.25 ppm for C-NMR; CD. OD: 3.31 ppm for H-NMR and 49.00 ppm for C-NMR or trimethylsilane (TMS) as an internal standard). Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad peak) and proton number. Mass spectrometry (LCMS) data were obtained on a Waters Acquity UPLC system in positive ESI mode.

[0168] Example 1: Representative synthesis of compounds of the present disclosure [ka] S1.R = Cl; S2.R = NO2 2-Aminobenzoic acid (10.0 mmol) and formaldehyde (1.8 g, 40.0 mmol) were combined in a pressure tube and heated at 150 °C overnight. The reaction was then cooled to room temperature. The solid was suspended in cold water, collected by vacuum filtration, and dried on a high vacuum line. Without further purification, 1.6 g (-Cl) and 1.8 g (-NO) of the product were isolated as pale brown solids in 88% (-Cl) and 95% (-NO) yields.

[0169] [ka] Sodium hydride (60% dispersion in mineral oil) (0.88 g, 22.0 mmol) was dissolved in 40 mL of anhydrous DMSO at 0 °C under N2. Trimethylsulfoxonium iodide (4.84 g, 22.0 mmol) was added portionwise to the solution. When the addition was complete, the mixture was warmed to room temperature and stirred for 40 minutes. 1-Boc-4-piperidone (3.98 g, 20.0 mmol) was then added portionwise. The reaction mixture was then stirred for 1 hour at room temperature and for an additional hour at 65 °C. The mixture was poured into 100 mL of ice. The aqueous phase was extracted with EtOAc (50 mL × 2). The combined organic phases were washed with brine, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude material was purified by flash column chromatography (50% EtOAc in hexanes) to give 2.98 g of product in 70% yield.

[0170] [ka] S4.R = Cl; S5.R = NO2 To an aqueous solution of S1 (2.13 g, 11.8 mmol) in 50 mL of DMF was added S3 (2.78 g, 13.0 mmol) and Cs2CO3 (11.54 g, 35.4 mmol). The mixture was heated at 80 °C overnight. The reaction was then cooled to room temperature and diluted with EtOAc. The solution was washed with saturated NH4Cl (50 mL × 2), and the aqueous phase was extracted with additional EtOAc. The combined organic phases were washed with brine, dried over MgSO4, filtered, and evaporated under reduced pressure. The crude material was purified by flash column chromatography (40% to 100% EtOAc in hexanes) to give 3.94 g of product in 85% yield.

[0171] [ka] S4.R=Cl;S5.R=NO2;S6.R=Cl;S7.R=NO2 S4 was taken up as a 1 M solution in trifluoroacetic acid (TFA) and stirred for 2 h at room temperature. The solution was concentrated under reduced pressure and then under high vacuum overnight. S6 was used directly as starting material for the following synthesis without further purification.

[0172] [ka] S6.R = Cl; S7.R = NO2 Amide formation by HATU-catalyzed coupling reaction S6 was taken up in DMF as a 1 M solution and 3 equivalents of Et3N were added. Equal concentrations of HATU (2 eq) and Et3N (5 eq) and carboxylic acid (1.2 eq) were premixed in DMF and stirred for 10 min at room temperature. The two solutions were then mixed and further stirred for 5 h at room temperature. The reaction was directly subjected to preparative HPLC purification. The isolated product was then further purified by normal-phase flash chromatography to yield the product with the desired purity for the following biological studies.

[0173] [ka] Amide formation by acylation with acid chlorides S6 (0.04 g, 0.1 mmol) was taken up in 2 mL of dichloromethane. EtN (0.07 mL, 0.5 mmol) was added, followed by acetyl chloride (0.015 mL, 0.2 mL). The reaction was stirred for 2 h and stored at 0 °C. The reaction was then quenched by dropwise addition of water and immediately purified by flash column chromatography. Product 6, isolated by HPLC, was further purified to give 19 mg of product in 57% yield.

[0174] [ka] Reduction of aromatic nitro groups S7 (0.49 g, 1.08 mmol) was dissolved in 10 mL of AcOH / EtOH (1:1). Iron powder (0.25 g, 4.39 mmol) was added in one portion. The reaction was then stirred at 50 °C for 1 h. The iron powder was removed by filtration. The filtrate was concentrated under reduced pressure. The crude material was purified by normal-phase flash column chromatography (10% to 40% MeOH in EtOAc) to give 0.22 g of product S8 in 53% yield after reverse-phase HPLC.

[0175] [ka] Introduction of soluble groups S8 (0.11 g, 0.25 mmol) was dissolved in 5 mL of dichloromethane. EtN (0.035 mL, 0.25 mmol) was added at -20 °C. 3-Bromopropionyl chloride (0.03 mL, 0.25 mmol) in 1 mL of DCM was added dropwise. The reaction was stirred for 3 h at 0 °C. After that, the reaction was quenched by the dropwise addition of water and concentrated under reduced pressure. The crude product was used in the next step without further purification.

[0176] The crude material from the last step (0.06 g, 0.1 mmol) was dissolved in 1 mL of DMF. To the solution was added N-methylpiperazine (0.016 mL, 0.12 mmol) and EtN (0.028 mL, 0.2 mmol). The reaction was stirred at 80 °C for 3 h. The solution was directly subjected to reverse-phase HPLC purification, and after normal-phase flash column chromatography (20% to 60% MeOH in EtOAc with 0.5% EtN), 0.043 g of product 10 was obtained in 75% yield. Using these procedures and variations thereof, the following compounds were synthesized:

[0177] [ka] 1 ((R)-7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 50% yield 1H NMR (500 MHz, DMSO) δ 8.27 (d, J = 12.8 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 1.7 Hz, 1H), 7.58 (dd, J = 8.6, 2.0 Hz, 1H), 7.26 (dd, J = 13.8, 6.7 Hz, 4H), 7.20-7.08 (m, 1H), 4.93 (d, J = 5.9 Hz, 1H), 4.04 (d, J = 13.6 Hz, 2H), 3.92 (q, J = 13.9 Hz, 1H), 3.65 (t, J = 12.5Hz, 1H), 3.28-3.06 (m, 2H), 2.86 (ddd, J = 13.9, 8.6, 3.2 Hz, 1H), 2.68-2.50 (m, 2H), 1.62-1.26 (m, 3H), 1.26-1.12 (m, 3H). 13 C NMR (126 MHz, DMSO) δ 168.92, 159.90, 150.16, 148.76, 146.43, 146.31, 138.70, 128.22, 127.99, 127.96, 126.98, 126.68, 126.64, 125.99, 125.74, 125.69, 120.12, 69.05, 69.00, 53.58, 40.82, 40.71, 39.99, 36.68, 36.01, 35.78, 34.78, 34.65, 34.09, 33.95, 21.83, 21.63.LCMS (ESI) m / z 440.29 [(M+H) + ;C 24 H 27 ClN3O3 + Calculated value for: 440.17].

[0178] [ka] 2 (7-chloro-3-((1-(3-phenylpropanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 33% yield 11H NMR (500 MHz, CDCl3) δ 8.21 (d, J = 8.6 Hz, 1H), 7.93 (s, 1H), 7.70 (d, J = 1.9 Hz, 1H), 7.46 (dd, J = 8.6, 2.0 Hz, 1H), 7.31 - 7.25 (m, 2H), 7.22 - 7.18 (m, 3H), 4.69 (d, J = 13.4 Hz, 1H), 3.88 - 3.73 (m, 3H), 3.00 - 2.92 (m, 2H), 2.91 - 2.82 (m, 1H), 2.60 (dp, J = 14.3, 7.3 Hz, 2H), 2.48 (td, J = 13.1, 2.4 Hz, 1H), 2.17 - 2.04 (m, 1H), 1.70 (d, J = 12.9 Hz, 1H), 1.62 (d, J = 12.8 Hz, 1H), 1.17 (qd, J = 12.5, 4.3 Hz, 1H), 0.97 (qd, J = 12.5, 4.2 Hz, 1H). 13 13C NMR (126 MHz, DMSO) δ 169.56, 159.76, 149.58, 148.95, 141.43, 138.88, 128.37, 128.18, 127.28, 126.30, 125.79, 120.37, 50.79, 44.55, 40.74, 38.22, 35.01, 33.98, 30.87, 29.54, 28.84. LCMS (ESI) m / z 410.29 [(M + H) + ; C 23 H 25 ClN3O2 + calculated for: 410.16].

[0179]

Chemical Structure

[0180] [ka] 4 (7-chloro-3-((4-hydroxy-1-(3-phenylpropyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 13% yield 1 H NMR (500 MHz, MeOD) δ 8.29 (s, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.54 (dd, J = 8.6, 2.0 Hz, 1H), 7.26 (q, J = 7.1 Hz, 2H), 7.21-7.13 (m, 3H), 4.11 (s, 2H), 2.97 (d, J = 12.0 Hz, 2H), 2.76-2.59 (m, 6H), 1.98-1.81 (m, 4H), 1.63 (d, J = 13.4 Hz, 2H). 13 C NMR (126 MHz, MeOD) δ 161.13, 149.97, 148.77, 140.38, 140.25, 128.24, 128.13, 128.00, 127.52, 126.00, 120.22, 67.60, 56.16, 53.81, 48.21, 32.27, 32.00, 25.90.LCMS (ESI) m / z 412.39 [(M+H) + ;C 23 H 27 ClN3O2 + Calculated value for: 412.18].

[0181] [ka] 5 (7-chloro-3-((3-hydroxy-1-(3-phenylpropanoyl)pyrrolidin-3-yl)methyl)quinazolin-4(3H)-one: white solid, 8% yield 1 H NMR (500 MHz, DMSO) δ 8.29 (s, 1H), 8.17 (dd, J = 8.6, 4.3 Hz, 1H), 7.76 (t, J = 2.3 Hz, 1H), 7.59 (ddd, J = 8.7, 7.5, 2.1 Hz, 1H), 7.29-7.19 (m, 4H), 7.16 (t, J = 6.8 Hz, 1H), 5.27 (s, 1H), 4.17 (s, 1H), 4.14 (d, J = 4.0 Hz, 1H), 3.51 (s, 2H), 3.32 (dt, J = 23.6, 11.7 Hz, 3H), 2.84-2.74 (m, 2H), 2.59-2.51 (m, 2H), 2.47-2.40 (m, 1H), 1.95 (ddt, J = 39.5, 12.7, 9.3 Hz, 1H), 1.76 (ddd, J = 12.9, 11.0, 5.7 Hz, 1H). 13 C NMR (126 MHz, DMSO) δ 169.93, 169.76, 160.22, 160.13, 150.33, 149.06, 149.04, 141.55, 141.51, 138.95, 128.45, 128.40, 128.35, 128.22, 127.24, 126.27, 125.81, 120.46, 78.33, 76.84, 55.97, 55.57, 51.10, 50.87, 44.46, 43.77, 36.06, 35.63, 35.06, 34.54, 30.29, 30.23.LCMS (ESI) m / z 412.29 [(M+H) + ;C 22 H 23 ClN3O3 + Calculated value for: 412.14].

[0182] [ka] 6 (3-((1-acetyl-4-hydroxypiperidin-4-yl)-7-chloroquinazolin-4(3H)-one: white solid, 57% yield) 1H NMR (500 MHz, DMSO) δ 8.40 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.6, 2.1 Hz, 1H), 4.09-3.96 (m, 4H), 3.58 (d, J = 13.4 Hz, 1H), 3.33-3.20 (m, 1H), 2.97-2.85 (m, 1H), 1.98 (s, 3H), 1.56 (td, J = 13.3, 4.3 Hz, 1H), 1.49-1.34 (m, 3H). 13 C NMR (126 MHz, DMSO) δ 167.66, 159.89, 150.25, 148.62, 138.65, 128.19, 126.92, 125.88, 120.05, 68.98, 53.39, 41.40, 36.44, 34.61, 33.93, 20.98.LCMS (ESI) m / z 336.18 [(M+H) + ;C 16 H 19 ClN3O3 + Calculated value for: 336.11].

[0183] [ka] 7(7-chloro-3-((4-hydroxy-1-(2-phenylacetyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, commercially available compound) 1 H NMR (500 MHz, DMSO) δ 8.27 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.6, 2.1 Hz, 1H), 7.30-7.24 (m, 2H), 7.24-7.15 (m, 3H), 4.07-4.03 (m, 1H), 3.97 (dd, J = 38.6, 13.8 Hz, 2H), 3.74-3.64 (m, 3H), 3.29-3.20 (m, 1H), 3.03-2.92 (m, 1H), 1.51-1.27 (m, 4H). 13 C NMR (126 MHz, DMSO) δ 168.31, 159.88, 150.12, 148.71, 138.66, 135.76, 128.57, 128.19, 127.99, 126.93, 126.00, 125.95, 120.07, 68.97, 53.45, 41.15, 39.35, 36.86, 34.61, 33.99.LCMS (ESI) m / z 412.29 [(M+H) + ;C 22 H 23 ClN3O3 + Calculated value for: 412.14].

[0184] [ka] 8 (7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, commercially available compound) 1 H NMR (500 MHz, DMSO) δ 8.29 (s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.56 (dd, J = 8.6, 2.1 Hz, 1H), 7.30-7.22 (m, 4H), 7.18-7.13 (m, 1H), 4.12-3.55 (m, 4H), 3.41-2.85 (m, 3H), 2.62 (dd, J = 14.9, 6.6 Hz, 1H), 2.54 (dd, J = 14.9, 7.6 Hz, 1H), 1.40 (t, J = 15.9 Hz, 4H), 1.25 (d, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 168.89, 159.94, 159.88, 150.11, 148.74, 146.42, 146.30, 138.68, 128.20, 127.97, 127.94, 126.94, 126.66, 126.63, 125.98, 125.72, 125.67, 120.10, 69.04, 68.99, 53.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77, 34.78, 34.64, 34.09, 33.95, 21.81, 21.61.LCMS (ESI) m / z 440.39 [(M+H) + ; C 24 H 27 ClN3O3 + Calculated value for: 440.17].

[0185] [ka] 9((S)-7-chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one: white solid, 57% yield 1 H NMR (500 MHz, DMSO) δ 8.27 (d, J = 12.8 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 1.6 Hz, 1H), 7.58 (dd, J = 8.6, 2.0 Hz, 1H), 7.26 (dd, J = 13.7, 6.6 Hz, 4H), 7.21-7.09 (m, 1H), 4.93 (d, J = 5.8 Hz, 1H), 3.99 (dd, J = 43.9, 13.7 Hz, 2H), 3.91 (s, 1H), 3.65 (t, J = 12.2 Hz, 1H), 3.28-3.09 (m, 2H), 2.85 (d, J = 13.2 Hz, 1H), 2.68-2.55 (m, 2H), 1.60-1.25 (m, 3H), 1.20 (dd, J = 6.9, 1.5 Hz, 3H).13 C NMR (126 MHz, DMSO) δ 168.89, 159.94, 159.88, 150.11, 148.74, 146.42, 146.30, 138.68, 128.20, 127.97, 127.94, 126.94, 126.66, 126.63, 125.98, 125.72, 125.67, 120.10, 69.04, 68.99, 53.57, 40.82, 40.71, 39.99, 36.67, 35.99, 35.77, 34.78, 34.64, 34.09, 33.95, 21.81, 21.61.LCMS (ESI) m / z 440.29 [(M+H) + ; C 24 H 27 ClN3O3 + Calculated value for: 440.17].

[0186] [ka] 10 ((R)—N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propanamide: off-white solid, 75% yield 11H NMR (500 MHz, DMSO) δ 10.57 (s, 1H), 8.20 (d, J = 13.1 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.25 (dd, J = 12.4, 6.2 Hz, 4H), 7.13 (dd, J = 18.7, 10.4 Hz, 1H), 4.96 (d, J = 5.8 Hz, 1H), 4.02 (d, J = 13.6 Hz, 1H), 3.90 (q, J = 14.0 Hz, 2H), 3.63 (dd, J = 29. *0*, 16.2 Hz, 1H), 3.27 - 3.12 (m, 2H), 2.86 (dd, J = 17.7, 14.8 Hz, 1H), 2.69 - 2.61 (m, 2H), 2.61 - 2.52 (m, 3H), 2.48 - 2.22 (m, 8H), 2.16 (s, 3H), 1.58 - 1.26 (m, 4H), 1.20 (d, J = 6.4 Hz, 3H). 13 13C NMR (126 MHz, DMSO) δ 170.76, 168.83, 159.92, 159.87, 149.10, 148.73, 146.41, 146.28, 144.06, 127.93, 127.90, 126.97, 126.62, 126.60, 125.69, 125.63, 118.04, 116.26, 114.45, 69.03, 68.98, 54.37, 53.22, 51.96, 45.30, 40.81, 40.71, 39.97, 36.67, 35.95, 35.74, 34.77, 34.64, 34.09, 34.01, 33.95, 21.79, 21.61.LCMS (ESI) m / z 575.32 [(M + H) + ; C 32 H 43 ClN6O4 + calculated value for: 575.33].

[0187]

Chemical Structure

[0188] [ka] 12 (N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propenamide: white solid, 16% yield 1 H NMR (500 MHz, DMSO) δ 10.84 (s, 1H), 8.25 (d, J = 13.2 Hz, 1H), 8.15-7.99 (m, 2H), 7.69 (d, J = 8.7 Hz, 1H), 7.28-7.25 (m, 4H), 7.19-7.10 (m, 1H), 4.05-3.88 (m, 3H), 3.65 (t, J = 12.8 Hz, 1H), 3.28-3.14 (m, 10H), 2.92-2,85 (m, 3H), 2.79 (s, 3H), 2.64-2.53 (m, 4H), 1.53-1.28 (m, 4H), 1.20 (d, J = 6.0, 3H). 13 C NMR (126 MHz, DMSO) δ 169.60, 160.66, 160.61, 158.82, 158.57, 149.95, 149.34, 147.15, 147.02, 144.63, 128.69, 128.66, 127.69, 127.38, 127.35, 126.44, 126.39, 118.90, 117.14, 115.37, 69.76, 69.71, 53.94, 41.57, 41.46, 40.88, 40.70, 37.42, 36.71, 36.49, 35.50, 35.37, 34.81, 34.66, 22.53, 22.34.LCMS (ESI) m / z 575.32 [(M+H) + ;C 32 H 43 ClN6O4 +Calculated value for: 575.33].

[0189] [ka] 13 (N-(3-((4-hydroxy-1-(4-methyl-3-phenylpentanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)propenamide: off-white solid, 40% yield 1 H NMR (500 MHz, DMSO) δ 10.65 (s, 1H), 8.21 (d, J = 16.9 Hz, 1H), 8.11 (dd, J = 8.7, 1.9 Hz, 1H), 8.05 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.23 (td, J = 7.6, 2.9 Hz, 2H), 7.15 (t, J = 6.5 Hz, 2H), 7.13-7.06 (m, 1H), 4.04-3.93 (m, 2H), 3.85 (s, 1H), 3.75-3.61 (m, 1H), 3.58-2.98 (m, 10H), 2.91-2.55 (m, 10H), 1.88-1.80 (m, 1H), 1.53-1.25 (m, 3H), 1.12-1.06 (m, 1H), 0.89 (t, J = 7.2 Hz, 3H), 0.70-0.61 (m, 3H). 13C NMR (126 MHz, DMSO) δ 169.94, 169.86, 160.61, 160.53, 149.95, 149.29, 144.51, 144.09, 144.03, 128.82, 128.79, 128.24, 128.16, 127.81, 126.31, 118.89, 117.21, 115.33, 69.76, 69.66, 54.04, 50.83, 49.24, 49.08, 48.75, 42.48, 41.51, 37.43, 37.33, 36.01, 35.94, 35.55, 35.31, 34.72, 34.65, 32.83, 32.63, 32.39, 21.24, 21.19, 20.76, 20.56.LCMS (ESI) m / z 603.43 [(M+H) + ;C 34 H 47 ClN6O4 + Calculated value for: 603.37]

[0190] [ka] 14 ((R)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-morpholinopropanamide: off-white solid, 65% yield 1 H NMR (500 MHz, DMSO) δ 10.52 (s, 1H), 8.20 (d, J = 13.3 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 4.96 (d, J = 6.4 Hz, 1H), 4.02 (d, J = 13.7 Hz, 1H), 3.90 (q, J = 14.0 Hz, 2H), 3.73-3.60 (m, 1H), 3.59-3.55 (m, 4H), 3.18 (ddd, J = 21.0, 18.3, 9.2 Hz, 2H), 2.93-2.80 (m, 1H), 2.65 (t, J = 6.9 Hz, 2H), 2.61-2.54 (m, 3H), 2.41 (m, 4H), 1.40 (dddd, J = 41.2, 26.0, 16.8, 9.5 Hz, 4H), 1.19 (dd, J = 6.9, 1.7 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 170.62, 168.81, 160.00, 149.15, 148.76, 146.44, 146.31, 144.09, 118.13, 116.29, 114.51, 69.06, 65.93, 53.73, 52.79, 40.85, 40.75, 39.98, 36.71, 36.00, 35.78, 34.79, 34.66, 34.11, 33.98, 33.81, 21.83, 21.64.LCMS (ESI) m / z 562.32 [(M+H) + ;C 31 H 40 ClN5O5 + Calculated value for: 562.30].

[0191] [ka] 15 ((R)-3-(dimethylamino)-N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)propanamide: off-white solid, 36% yield 1 H NMR (500 MHz, DMSO) δ 10.47 (s, 1H), 8.18 (dd, J = 13.0, 7.3 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.63-7.59 (m, 1H), 7.24 (dd, J = 12.6, 6.3 Hz, 4H), 7.17-7.09 (m, 1H), 4.92 (s, 1H), 4.07-3.95 (m, 1H), 3.89 (q, J = 14.0 Hz, 2H), 3.63 (t, J = 13.0 Hz, 1H), 3.18 (ddd, J = 20.8, 18.0, 9.1 Hz, 2H), 2.86 (ddd, J = 13.8, 10.5, 5.5 Hz, 1H), 2.60-2.54 (m, 3H), 2.54-2.47 (m, 2H), 2.17 (s, 6H), 1.56-1.23 (m, 4H), 1.24-1.15 (m, 3H). 13 C NMR (126 MHz, DMSO) δ 170.76, 168.83, 159.89, 149.09, 148.74, 146.42, 146.29, 144.07, 127.94, 127.92, 126.97, 126.61, 125.70, 125.64, 118.06, 116.26, 114.46, 68.99, 54.59, 53.29, 44.63, 40.82, 40.72, 39.98, 39.96, 36.68, 35.97, 35.75, 34.78, 34.65, 34.61, 34.11, 33.96, 21.80, 21.61.LCMS (ESI) m / z 520.31 [(M+H) + ;C 29 H 38 ClN5O4 + Calculated value for: 520.29].

[0192] [ka] 16 (N-(3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(1H-imidazol-1-yl)propenamide: white solid, 19% yield1 1H NMR (500 MHz, DMSO) δ 10.62 (s, 1H), 9.16 (s, 1H), 8.22 (d, J = 12.9 Hz, 1H), 8.09 (d, J = 8.7 Hz, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.67 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.28 - 7.25 (m,,4H), 7.15 (d, J = 3.6 Hz, 1H), 4.03 - 3.87 (m, 3H), 3.65 (t, J = 12.4 Hz, 1H), 3.26 - 3.14 (m, 2H), 3.08 (t, J = 6.3 Hz, 2H), 2.90 - 2.85 (m, 1H), 2.64 - 2.49 (m, 4H), 1.54 - 1.25 (m, 4H), 1.20 (d, J = 5.4 Hz, 3H). 13 13C NMR (126 MHz, DMSO) δ 169.60, 169.43, 160.60, y160.55, 158.88, 158.60, 149.99, 149.25, 147.15, 147.03, 144.33, 136.33, 128.68, 128.66, 127.85, 127.37, 127.35, 126.44, 126.39, 122.58, 120.32, 118.86, 117.25, 115.33, 69.77, 69.71, 54.04, 44.94, 41.55, 41.44, 40.71, 37.41, 36.70, 36.57, 36.49, 35.52, 35.39, 34.80, 34.66, 22.53, 22.35. LCMS (ESI) m / z 543.22 [(M + H) + ; C 30 H 35 [[ID=□]]ClN6O4 + Calculated value for: 543.27].

[0193]

Chemical formula

[0194] Example 3: Bioassay USP7 enzyme expression and purification A construct spanning residues 208–560 of human USP7 in the pET28aLIC vector was overexpressed in E. coli BL21(DE3) in terrific broth (TB) medium in the presence of 50 μg / mL kanamycin. Cells were grown at 37°C to an OD of 0.8, cooled to 17°C, induced with 500 μM isopropyl alcohol-1-thio-D-galactopyranoside (IPTG), incubated overnight at 17°C, harvested by centrifugation, and stored at −80°C. Cell pellets were sonicated in Buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM imidazole, and 3 mM BME), and the resulting lysate was centrifuged at 30,000 × g for 40 min. Ni-NITA beads (Qiagen) were mixed with the lysate supernatant for 30 min and washed with Buffer A. The beads were transferred to an FPLC-compatible column, and the bound protein was washed with 15% Buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 300 mM imidazole, and 3 mM BME) and eluted with 100% Buffer B. Thrombin was added to the eluted protein and incubated overnight at 4°C. The sample was concentrated and passed through a Superdex200 16 / 60 column (GE Healthcare) in a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, and 1 mM TCEP. Fractions were pooled, concentrated, and frozen at -80°C.

[0195] The full-length USP7 (aa 0001.1102) in pET28aLIC was transformed into BL21(DE3) cells. An overnight culture was used to inoculate 1 L of TB supplemented with 50 μg / mL kaomycin. Cells were grown at 37°C until they reached an optical density at 600 nm (OD) of approximately 0.6. Protein expression was initiated by the addition of 0.4 mM IPTG. Cells were then grown at 17°C for 16–20 h before harvesting by centrifugation. The cell pellet was washed in PBS, resuspended in 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, and 1 mM TCEP, 10 mM imidazole, 0.1% IGEPAL, sonicated, and incubated with Ni-Nta beads (Quiagen) for 30 min at 4°C. The beads were washed with 10% Buffer B (25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, and 1 mM TCEP, 250 mM imidazole) and eluted with 100% Buffer B. The protein-containing fractions were concentrated and loaded onto a Superdex200 10 / 300GL column in a buffer containing 20 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, and 1 mM TCEP. The fractions were pooled, concentrated, and frozen at -80°C.

[0196] Site-directed mutagenesis Amino acid mutations in the catalytic domain and full-length USP7 were introduced by PCR using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Table 1 lists the primers used for each mutation generated. [Table 1-1] [Table 1-2]

[0197] Isothermal Titration Calorimetry Protein / ligand binding affinity was measured by adding 0.02 mM protein to cells and titrating with 0.2 mM ligand in a syringe using an Auto-ITC200 microcalorimeter (Malvern) at 20 °C (Figure 2C). Protein and ligand were prepared in ITC buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, and 2% DMSO. Data were fitted using Origin 7.0 software. The ITC results are summarized in Table 2. [Table 2]

[0198] Selectivity Profiling Selectivity profiling (DUBProfiler) was performed by Ubiquigent using the manufacturer's protocol. Figure 2B shows the dose-dependent inhibition of the USP7 catalytic domain by compounds A, 10, and 11. Figure 2D shows the inhibitory activity of compound 10 across a panel of 41 purified DUBs using ubiquitin-rhodamine (Ub-Rho) as a substrate.

[0199] Antagonistic activity-based protein profiling HEK293T cells were pelleted, washed with PBS, and lysed on ice (50 mM Tris pH 7.6, 150 mM NaCl, 5 mM MgCl, 0.5 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM TCEP phosphatase inhibitor cocktail (Sigma P5726 and Calbiochem 524624) and protease inhibitors (pepstatin, leupeptin, PMSF, and aprotinin)) and cleared by centrifugation. Protein content was quantified by BCA, and 50 μg of lysate was diluted into 30 μL of labeling buffer (50 mM Tris pH 7.6, 5 mM MgCl, 0.5 mM EDTA, 250 mM sucrose, 1 mM TCEP) and incubated for 30 min at room temperature with shaking in the indicated inhibitors. Samples were then diluted with 1 mM TCEP. The mixture was supplemented with HA-Ub-Bs and incubated for 15 minutes at room temperature with shaking. The reaction was stopped with 4x LDS sample buffer (Thermo Fisher B0007) supplemented with 10% BME, vortexed vigorously, and heated to 95°C for 5 minutes. Samples were resolved by SDS-PAGE and analyzed by Western blot using the indicated antibodies (Figure 5C).

[0200] Cell treatment MCF7 and MM.1S cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were treated with DMSO or various concentrations of compounds 10 and 11 for 6 hours (MM1S) or 16 hours (MCF7) in the presence or absence of cycloheximide. For experiments in which cycloheximide was used, cells were treated with the compounds for 4 hours (MM1S) or 14 hours (MCF7) before the addition of 50 μg / mL cycloheximide. At the 6-hour or 16-hour time point, cells were washed with PBS and resuspended in modified RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM MgCl, 1% phosphate-buffered saline ... The protein concentration was quantified using a BCA protein assay kit (Pierce) and analyzed by ELISA using the ELISA kit (Santa Claus). Samples were verified by immunoblotting using cruz (sc-965), p53 (Cell Signaling 9282), p21 (Cell Signaling 2947), GAPDH (Cell Signaling 2118), and USP7 (Cell Signaling 4833) antibodies (Figure 6A-D).

[0201] Peripheral blood mononuclear cell test Peripheral blood mononuclear cells (PBMCs) were generously provided by Dr. Steven Treon and Dr. Guang Yang. PBMCs from normal individuals were isolated by density gradient centrifugation with Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden) at 400 x g for 25 minutes and washed twice in PBS. Cells were maintained in RPMI + 10% FBS, supplemented with 10% FBS. Primary cells were obtained via written consent under approval of the Dana-Farber Cancer Institute Institutional Review Board. Trypan blue dye exclusion tests, as previously described (Weisberg et al., 2002), were used to quantify PBMCs before plating for the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). These assays were used for the studied proliferation and were performed according to the manufacturer's instructions. Cell viability is reported as a percentage of control (untreated) cells, and error bars indicate the standard deviation for each data point.

[0202] Ub-AMC assay USP7 and mutants were tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors. For this assay, the USP7 catalytic domain WT or mutants were used at concentrations of 250 nM USP7 WT, M407K, M407K / M410S, or Q351S, 125 nM H461A, 600 nM Y514A, and 10 nM M410S. For this assay, USP7 full-length WT and Q351 mutant were used at 50 nM. USP7 mutants were preincubated with various concentrations of inhibitors or DMSO as a control in 50 mM HEPES pH 7.6, 0.5 mM EDTA, 11 μM ovalbumin, and 5 mM DTT. Reactions were incubated at room temperature for 30 minutes before the addition of 2 μM Ubiquitin-AMC (Boston Biochem) substrate. The initial velocity of the reaction was measured by collecting fluorescence data at 1-minute intervals over a 30-minute period using a Clariostar fluorescent plate reader at an excitation wavelength of 345 nm and an emission wavelength of 445 nm. The calculated initial velocity values ​​were plotted against the inhibitor concentration to obtain the IC. 50 The s was determined. All experimental data were plotted using Prism GraphPad. Figure 2A shows the structure-based optimization for USP7 inhibition. Compound A (WO2013 / 030218) shown below was optimized to arrive at compounds 10 and 11.

[0203] [ka] Figures 3A-C show that compound 10 binds to USP7. Figures 4A-D show the binding and dose-responsive inhibition of compounds A and 10. Figures 5A-C show the microsomal stability and USP7 binding ability of compound A and compounds of the present disclosure.

[0204] Table 3: USP7 activity of exemplary compounds in the USP7 assay. ++++ indicates an IC of less than about 0.2 μM 50 +++ indicates IC of approximately 0.2 μM to approximately 1 μM 50 ++ indicates IC of approximately 1 μM to approximately 10 μM 50 + indicates IC above approximately 10 μM50 ND means not disclosed. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14]

[0205] Further exemplary compounds include, but are not limited to, those provided in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0206] Incorporation by Reference All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0207] Equivalent forms While specific embodiments of the subject invention are discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and by reference to the specification, along with variations thereof.

Claims

[Claim 1] The invention as described in the drawings.

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