US009 1750 18B2

(12) United States Patent (10) Patent No.: US 9,175,018 B2 Elliott et al. (45) Date of Patent: Nov. 3, 2015

(54) BORONATE ESTER COMPOUNDS AND (56) References Cited PHARMACEUTICAL COMPOSITIONS THEREOF U.S. PATENT DOCUMENTS 4,499,082 A 2f1985 Shenviet al. (71) Applicant: Millennium Pharmaceuticals, Inc., 5,106,948 A 4, 1992 RE al. Cambridge, MA (US) 5,159,060 A 10, 1992 Kinder et al. 5,169,841 A 12/1992 Kleeman et al. (72) Inventors: Eric L. Elliott, Brighton, MA (US); Abu 5,242,9045,187,157 A 9,2f1993 1993 Kettner et al. J. Ferdous, Winchester, MA (US); 5,250,720 A 10/1993 Kettner et al. Michael J. Kaufman, Lexington, MA 5.492.900 A 2/1996 LaHann (US); Sonja A. Komar, Belmont, MA 3. A AE t tal (US); Debra L. Mazaik, Holliston, MA 5,780,454- ww. A 7/1998 Adamsabeque et et al. al. (US); Quentin J. Mccubbin, Belmont, 5,834,487. A 1 1/1998 Lumet al. MA (US); Phuong M. Nguyen, 5,837,531. A 1 1/1998 Dedieu et al. Cambridge, MA (US); Vaithianathan 5,935,944 A 8, 1999 LaHann Palaniappan,ppan, Andover,s MA (US);s 6,060,4625.990,083. A 1 5/20001/1999 IqbalGalemmo, et al. Jr. et al. Raymond D. Skwierczynski, Andover, 6,066,730 A 5, 2000 Adams et al. MA (US); Nobel T. Truong, Milford, '39. f 29s ST stal MA (US); Csanad M. Varga, Newton, 6,297.217J. W-1 B1 10/2001 Adamsul et et al. al. MA (US); Peter N. Zawaneh, 6,699,835 B2 3/2004 Plamondon et al. Cambridge, MA (US) 6,713,446 B2 3/2004 Gupta 6,781,000 B1 8, 2004 Wang et al. (*) Notice: Subject to any disclaimer, the term of this gigs R: 1 858 Eley patent is extended or adjusted under 35 7,109,323- - w B2 9/2006 Plamondonupta et al. U.S.C. 154(b) by 0 days. 7,223,745 B2 5/2007 Chatterjee et al. 7,442,830 B1 10/2008 Olhava et al. (21) Appl. No.: 14/449,328 7,687,662 B2 3/2010 Olhava et al. 8,003,819 B2 8/2011 Olhava et al. 8,859,504 B2 * 10/2014 Elliott et al...... 514, 19.3 (22) Filed: Aug. 1, 2014 2002/0188100 A1 12/2002 Plamondon et al. (65) Prior Publication Data (Continued) US 2014/0343314 A1 Nov. 20, 2014 FOREIGN PATENT DOCUMENTS

AU 395.1389 A 3, 1990 O O EP 0092999 B1 4f1992 Related U.S. Application Data EP 2 178888 B1 4/2012 (60) Division- - - of application No. 13/667,164, filed O Nov. WOJP WO96,2002145848 1326.6 AA1 5, 20021996 2. 2012, OW Pat. No. 8,859,504, which is a WO WO96, 14857 A1 5, 1996 continuation of application No. 12/485,344, filed on WO WO 98.35691 A1 8/1998 Jun. 16, 2009, now abandoned. WO WO99, 15183 A1 4f1999 WO WO99,30707 A1 6, 1999 (60) Provisional application No. 61/211,499, filed on Mar. WO WOOO,57887 A1 10, 2000 31, 2009, provisional application No. 61/132,244, (Continued) filed on Jun. 17, 2008. OTHER PUBLICATIONS (51) Int. Cl. C07F 5/04 (2006.01) Berge. et al., J. Pharm. Sci., 1977, vol. 66, 1-19. C07F 5/02 (2006.01) (Continued) C07K 5/078 (2006.01) C07K5/06 (2006.01) CD7C 23.3/83 (2006.01) Primary Examiner — Rei-Tsang Shiao A6 IK3I/98 (2006.01) (74) Attorney, Agent, or Firm — Honigman Miller Schwartz A6 IK3 L/454 (2006.01) and Cohn LLP; Lucy X. Yang; Jonathan P. O'Brien A6 IK3I/69 (2006.01) (52) U.S. Cl. (57) ABSTRACT CPC ...... C07F5/04 (2013.01); A61 K31/198 (2013.01); A61 K3I/454 (2013.01); A61 K The present invention provides novel compounds useful as 31/69 (2013.01); C07C233/83 (2013.01); proteasome inhibitors. The invention also provides pharma C07F 5/025 (2013.01); C07K5/06139 ceutical compositions comprising the compounds of the (2013.01); C07K5/0619.1 (2013.01) invention and methods of using the compositions in the treat (58) Field of Classification Search ment of various diseases. USPC ...... 558/288 See application file for complete search history. 40 Claims, 8 Drawing Sheets US 9,175,018 B2 Page 2

(56) References Cited Mullard, A., “Next-generation proteasome blockers promise safer cancer therapy'. Nature Medicine, vol. 18 No. 1, Jan. 2012, p. 7. U.S. PATENT DOCUMENTS Pachnis, V., et al., “The structure and expression of a novel gene activated in early mouse embryogenesis.” The EMBO Journal, Mar. 2004/0171556 A1 9, 2004 Purandare et al. 2005/0107307 A1 5, 2005 Bernadini et al. 1988, vol. 7, No. 3, pp. 673-681. 2005/0282742 A1 12/2005 Plamondon et al. Ruggeri, B., “The Development and Pharmacology of Proteasome 2006, OO84592 A1 4/2006 Boucher Inhibitors for the Management and Treatment of Cancer.” Advances 2006/025274.0 A1 11/2006 Johnson, Jr. et al. in Pharmacology, vol. 57, 2009, pp. 91-135. 2010.0081633 A1 4/2010 Fleming et al. Yoo-Warren, H. et al., “Two regulatory Domains Flank the Mouse 2010/0204180 A1 8, 2010 Olhava et al. H19 Gene'. Molecular and Cellular Biology, vol. 8, No. 11, 1988, pp. 2011/0245203 A1 10/2011 Fleming et al. 4707-4715. 2012/0041196 A1 2/2012 Bernardini Zhang, Y. et al., “Imprinting of human H19: allele-spedfic CpG methylation, loss of the active allele in Wilms tumor, and potential for FOREIGN PATENT DOCUMENTS somatic allele Switching”. Am. J. Hum. Genet., vol. 53, 1993, pp. WO WO 01/02424 A2 1, 2001 113-124. WO O2O5923 1, 2002 Opponent's Notice of Opposition in EP1660507 Opposition (Euro WO WO O2/O591.30 A1 8, 2002 pean Patent Office Date: May 4, 2010). WO WO O2/O591.31 A1 8, 2002 Proprietor's Response to the Notice of Opposition with Amended WO O3O33507 4/2003 Claims in EP1660507 Opposition (European Patent Office Date: Jan. WO WOO3,059898 A2 T 2003 17, 2011). WO 2005O16859 2, 2005 WO WO 2005/021558 A2 3, 2005 Summons to Attend Oral Proceedings with Annex in EP1680507 WO WO 2005/097809 A2 10/2005 Opposition (European Patent Office Date: Apr. 1, 2011). WO 2005111008 11, 2005 Proprietor's Submissions and Amendments in EP1660507 Opposi WO 2006052733 5, 2006 tion (European Patent Office Date: Sep. 7, 2011). WO WO 2007/005991 A1 1, 2007 Opponent's Reply to Proprietor's Sep. 5, 2011 Submission in WO 20070896.18 8, 2007 EP1660507 Opposition (European Patent Office Date: Sep. 19, WO 2009006473 1, 2009 WO WO 2009/020448 A1 2, 2009 2011). WO WO 2010/036357 A1 4/2010 Proprietor's Letters Regarding Oral Proceedings in EP1680507 WO WO 2011/1235O2 A1 10, 2011 Opposition (European Patent Office Date: Sep. 21, 2011). Oral Proceedings Minutes and Results in EP1660507 Opposition OTHER PUBLICATIONS (European Patent Office Date: Nov. 2, 2011). Opponent's Request for Correction of Minutes of Oral Proceedings Cancer (online), Retrieved on Jul. 6, 2007: MedlinePlus, a service of in EP1660507 Opposition (European Patent Office Date: Nov. 10, Medicine and the National Institutes of Health, Retrieved from the 2011). Internet, URL: http://www.nlm.nih.gov/medlineplus/cancer.html, 10 Proprietor's Request Regarding Minutes of Oral Proceedings in pageS. EP1660507 Opposition (European Patent Office Date: Nov. 29, Capitulo 28: Iiotilizacion, Farmacotecnia: teoria y practicatomo 3. 2011). 1982. MX, pp. 1038-1041. Correction of Minutes of Oral Proceedings in EP1660507 Opposition Dick. L.R., et al., “Building on bortezomib: second-generation (European Patent Office Date: Dec. 5, 2011). proteasome inhibitors as anti-cancer therapy'. Elsevier, Drug Dis Opponent's Letter Further to Proprietor's Representative's Letter covery Today, vol. 15, No. 5. Mar. 6, 2010, pp. 243-249. Dated Nov. 29, 2011 in EP1660507 Opposition (European Patent Office Date: Dec. 7, 2011). EP1880607 Opposition. Jan. 11, 2013, Proprietor's Submission, 10 Annex to Opposition Letter Dated Nov. 2, 2011 in EP1660507 Oppo pageS. sition (European Patent Office Date: Dec. 7, 2011). EP2178888 Opposition. Oct. 17, 2011, Summons to Attend Oral Interlocutory Decision in Opposition Proceedings in EP1660507 Proceedings with Annex, 3 pages. Opposition (European Patent Office Date: Dec. 14, 2011). EP2178888 Opposition, Dec. 30, 2011, Annex to Proprietor's Letter Proprietor's Notice of Appeal Against Decision of the Opposition dated Dec. 30, 2011, 3 pages. Division in EP1660507 Opposition (European Patent Office Date: EP2178888 Opposition. Dec. 30, 2011, Proprietor's Submission. 3 Feb. 23, 2012). pageS. Opponent's Notice of Appeal Against Decision of the Opposition EP2178888 Opposition, Apr. 4, 2013, Opponent's Submission, 25 Division in EP1660507 Opposition (European Patent Office Date: pageS. Feb. 23, 2012). Golub, T.R., et al., “Molecular Classification of Cancer: Class Dis Opponent's Statement of Grounds of Appeal in EP1660507 Opposi covery and Class Prediction by Gene Expression Monitoring.” Sci tion (European Patent Office Date: Apr. 23, 2012). ence, vol. 288, pp. 531-537 (Oct. 15, 1999). Proprietor's Submissions and Amendments in EP1680507 Opposi http://goldbookiupac.org/A00228.html. Retrieved from IUPAC, tion (European Patent Office Date: Apr. 24, 2012). Compendium of Chemical Terminology, 2nded. (the "Gold Book”). Opponent's Reply to Proprietor's Grounds of Appeal of Apr. 24, 2012 Compiled by A. D. McNaught and A. Wilkinson, Blackwell Scientific in EP1660507 Opposition (European Patent Office Date: Sep. 12, Publications. Oxford (1997), XML on-line corrected version: http:// 2012). goldbookiupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; Proprietor's Response to Opponent's Grounds of Appeal in updates compiled by A. Jenkins. ISBN 0-9678550-9-8. doi:10.1351/ EP1660507 Opposition (European Patent Office Date: Sep. 12, goldbook, 1 page. 2012). Lala, P.K. et al., “Role of Nitric Oxide in Tumor Progression: Lessons U.S. Office Action Mailed Mar. 7, 2012 in U.S. Appl. No. from Experimental Tumors.” Cancer and Metastasis Reviews, vol. 13/249,738. 17, pp. 91-106 (1998). Response Filed Jul. 9, 2012 to Mar. 7, 2012 Office Action in U.S. Lieberman, H., et al., “Tablet formulation and design.” Pharmaceu Appl. No. 13/249,739. tical Dosage Forms: tablets vol. 1, 1989, US, pp. 91-127. U.S. Office Action (made final) Mailed Sep. 7, 2012 in U.S. Appl. No. Matteson, D.S., et al., "99% Chirally Selective Syntheses via 13/249,738. Pinanediol Boronic Esters: Insect Pheromones, Diols, and an Amino International Search Report dated Jan. 19, 2010 in International Alcohol.” Journal of American Chemical Society, vol. 108, No. 4. Application No. PCT/US2009/005324 which corresponds with U.S. 1986, pp. 810-819. Appl. No. 12/586,650. US 9,175,018 B2 Page 3

(56) References Cited Kinder, David H., et al., “Acylamino boronic acids and dif fluoroborane analogues of amino acids: potent inhibitors of OTHER PUBLICATIONS chymotrypsin and elastase.” Journal of Medicinal Chemistry, vol. 28. No. 12 (1985) pp. 1917-1925. International Search Reportdated Jun. 7, 2011 in International Appli Kisselev, Alexei F., et al., “Proteasome inhibitors: from research tools cation No. PCT/US2011/30455 which corresponds with U.S. Appl. to drug candidates.” Chemistry & Biology, vol. 8, No. 8 (2001) pp. No. 13/075,306. Andersen, Marc W. et al., “E- and Z-Pentenyiboronates, Reagents for 739-758. Simple Diasteroselection on Addition to Aldehydes.” Chemische Korcek, S., et al., “Absolute rate constants for the autoxidation of Berichte, vol. 122 (1989), pp. 1777-1782. organometallic compounds. Part II. Benzylboranes and Armstrong, Terriet al., “Central Nervous SystemToxicity from Can 1-phenylethylboranes,” Journal of the Chemical Society, Perkin cer Treatment.” Current Oncology Reports, vol. 5 (2004), pp. 11-19. Transaction 2. (1972) pp. 242-248. Groll, Michael et al., “Structure of 20S Proteasome from Yeast at Loidl, Gunther, et al., “Bifunctional inhibitors of the trypsin-like 24A Resolution.” Nature, vol. 386 (Apr. 3, 1997), pp. 463-471. activity of eukaryotic proteasomes.” Chemistry & Biology, vol. 6, No. Gross, Erhard et al., “The Peptides: Analysis, Synthesis, Biology.” 4 (1999) pp. 197-204. Protection of Functional Groups in Peptide Synthesis, vol. 3. (Aca Richardson, Paul G. et al., “A phase 2 study of bortezomib in demic Press New York) (1981), pp. 3-88. relapsed, refractory myeloma.” The New England Journal of Medi Harris, Jennifer L. et al., “Substrate Specificity of the Human Protea cine, vol. 348, No. 26 (Jun. 28, 2003) pp. 2609-2617. some.” Chemistry and Biology, vol. 8 (2001), pp. 1131-1141. Snyder, H.R., et al., "Aryl botanic acids. II. Aryl boronic anhydrides Hoffmann, Reinhard W. et al., “Towards an Understanding of Cram? and their amine complexes,” Journal of the American Chemical Soci anti-Cram Selectivity on Addition of Crotylboronates to ety, vol. 80 (Jul 20, 1958) pp. 3611-3615. O-Methylbutyraldehyde.” Chemische Berichte, vol. 123 (1990), pp. Stella, V.J., et al., “Development ofparenteral formulations of experi 2387-2394. mental cytotoxic agents. I. Rhizoxin (NSC-332598).” International Kettner, Charles A. et al., “Inhibition of the Serine Proteases Journal of Pharmaceutics, vol. 43 (1988) pp. 191-199. Leukocyte Elastase, Pancreatic Elastase, Cathepsin G, and Tran, Thuy, et al., “Synthesis and structure-activity relationship of Chymotrypsin by Peptide Boronic Acids.” The Journal of Biological N-acyl-Gly-, N-acyl-Sar- and N-blocked-boroPro Inhibitors of FAP, Chemistry, vol. 259, No. 24 (Dec. 25, 1984), pp. 15106-15114. King, Randall W. et el. “How Proteolysils Drives the Cell Cycle.” DPP4, and POP” Bioorganic & Medicinal Chemistry Letters, vol. 17 Science, vol. 274 (Dec. 6, 1996), pp. 1652-1659. (2007) pp. 1438-1442. Kupperman, Erik et al., “Evaluation of the Proteasome Inhibitor Williams, N. Adeyinka, et al., “The effects of cooling rate on solid MLN9708 in Preclinical Models of Human Cancer, Cancer phase transitions and associated vial breakage occurring in frozen Research, vol. 70, No. 5 (Mar. 1, 2010), pp. 1970-1980. mannitol solutions,” Journal of Parenteral Science & Technology, Palombella, Vito J. et al., “The Ubiquitin-Proteasome Pathway Is vol. 40, No. 4 (Jul.-Aug. 1986) pp. 135-141. Required for Processing the NF-kB1, Precursor Protein and the Acti Williams, N. Adeyinka, et al., “Vial breakage by frozen mannitol vation of NF-kB.” Cell, vol. 78, (Sep. 9. 1994), pp. 773-785. solutions: correlation with thermal characteristics and effect of Protective Groups in Organic Synthesis, 3 edition, edited by Stereoisomerism, additives, and vial configuration.” Journal of Theodora W. Greene and Peter G.M. Wuts, (A Wiley-Interscience Parenteral Science & Technology, vol. 45, No. 2 (Mar.-Apr. 1991) pp. Publication John Wiley & Sons, Inc.) (1999), pp. 531-537. 94-100. Wallace, Richard H. et al., “Preparation and 1-Carbon Homologation Houston, Todd A., et al., “Boric acid catalyzed chemoselective of Boronic Ester Substituted A*-isoxazolines: The 1.3 Dipolar esterification of O-hydroxycarboxylic acids' Organic Letters, vol. 6, Cycloaddition of Nitrile Oxides to Vinyl Boronic Esters.” Tetrahedron No. 5 (2004) pp. 679-681. Letters, vol. 33, No. 46 (1992), pp. 6941-6944. Meilend, Marcel, et al., “Seven-membered ring boronates at trans Zenk, Rolandet al., “Organic Boronic Acids and Boronic Acid Esters diol moieties of carbohydrates.” Tetrahedron Letters, vol. 50 (2009) in Industrial Synthesis.” Chimica Oggi (Chemistry Today) (May pp. 469-472. 2003), pp. 70-73. International Search Report and Written Opinion of the International Lorand, John P. et al., “Polyol complexes and structure of the Searching Authority dated Oct. 5, 2009 issued in International Appli benzeneboronate ion.” Journal of Organic Chemistry, vol. 24 (1958) cation No. PCT/US09/003602, which corresponds to U.S. Appl. No. pp. 769-774. 12/485,344. Scheibe, Edmund, “The borocitrates and their preparation.” The International Search Report and Written Opinion of the International Pharmaceutical Journal and Transactions, Third Series, No. 542 Searching Authority dated Dec. 3, 2007 issued in International Appli (Nov. 18, 1880) p. 389. cation No. PCT/US07/017.440, which corresponds to U.S. Appl. No. Prasad Sarju, et al., “Studies on the formation of some borocitrates' 1 1/890,412. Journal of the Indian Chemistry Society, vol. 44, No. 3 (1967) pp. Ciechanover, Aaron, “The ubiquitin-proteasome proteolytic path 219-220. way,” Cell, vol. 79 (Oct. 7, 1994) pp. 13-21. Bartusek, M., et al., “Boronchelates with citrate.” Scripta-Chemistry, Gardner, Robert C. et al., “Characterization of peptidylboronic acid vol. 21 (1991) pp. 63-67. Inhibitors of mammalian 20 S and 26 Sproteasomes and their Inhi Gray, Charles W. “Boronic acid receptors for bition of proteasomes in cultured cells.” Biochemistry Journal, vol. O-hydroxycarboxylates: high affinity of Shinkai's glucose receptor 346 (2000) pp. 447-454. for tartrate.” Journal of Organic Chemistry, vol. 67, No. 15 (2002)pp. Gennaro, Algonso R. (editor), “Remington: The Science and practice 5426-5428. of pharmacy,” 20" Edition, Chapter 42, Lippincott Williams & Van Duin, M., et al., “Studies onborate esters I.” Tetrahedron, vol. 40, Wilkins (publishers), Baltimore, MD (2000) pp. 802-803. No. 15 (1984) pp. 2901-2911. Kataoka, Kazunori, et al., “Totally synthetic polymer gels responding Van Duin, M., et al., “Studies on borate esters II.” Tetrahedron, vol. to external glucose concentration: their preparation and application 41, No. 16 (1986) pp. 3411-3421. to on-off regulation of insulin release.” Journal of the American Communication pursuant to Article 94(3) EPC (Examination Report) Chemical Society, vol. 120 (1998) pp. 12694-12695. dated Oct. 25, 2011 from corresponding European patent application Kibbe, Arthur H. (editor), “Handbook of Pharmaceutical O9 767 0509-2117. Excipients.”3 Edition, American Pharmaceutical Association (pub lishers), Washington, D.C. (2000) pp. 324-328. * cited by examiner U.S. Patent Nov. 3, 2015 Sheet 1 of 8 US 9,175,018 B2

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C s r s (3/M) AOI: JeeH US 9, 175,018 B2 1. 2 BORONATEESTER COMPOUNDS AND core of the 26S proteasome is the 20S proteasome. McCor PHARMACEUTICAL COMPOSITIONS mack et al., Biochemistry 37:7792 (1998), teaches that a THEREOF variety of peptide Substrates, including Suc-Leu-Leu-Val Tyr-AMC, Z-Leu-Leu-Arg-AMC, and Z-Leu-Leu-Glu-2NA, PRIORITY wherein Suc is N-succinyl, AMC is 7-amino-4-methylcou marin, and 2NA is 2-naphthylamine, are cleaved by the 20S This application is a divisional of U.S. Utility patent appli proteasome. cation Ser. No. 13/667,164, filed Nov. 2, 2012, which is a Proteasome inhibition represents an important new strat continuation of U.S. utility patent application Ser. No. egy in cancer treatment. King et al., Science 274: 1652-1659 12/485,344, filed Jun. 16, 2009, which claims priority from 10 (1996), describes an essential role for the ubiquitin-protea U.S. Provisional Patent Application Ser. No. 61/132,244. Some pathway in regulating cell cycle, neoplastic growth and filed Jun. 17, 2008, and U.S. Provisional Patent Application metastasis. The authors teach that a number of key regulatory Ser. No. 61/211,499, filed Mar. 31, 2009; both which are proteins, including, cyclins, and the cyclin-dependent kinases hereby incorporated by reference in their entirety. p21 and p27', are temporally degraded during the cell 15 cycle by the ubiquitin-proteasome pathway. The ordered deg FIELD OF THE INVENTION radation of these proteins is required for the cell to progress through the cell cycle and to undergo mitosis. The present invention relates to boronate ester compounds Furthermore, the ubiquitin-proteasome pathway is useful as proteasome inhibitors. The invention also provides required for transcriptional regulation. Palombella et al., Cell, pharmaceutical compositions comprising the compounds of 78:773 (1994), teaches that the activation of the transcription the invention, and methods of using the compositions in the factor NF-kB is regulated by proteasome-mediated degrada treatment of various diseases. tion of the inhibitor protein IKB. In turn, NF-kB plays a central role in the regulation of genes involved in the immune BACKGROUND OF THE INVENTION and inflammatory responses. Readet al., Immunity 2:493-506 25 (1995), teaches that the ubiquitin-proteasome pathway is Boronic acid and boronate ester compounds display a vari required for expression of cell adhesion molecules, such as ety of pharmaceutically useful biological activities. Shenviet E-selectin, ICAM-1, and VCAM-1. Zetter, Seminars in Can al., U.S. Pat. No. 4,499,082 (1985), discloses that peptide cer Biology 4:219–229 (1993), teaches that cell adhesion mol boronic acids are inhibitors of certain proteolytic enzymes. ecules are involved in tumor metastasis and angiogenesis in Kettner and Shenvi, U.S. Pat. No. 5,187,157 (1993), U.S. Pat. 30 Vivo, by directing the adhesion and extravastation of tumor No. 5,242,904 (1993), and U.S. Pat. No. 5,250,720 (1993), cells to and from the vasculature to distant tissue sites within describe a class of peptide boronic acids that inhibit trypsin the body. Moreover, Beg and Baltimore, Science 274:782 like proteases. Kleeman et al., U.S. Pat. No. 5,169,841 (1996), teaches that NF-kB is an anti-apoptotic controlling (1992), discloses N-terminally modified peptide boronic factor, and inhibition of NF-kB activation makes cells more acids that inhibit the action of renin. Kinder et al., U.S. Pat. 35 sensitive to environmental stress and cytotoxic agents. No. 5,106,948 (1992), discloses that certain boronic acid The proteasome inhibitor VELCADE(R) (bortezomib: N-2- compounds inhibit the growth of cancer cells. Magde et al., pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid) is WO 04/022070 discloses peptide boronic acid compounds the first proteasome inhibitor to achieve regulatory approval. that inhibit thrombin. Boucher, U.S. Patent Application Pub. Mitsiades et al., Current Drug Targets, 7:1341 (2006), No. 20060084592 discloses various basic addition salts of 40 reviews the clinical studies leading to the approval of bort peptide boronic acid compounds. Bachovchin et al., WO eZomib for the treatment of multiple myeloma patients who 07/005991, discloses peptide boronic acid compounds that have received at least one prior therapy. Fisher et al., J. Clin. inhibit fibroblast activating protein. Oncol., 30:4867 (2006), describes an international multi-cen Boronic acid and ester compounds hold particular promise ter Phase II study confirming the activity of bortezomib in as inhibitors of the proteasome, a multicatalytic protease 45 patients with relapsed or refractory mantle cell lymphoma. responsible for the majority of intracellular protein turnover. Ishii et al., Anti-Cancer Agents in Medicinal Chemistry, 7:359 Adams et al., U.S. Pat. No. 5,780,454 (1998), describes pep (2007), and Roccaro et al., Curr: Pharm. Biotech., 7:1341 tide boronic ester and acid compounds useful as proteasome (2006), discuss a number of molecular mechanisms that may inhibitors. The reference also describes the use of boronic contribute to the antitumor activities of bortezomib. ester and acid compounds to reduce the rate of muscle protein 50 Structural analysis reported by Voges et al., Annu. Rev. degradation, to reduce the activity of NF-KB in a cell, to Biochem., 68: 1015 (1999) reveals that the 20S proteasome reduce the rate of degradation of p53 protein in a cell, to comprises 28 Subunits, with the catalytic subunits f1, f2, and inhibit cyclin degradation in a cell, to inhibit the growth of a B5 being responsible for peptidylglutamyl, tryptic, and chy cancer cell, and to inhibit NF-kB dependent cell adhesion. motryptic peptidase activity, respectively. Rivett et al., Curr: Furetet al., WO 02/096933, Chatterjee et al., WO 05/016859, 55 Protein Pept. Sci., 5:153 (2004) discloses that when the pro and Bernadini et al., WO 05/021558 and WO 06/08660, dis teasome is exposed to certain cytokines, including IFN-Y and close additional boronic ester and acid compounds that are TNF-C., the B1, B2, and B5 subunits are replaced with alter reported to have proteasome inhibitory activity. nate catalytic Subunits, Bli, 32i, and B5i, to form a variant Ciechanover, Cell, 79:13-21 (1994), discloses that the pro form of the proteasome known as the immunoproteasome. teasome is the proteolytic component of the ubiquitin-protea 60 Orlowski, Hematology (Am. Soc. Hematol. Educ. Pro Some pathway, in which proteins are targeted for degradation gram) 220 (2005), discloses that the immunoproteasome also by conjugation to multiple molecules of ubiquitin. Ciecha is expressed constitutively in some cells derived from nover also discloses that the ubiquitin-proteasome pathway hematopoietic precursors. The author Suggests that inhibitors plays a key role in a variety of important physiological pro specific for the immunoproteasome may allow for targeted cesses. Rivett et al., Biochem. J. 291:1 (1993) discloses that 65 therapy against cancers arising from hematologic origins, the proteasome displays tryptic-, chymotryptic-, and pepti thereby potentially sparing normal tissues, such as gas dylglutamyl-peptidase activities. Constituting the catalytic trointestinal and neurological tissues, from side effects. US 9, 175,018 B2 3 Unfortunately, boronic acid compounds are relatively dif ficult to obtain in analytically pure form. For example, Snyder (I) et al., J. Am. Chem. Soc. 80: 3611 (1958), teaches that aryl O Ral Zl boronic acid compounds readily form cyclic trimeric anhy S. drides under dehydrating conditions. Also, alkylboronic acids 5 P N N1 NZ2 and their boroxines are often air-sensitive. Korcek et al., J. H Chem. Soc., Perkin Trans. 2 242 (1972), teaches that butyl Ra2 A. O boronic acid is readily oxidized by air to generate 1-butanol and boric acid. These difficulties limit the pharmaceutical 10 or a pharmaceutically acceptable salt or thereof, wherein: utility of boronic acid compounds, complicating the charac A is 0, 1, or 2: terization of pharmaceutical agents comprising boronic acid P is hydrogen or an amino-group-blocking moiety; compounds and limiting their shelf-life. R" is hydrogen, C aliphatic, C. fluoroaliphatic, Plamondon et al., WO 02/059131 discloses stable, phar -(CH2), CH2 R. —(CH), CH NHC maceutically acceptable compositions prepared from boronic 15 (—NR)NH Y, —(CH), CH CONCR), acid compounds and Sugars. There remains a need for addi —(CH), CH, N(R)CON(R), -(CH), CH tional stable formulations of boronic acid compounds. (R)N(R), (CH), CH(R) OR, or —(CH), CH(R) SR; BRIEF DESCRIPTION OF THE DRAWINGS R" is hydrogen, Ce aliphatic, Ce fluoroaliphatic, -(CH2), CH2 R. —(CH), CH NHC (—NR)NH Y, —(CH), CH CONCR), FIG. 1 is a powder X-ray diffractogram of 4-(R.S)-(car —(CH), CH, N(R)CON(R), -(CH), CH boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta (R)N(R), (CH), CH(R) OR, or mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-car —(CH), CH(R) SR; boxylic acid (I-1) Form 1. 25 each R' independently is hydrogen, Caliphatic, C. FIG. 2 is a differential scanning calorimetry (DSC)/ther fluoroaliphatic, (CH), CH. R. (CH2), mal gravimetric analysis (TGA) profile for 4-(R.S)-(car boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta CH NHC(=NR)NH Y, -(CH), CH, CON mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4- (R), -(CH), CH, N(R)CON(R), carboxylic acid (I-1) Form 1. 30 —(CH), CH(R)N(R), -(CH), CH(R)- FIG. 3 is a powder X-ray diffractogram of 4-(R.S)-(car OR, or -(CH), CH(R) SR; boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta each R” independently is a substituted or unsubstituted mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-car mono- or bicyclic ring system; boxylic acid (I-1) Form 2. each R independently is hydrogen or a substituted or 35 unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl FIG. 4 is a differential scanning calorimetry (DSC)/ther group; or two R' on the same nitrogen atom, taken mal gravimetric analysis (TGA) profile for 4-(R.S)-(car together with the nitrogen atom, form a Substituted or boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta unsubstituted 4- to 8-membered heterocyclyl ring hav mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4- ing, in addition to the nitrogen atom, 0-2 ring heteroat carboxylic acid (I-1) Form 2. 40 oms independently selected from the group consisting of FIG. 5 is a powder X-ray diffractogram of 2,5-dichloro-N- N, O, and S; 2-({(1R)-3-methyl-1-(4S)-4-methyl-5-oxo-1,3,2-diox each R independently is hydrogen or a substituted or aborolan-2-ylbutylamino)-2-oxoethylbenzamide (I-7). unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl FIG. 6 is a powder X-ray diffractogram of 2,5-dichloro-N- group; (2-(1R)-3-methyl-1-(4-oxo-4H-1,3,2-benzodioxaborinin 45 each R" independently is hydrogen or a substituted or 2-yl)butylamino-2-oxoethyl)benzamide (I-13). unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl FIG. 7 is a powder X-ray diffractogram of 4-(R.S)-(car group; boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta each R' independently is hydrogen or a substituted or mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-car unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl 50 group; boxylic acid (I-1) Form 2. each R' independently is a substituted or unsubstituted FIG. 8 is a differential scanning calorimetry (DSC) profile aliphatic, aryl, or heteroaryl group; of 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichloroben Y is hydrogen, —CN, or—NO; Zamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori m is 0, 1, or 2; and nane-4-carboxylic acid (I-1) Form 2. 55 Z" and Z together form a moiety derived from an alpha hydroxy carboxylic acid, wherein the atom attached to boron in each case is an oxygen atom; or Z' and Z DESCRIPTION OF THE INVENTION together form a moiety derived from a beta-hydroxy carboxylic acid, wherein the atom attached to boron in The present invention provides novel boronate ester com 60 each case is an oxygen atom. pounds and stable pharmaceutically acceptable compositions In another aspect, the present invention provides pharma comprising them. These compounds and compositions are ceutical compositions comprising the compound of formula useful for inhibiting proteasome activity in vitro and in vivo, (I), or a crystalline form thereof, and additional excipients and are especially useful for the treatment of various cell described herein, suitable for the production of an oral phar proliferative diseases. 65 maceutical dosage form. In one aspect, the invention provides compounds of the In another aspect, the invention provides a pharmaceutical general formula (I): composition comprising the compound of formula (I), or a US 9, 175,018 B2 5 6 crystalline form thereof, and additional excipients described wherein the aliphatic ring system is optionally substituted. In herein, suitable for the production of a lyophilized powder Some embodiments, the cycloaliphatic is a monocyclic pharmaceutical dosage form. hydrocarbon having 3-8 or 3-6 ring carbon atoms. Nonlimit In another aspect, the invention provides a pharmaceutical ing examples include cyclopropyl, cyclobutyl, cyclopentyl, composition comprising the compound of formula (I), or a cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclo crystalline form thereof, and additional excipients described heptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In herein, Suitable for the production of a liquid pharmaceutical Some embodiments, the cycloaliphatic is a bridged or fused dosage form. bicyclic hydrocarbon having 6-12, 6-10, or 6-8 ring carbon In another aspect, the invention provides a pharmaceutical atoms, wherein any individual ring in the bicyclic ring system composition, comprising the compound of formula (I), or a 10 has 3-8 members. crystalline form thereof, a filler, and optionally a lubricant. In some embodiments, two adjacent Substituents on the In another aspect, the invention provides a pharmaceutical cycloaliphatic ring, taken together with the intervening ring composition, comprising the compound of formula (I), or a atoms, forman optionally substituted fused 5- to 6-membered crystalline form thereof, a filler, optionally a lubricant, aromatic or 3- to 8-membered non-aromatic ring having 0-3 optionally a flow-aid, and optionally a buffer. 15 ring heteroatoms selected from the group consisting of O. N. In another aspect, the invention provides a pharmaceutical and S. Thus, the term “cycloaliphatic' includes aliphatic rings composition, comprising the compound of formula (I), or a that are fused to one or more aryl, heteroaryl, or heterocyclyl crystalline form thereof, a bulking agent, and a buffer. rings. Nonlimiting examples include indanyl, 5,6,7,8-tet In another aspect, the invention provides processes for the rahydroquinoxalinyl, decahydronaphthyl, or tetrahydronaph production of the pharmaceutical compositions of the inven thyl, where the radical or point of attachment is on the ali tion. phatic ring. In another aspect, the invention provides methods for the The terms “aryland “ar-”, used alone or as part of a larger use of the pharmaceutical compositions of the invention, for moiety, e.g., “aralkyl”, “aralkoxy’, or “aryloxyalkyl, refer to treating a patient having, or at risk of developing or experi a C to C aromatic hydrocarbon, comprising one to three encing a recurrence of a proteasome-mediated disorder. 25 rings, each of which is optionally substituted. Preferably, the In another aspect, the invention provides methods for the aryl group is a Co aryl group. Aryl groups include, without use of the pharmaceutical compositions of the invention for limitation, phenyl, naphthyl, and anthracenyl. In some the treatment of cancer. embodiments, two adjacent Substituents on the aryl ring, taken together with the intervening ring atoms, form an DEFINITIONS 30 optionally substituted fused 5- to 6-membered aromatic or 4 to 8-membered non-aromatic ring having 0-3 ring heteroat Unless otherwise explicitly stated, the term “proteasome' oms selected from the group consisting of O, N, and S. Thus, is intended to refer to both constitutive proteasome and the term “aryl', as used herein, includes groups in which an immunoproteasome. aryl ring is fused to one or more heteroaryl, cycloaliphatic, or The term “aliphatic' or “aliphatic group', as used herein, 35 heterocyclyl rings, where the radical or point of attachment is means a Substituted or unsubstituted Straight-chain, on the aromatic ring. Nonlimiting examples of Such fused branched, or cyclic C2 hydrocarbon, which is completely ring systems include indolyl, isoindolyl, benzothienyl, ben saturated or which contains one or more units of unsaturation, Zofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benz but which is not aromatic. For example, suitable aliphatic thiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, groups include Substituted or unsubstituted linear, branched 40 quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, or cyclic alkyl, alkenyl, or alkynyl groups and hybrids phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahy thereof. Such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cy droisoquinolinyl, fluorenyl, indanyl, phenanthridinyl, tet cloalkyl)alkenyl. In various embodiments, the aliphatic group rahydronaphthyl, indolinyl, phenoxazinyl, benzodioxanyl. has 1 to 12, 1 to 8, 1 to 6, 1 to 4, or 1 to 3 carbons. and benzodioxolyl. An aryl group may be mono-, bi-, tri-, or The terms “alkyl”, “alkenyl', and “alkynyl', used alone or 45 polycyclic, preferably mono-, bi-, or tricyclic, more prefer as part of a larger moiety, refer to a straight or branched chain ably mono- or bicyclic. The term “aryl” may be used inter aliphatic group having from 1 to 12 carbon atoms. For pur changeably with the terms “aryl group”, “aryl moiety', and poses of the present invention, the term “alkyl will be used “aryl ring”. when the carbon atom attaching the aliphatic group to the rest An “aralkyl or “arylalkyl group comprises an aryl group of the molecule is a saturated carbonatom. However, an alkyl 50 covalently attached to an alkyl group, either of which inde group may include unsaturation at other carbon atoms. Thus, pendently is optionally substituted. Preferably, the aralkyl alkyl groups include, without limitation, methyl, ethyl, pro group is Co-o aryl(C-6)alkyl, Co-o aryl(C-4 alkyl, or Co-o pyl, allyl, propargyl, butyl, pentyl, and hexyl. aryl(C)alkyl, including, without limitation, benzyl, phen For purposes of the present invention, the term “alkenyl ethyl, and naphthylmethyl. will be used when the carbon atom attaching the aliphatic 55 The terms "heteroaryl' and "heteroar-”, used alone or as group to the rest of the molecule forms part of a carbon part of a larger moiety, e.g., heteroaralkyl, or "het carbon double bond. Alkenyl groups include, without limita eroaralkoxy’, refer to groups having 5 to 14 ring atoms, tion, vinyl, 1-propenyl, 1-butenyl, 1-pentenyl, and 1-hexenyl. preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 Ju For purposes of the present invention, the term “alkynyl electrons shared in a cyclic array; and having, in addition to will be used when the carbon atom attaching the aliphatic 60 carbon atoms, from one to four heteroatoms. The term "het group to the rest of the molecule forms part of a carbon eroatom” refers to nitrogen, oxygen, or Sulfur, and includes carbon triple bond. Alkynyl groups include, without limita any oxidized form of nitrogen or Sulfur, and any quaternized tion, ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, and 1-hexy form of a basic nitrogen. Thus, when used in reference to a nyl. ring atom of a heteroaryl, the term "nitrogen' includes an The term "cycloaliphatic', used alone or as part of a larger 65 oxidized nitrogen (as in pyridine N-oxide). Certain nitrogen moiety, refers to a saturated or partially unsaturated cyclic atoms of 5-membered heteroaryl groups also are Substitut aliphatic ring system having from 3 to about 14 members, able, as further defined below. Heteroaryl groups include, US 9, 175,018 B2 7 8 without limitation, radicals derived from thiophene, furan, In some embodiments, two adjacent Substituents on a het pyrrole, imidazole, pyrazole, triazole, tetrazole, oxazole, erocyclic ring, taken together with the intervening ring atoms, isoxazole, oxadiazole, thiazole, isothiazole, thiadiazole, pyri form an optionally substituted fused 5- to 6-membered aro dine, pyridazine, pyrimidine, pyrazine, indolizine, naphthy matic or 3- to 8-membered non-aromatic ring having 0-3 ring ridine, pteridine, pyrrolopyridine, imidazopyridine, oxazol heteroatoms selected from the group consisting of O, N, and opyridine, thiazolopyridine, triazolopyridine, S. Thus, the terms "heterocycle”, “heterocyclyl, "heterocy pyrrolopyrimidine, purine, and triazolopyrimidine. As used clyl ring, "heterocyclic group”, “heterocyclic moiety', and herein, the phrase “radical derived from means a monovalent "heterocyclic radical', are used interchangeably herein, and radical produced by removal of a hydrogen radical from the include groups in which a heterocyclyl ring is fused to one or 10 more aryl, heteroaryl, or cycloaliphatic rings, such as indoli parent heteroaromatic ring system. The radical (i.e., the point nyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydro of attachment of the heteroaryl to the rest of the molecule) quinolinyl, where the radical or point of attachment is on the may be created at any Substitutable position on any ring of the heterocyclyl ring. A heterocyclyl group may be mono-, bi-, parent heteroaryl ring system. tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more In some embodiments, two adjacent Substituents on the 15 preferably mono- or bicyclic. The term "heterocyclylalkyl heteroaryl, taken together with the intervening ring atoms, refers to an alkyl group substituted by a heterocyclyl, wherein form an optionally substituted fused 5- to 6-membered aro the alkyl and heterocyclyl portions independently are option matic or 4- to 8-membered non-aromatic ring having 0-3 ring ally substituted. heteroatoms selected from the group consisting of O, N, and As used herein, the term “partially unsaturated’ refers to a S. Thus, the terms "heteroaryl' and "heteroar-, as used ring moiety that includes at least one double or triple bond herein, also include groups in which a heteroaromatic ring is between ring atoms. The term “partially unsaturated' is fused to one or more aryl, cycloaliphatic, or heterocyclyl intended to encompass rings having multiple sites of unsat rings, where the radical or point of attachment is on the uration, but is not intended to include aryl or heteroaryl moi heteroaromatic ring. Nonlimiting examples include indolyl, eties, as herein defined. isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, 25 The terms “haloaliphatic”, “haloalkyl, "haloalkenyl and indazolyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, “haloalkoxy' refer to an aliphatic, alkyl, alkenyl or alkoxy quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, group, as the case may be, which is substituted with one or quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phena more halogen atoms. As used herein, the term "halogen' or Zinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, “halo' means F, Cl, Br, or I. The term “fluoroaliphatic” refers tetrahydroisoquinolinyl, and pyrido2.3-b-1,4-oxazin-3 30 to a haloaliphatic wherein the halogen is fluoro, including (4H)-one. A heteroaryl group may be mono-, bi-, tri-, or perfluorinated aliphatic groups. Examples of fluoroaliphatic polycyclic, preferably mono-, bi-, or tricyclic, more prefer groups include, without limitation, fluoromethyl, difluorom ably mono- or bicyclic. The term "heteroaryl may be used ethyl, trifluoromethyl 2-fluoroethyl, 2.2.2-trifluoroethyl, 1.1, interchangeably with the terms "heteroaryl ring, or "het 2-trifluoroethyl, 1.2.2-trifluoroethyl, and pentafluoroethyl. eroaryl group', any of which terms include rings that are 35 The term “linker group' or “linker” means an organic optionally substituted. The term "heteroaralkyl refers to an moiety that connects two parts of a compound. Linkers typi alkyl group substituted by a heteroaryl, wherein the alkyland cally comprise an atom Such as oxygen or Sulfur, a unit Such heteroaryl portions independently are optionally Substituted. as NH , —CH2—, —C(O)— —C(O)NH , or a chain As used herein, the terms 'aromatic ring and 'aromatic of atoms, such as an alkylene chain. The molecular mass of a ring system” refer to an optionally Substituted mono-, bi-, or 40 linker is typically in the range of about 14 to 200, preferably tricyclic group having 0-6, preferably 0-4 ring heteroatoms, in the range of 14 to 96 with a length of up to about six atoms. and having 6, 10, or 14 JC electrons shared in a cyclic array. In some embodiments, the linker is a C- alkylene chain. Thus, the terms 'aromatic ring and 'aromatic ring system” The term “alkylene' refers to a bivalent alkyl group. An encompass both aryl and heteroaryl groups. “alkylene chain” is a polymethylene group, i.e., -(CH2), , As used herein, the terms "heterocycle”, “heterocyclyl, 45 whereiny is a positive integer, preferably from 1 to 6, from 1 "heterocyclic radical, and "heterocyclic ring are used inter to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted changeably and refer to a stable 3- to 7-membered monocy alkylene chain is a polymethylene group in which one or more clic, or to a fused 7- to 10-membered or bridged 6- to methylene hydrogen atoms is replaced with a Substituent. 10-membered bicyclic heterocyclic moiety that is either satu Suitable substituents include those described below for a rated or partially unsaturated, and having, in addition to car 50 Substituted aliphatic group. An alkylene chain also may be bonatoms, one or more, preferably one to four, heteroatoms, Substituted at one or more positions with an aliphatic group or as defined above. When used in reference to a ring atom of a a Substituted aliphatic group. heterocycle, the term "nitrogen' includes a substituted nitro An alkylene chain also can be optionally interrupted by a gen. As an example, in a heterocyclyl ring having 1-3 het functional group. An alkylene chain is “interrupted by a eroatoms selected from the group consisting of oxygen, Sulfur 55 functional group when an internal methylene unit is replaced or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H with the functional group. Examples of suitable “interrupting pyrrolyl), NH (as in pyrrolidinyl), or “NR (as in N-substituted functional groups' include —C(R)=C(R*)— —C=C pyrrolidinyl). A heterocyclic ring can be attached to its pen dant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally 60 Substituted. Examples of Such saturated or partially unsatur ated heterocyclic radicals include, without limitation, tet rahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolido nyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, 65 piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, —N(R) N(R')—. Each R" independently is hydrogen or thiazepinyl, morpholinyl, and quinuclidinyl. an optionally Substituted aliphatic, aryl, heteroaryl, or hetero US 9, 175,018 B2 cyclyl group, or two R' on the same nitrogen atom, taken together with the nitrogen atom, form a 5-8 membered aro matic or non-aromatic ring having, in addition to the nitrogen -P(O)(NR) N(R"), wherein R, R", and R* are as atom, 0-2 ring heteroatoms selected from the group consist defined above, or two adjacent Substituents, taken together ing of N, O, and S. Each R* independently is hydrogen or an with their intervening atoms, form a 5-6 membered unsatur optionally substituted aliphatic, aryl, heteroaryl, or heterocy ated or partially unsaturated ring having 0-3 ring atoms clyl group. Each R- independently is an optionally Substi selected from the group consisting of N, O, and S. tuted aliphatic, aryl, or heteroaryl group. An aliphatic group or a non-aromatic heterocyclic ring Examples of C alkylene chains that have been “inter may be substituted with one or more substituents. Examples rupted with —O— include —CHOCH2—, —CHO 10 (CH) , —CH2O(CH) , —CH2O(CH) , of suitable substituents on the saturated carbon of an aliphatic —(CH2)2OCH2—, —(CH2)2O(CH) , —(CH2)2O group or of a non-aromatic heterocyclic ring include, without (CH2). , —(CH2)5O(CH)— —(CH2)5O(CH) , and limitation, those listed above for the unsaturated carbon of an —(CH)O(CH)—. Other examples of alkylene chains that aryl or heteroaryl group and the following: —O. —S. are “interrupted with functional groups include 15 =C(R*) =N N(R*) =N OR*, N. NHC(O)R*, —CHZ*CH , —CH2*(CH) , —CH-Z*(CH) , =N NHCOR, =N. NHSOR, or =N R*, where —CH-Z*(CH) , —(CH)Z*CH , —(CH)Z* each R* and R is as defined above. (CH) , —(CH2)2(CH) , —(CH)Z*(CH)—, Suitable substituents on a substitutable nitrogen atom of a —(CH2)—Z*(CH) , and —(CH2)4Z*(CH2)—, wherein heteroaryl or non-aromatic heterocyclic ring include —R*, Z* is one of the “interrupting functional groups listed above. N(R*), C(O)R*, COR*, C(O) C(O)R* = C(O) One of ordinary skill in the art will recognize that when an CHC(O)R*, -SOR*, -SON(R*), C(=S)N(R*), alkylene chain having an interruption is attached to a func - C(-NH) N(R*), and - NR*SOR*; wherein each R* tional group, certain combinations would not be sufficiently is as defined above. A ring nitrogen atom of a heteroaryl or stable for pharmaceutical use. Only stable or chemically fea non-aromatic heterocyclic ring also may be oxidized to form sible compounds are within the scope of the present inven 25 the corresponding N-hydroxy or N-oxide compound. A non tion. A stable or chemically feasible compound is one which limiting example of such a heteroaryl having an oxidized ring maintains its integrity long enough to be useful for therapeu nitrogen atom is N-oxidopyridyl. tic or prophylactic administration to a patient. Preferably, the The term “about is used herein to mean approximately, in chemical structure is not substantially altered when kept at a the region of roughly, or around. When the term “about is temperature below -70° C., below -50° C., below -20°C., 30 used in conjunction with a numerical range, it modifies that below 0°C., or below 20° C., in the absence of moisture or range by extending the boundaries above and below the other chemically reactive conditions for at least a week. numerical values set forth. In general, the term “about” is The term “substituted, as used herein, means that a hydro used herein to modify a numerical value above and below the gen radical of the designated moiety is replaced with the stated value by a variance of 10%. radical of a specified substituent, provided that the substitu 35 As used herein, the term “comprises' means “includes, but tion results in a stable or chemically feasible compound. The is not limited to.” term “substitutable', when used in reference to a designated It will be apparent to one skilled in the art that certain atom, means that attached to the atom is a hydrogen radical, compounds of this invention may exist in tautomeric forms, which can be replaced with the radical of a suitable substitu all such tautomeric forms of the compounds being within the ent. 40 scope of the invention. Unless otherwise stated, structures The phrase “one or more substituents', as used herein, depicted herein are also meant to include all geometric (or refers to a number of substituents that equals from one to the conformational) isomers, i.e., (2) and (E) double bond iso maximum number of Substituents possible based on the num mers and (Z) and (E) conformational isomers, as well as all ber of available bonding sites, provided that the above con stereochemical forms of the structure; i.e., the R and S con ditions of stability and chemical feasibility are met. Unless 45 figurations for each asymmetric center. Therefore, single Ste otherwise indicated, an optionally substituted group may reochemical isomers as well as enantiomeric and diastereo have a Substituent at each Substitutable position of the group, meric mixtures of the present compounds are within the scope and the substituents may be either the same or different. of the invention. When a mixture is enriched in one stereoi As used herein, the terms “independently” or “indepen Somer relative to another stereoisomer, the mixture may con dently selected” means that the same or different values may 50 tain, for example, an enantiomeric excess of at least 50%, be selected for multiple instances of a given variable in a 75%, 90%, 99%, or 99.5%. single compound. Unless otherwise stated, structures depicted herein are also An aryl (including the aryl moiety in aralkyl, aralkoxy, meant to include compounds which differ only in the pres aryloxyalkyl and the like) or heteroaryl (including the het ence of one or more isotopically enriched atoms. For eroaryl moiety in heteroaralkyl and heteroaralkoxy and the 55 example, compounds having the present structure except for like) group may contain one or more Substituents. Examples the replacementofahydrogenatom by a deuterium or tritium, of Suitable Substituents on the unsaturated carbon atom of an or the replacement of a carbonatom by a C- or 'C-enriched aryl or heteroaryl group include -halo. —NO. —CN, R*, carbon are within the scope of the invention. C(R*)=C(R*), -C=C R*, OR*, SR, S(O) As used herein, the term “seeding is used to refer to the R, SOR, SOR*, SON(R), N(R), NRC 60 addition of a crystalline material to initiate crystallization or (O)R*, NRC(O)N(R), N(R) C(=NR) N(R'), recrystallization. N(R')C(-NR) R, NR"COR, NRSOR, When a compound crystallizes from a solution or slurry, it NRSON(R), O C(O)R*, O COR*, OC(O) may crystallize with different spatial lattice arrangements, a N(R), C(O)R*, COR*, C(O) C(O)R*, C(O)N property referred to as “polymorphism. Each of the crystal (R), C(O)N(R') OR*, C(O)N(R)C(=NR) N 65 forms is a “polymorph.” While polymorphs of a given sub (R), N(R')C(-NR") N(R) C(O)R*, stance have the same chemical composition, they may differ C(=NR) N(R), C(=NR) OR*, N(R) N from each other with respect to one or more physical proper US 9, 175,018 B2 11 12 ties, such as Solubility and dissociation, true density, melting butylboronic acid are used interchangeably. The compound point, crystal shape, compaction behavior, flow properties, of formula (VIII-1) is disclosed in U.S. Pat. No. 7,442,830 and/or solid state stability. and WO 09/020448. As used herein, the term "solvate or solvated” means a As used herein, the terms “compound of formula (I-15). physical association of a compound with one or more solvent “compound (I-15) and "(I-15) are used interchangeably molecules. This physical association includes hydrogen and are used to refer to the citrate ester of the compound bonding. In certain instances the solvate will be capable of (VIII-15), and the compound produced in Example 15 of the isolation, for example when one or more solvent molecules Examples below. are incorporated in the crystal lattice of the crystalline solid. As used herein, the term “anhydride' used in reference to a “Solvate or solvated encompasses both solution-phase and 10 boronic acid such as the compound of formula (VIII), refers isolable solvates. Representative solvates include, for to a chemical compound formed by combination of two or example, hydrates, ethanolates, or methanolates. The physi more molecules of a boronic acid compound, with loss of one cal properties of a solvate typically differ from other solvates, or more water molecules. When mixed with water, the and from unsolvated forms of the compound. Because the 15 boronic acid anhydride compound is hydrated to release the chemical composition also differs between solvates these free boronic acid compound. In various embodiments, the forms are referred to as “pseudo-polymorphs. boronic acid anhydride can comprise two, three, four, or more As used herein, the term “hydrate” is a solvate wherein the boronic acid units, and can have a cyclic or linear configura solvent molecule is HO that is present in a defined stoichio tion. Non-limiting examples of oligomeric boronic acid anhy metric amount, and may, for example, include hemihydrate, drides of peptide boronic acids compound of the invention are monohydrate, dihydrate, or trihydrate. As used herein, the illustrated below: term “anhydrate is a compound of the invention that contains no H2O incorporated in its crystal lattice. As used herein, “crystalline' refers to a solid having a (1) highly regular chemical structure. In particular, a crystalline 25 W W W compound may be produced as one or more single crystalline forms of the compound. For the purposes of this application, Ho1 B No-No-1B B Noh the terms “single crystalline form” or “crystalline form” are (2) used interchangeably and distinguish between crystals that W have different properties (e.g., different XRPD patterns, dif 30 ferent DSC scan results). Thus, each distinct polymorph and B pseudopolymorph of a compound is considered to be a dis O O tinct single crystalline form herein. \. “Substantially crystalline' refers to a compound that may ( w1. No) Yw be at least a particular weight percent crystalline. Particular 35 weight percentages are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, In formula (1) and (2), the variable nn is an integer from 0 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or to about 10, preferably 0, 1, 2, 3, or 4. In some embodiments, any percentage between 10% and 100%. In some embodi the boronic acid anhydride compound comprises a cyclic ments, Substantially crystalline refers to compounds that are 40 trimer (“boroxine') of formula (2), wherein nn is 1. The at least 70% crystalline. In other embodiments, substantially variable W has the formula (3): crystalline refers to compounds that are at least 90% crystal line. (3) “Substantially pure” refers to a compound that may be at O Ra least a particular weight percent of the compound. Particular 45 H H weight percentages are about 80%, about 85%, about 90%, N N P N about 91%, about 92%, about 93%, about 94%, about 95%, H about 96%, about 97%, about 98%, about 99%, or about Ra2 A. O 99.5%. Unless otherwise explicitly stated, structures depicted 50 herein are meant to include all hydrates, anhydrates, Solvates wherein P. R', A. R'' and Rare as defined herein. and polymorphs thereof. As used herein, the total weight of a single oral pharma As used herein, the terms “compound (I-1) and “4-(R.S)- ceutical dosage form is determined by adding all the weights (carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)ac of the components in the oral pharmaceutical dosage form, etamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-car 55 and does not include the weight of any coatings which may be boxylic acid are used interchangeably, and include all optionally applied to the oral pharmaceutical dosage form crystalline forms. Both terms refer to the compounds pro after it is formed. The total weight of a single oral pharma duced in Example 1 and Example 1A in the Examples below ceutical dosage form is used as the basis for calculating the including both Form 1 and Form 2. weight percentage of each of the components that comprise As used herein, the terms “compound (I-1) Form 2 and 60 the oral pharmaceutical dosage form. “4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenza As used herein, “low-moisture' used in reference to an mido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori excipient Such as a filler, refers to an excipient that has a water nane-4-carboxylic acid Form 2' are used interchangeably. content of about 0.5% to about 4%. The term “low-moisture' Both terms refer to the crystalline form 2 produced in may be used interchangeably with the term “low-water. Example 1 Form 2 and Example 1A in the Examples below. 65 As used herein, the term “lyophilized powder”, “cake', or As used herein, the terms “compound of formula (VIII-1). “lyophilized cake” refers to any solid material obtained by and “(R)-1-((2,5-dichlorobenzamido)acetamido)-3-methyl lyophilization of an aqueous mixture. US 9, 175,018 B2 13 14 As used herein, the term “tonicity modifier refers to In some embodiments, the alpha-hydroxy carboxylic acid agents which contribute to the osmolality of a liquid or solu is selected from the group consisting of glycolic acid, malic tion. acid, hexahydromandelic acid, citric acid, 2-hydroxyisobu As used herein, the terms “boronate ester” and “boronic tyric acid, mandelic acid, lactic acid, 2-hydroxy-3,3-dimeth ester are used interchangeably and refer to a chemical com ylbutyric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxy pound containing a B(Z')(Z) moiety, wherein Z' and Z isocaproic acid, and benzilic acid. In some other together form a moiety where the atom attached to boron in embodiments, the alpha-hydroxy carboxylic acid is selected each case is an oxygen atom. from the group consisting of glycolic acid, malic acid, In some embodiments, the boronate ester moiety is a hexahydromandelic acid, citric acid, 2-hydroxyisobutyric 5-membered ring. In some other embodiments, the boronate 10 acid, mandelic acid, lactic acid, 2-hydroxy-3.3-dimethylbu ester moiety is a 6-membered ring. In some other embodi tyric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxyisoca ments, the boronate ester moiety is a mixture of a 5-mem bered ring and a 6-membered ring. proic acid, tartaric acid, and benzilic acid. In certain embodi As used herein, the term “alpha-hydroxy carboxylic acid ments, the alpha-hydroxy carboxylic acid is citric acid. Some refers to a compound that contains a hydroxyl group directly 15 other non-limiting examples of alpha-hydroxy carboxylic attached to a carbon atom in an alpha position relative to a acids include glucoheptonic acid, maltonic acid, lactobionic carboxylic acid group. As used herein, the term “alpha-hy acid, and galactaric acid. droxy carboxylic acid' is not intended to be limited to com In some embodiments the beta-hydroxy acid is character pounds having only one hydroxyl group and one carboxylic ized by formula (VI): acid group. As used herein, the term “beta-hydroxy carboxylic acid refers to a compound that contains a hydroxyl group directly (VI) attached to a carbon atom in a beta position relative to a carboxylic acid group. As used herein, the term “beta-hy droxy carboxylic acid' is not intended to be limited to com 25 pounds having only one hydroxyl group and one carboxylic acid group. As used herein, the term "moiety derived from an alpha hydroxy carboxylic acid refers to a moiety formed by wherein each of R'' and R' independently is hydrogen, removing a hydrogen atom from a carboxylic acid within an 30 —COH, -OH, or a substituted or unsubstituted ali alpha-hydroxy carboxylic acid and by removing a hydrogen phatic, aryl, heteroaryl or heterocyclyl group; each of atom from a hydroxyl group directly attached to a carbon R” and e independently is hydrogen, CO.H, or a atom in an alpha position relative to the carboxylic acid substituted or unsubstituted aliphatic, aryl, heteroaryl or group. As used herein, the term "moiety derived from a beta heterocyclyl group; hydroxy carboxylic acid refers to a moiety formed by removing a hydrogen atom from a carboxylic acid within a 35 or R'' and Rare each independently hydrogen, and R' beta-hydroxy carboxylic acid and by removing a hydrogen and R', taken together with the carbon atoms to which atom from a hydroxyl group directly attached to a carbon they are attached, forman unsubstituted or substituted 4 atom in a beta position relative to the carboxylic acid group. to 8-membered non-aromatic ring having 0-3 ring het eroatoms selected from the group consisting of O, N, DETAILED DESCRIPTION OF THE INVENTION 40 and S, wherein said ring may optionally be fused to an unsubstituted or substituted 4- to 8-membered non-aro In Some embodiments the alpha-hydroxy acid is character matic ring or 5- to 6-membered aromatic ring having 0-3 ized by formula (V): ring heteroatoms selected from the group consisting of O, N, and S; 45 or R and Rare absent, and R'' and R, taken together (V) with the carbon atoms to which they are attached, form an unsubstituted or substituted 5- to 6-membered aro matic ring having 0-3 ring heteroatoms selected from the OH group consisting of O, N, and S, wherein said ring may 50 optionally be fused to an unsubstituted or substituted 4 OH to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from wherein each of R and R' independently is hydrogen, the group consisting of O, N, and S. —COH, or a substituted or unsubstituted aliphatic, In some embodiments each of RandR' independently is aryl, heteroaryl or heterocyclyl group. 55 hydrogen, Caliphatic, -(CH2), OH, or -(CH2) - In some embodiments, each of RandR independently COH, and p is 0, 1 or 2. In some such embodiments, each of is hydrogen, Caliphatic, or -(CH2)CO.H. and p is 0, R'' and R is hydrogen. In some other such embodiments, 1 or 2. In some embodiments, each of R and R' indepen R’ is OH and R is hydrogen. dently is hydrogen or Caliphatic. In certain Such embodi In some embodiments each of RandR independently is ments, each of RandR' independently is selected from the 60 hydrogen, Caliphatic, or—(CH2), COH, and p is 0, 1 or group consisting of hydrogen, methyl, ethyl, isopropyl. 2. In some embodiments, each of RandR independently isobutyl, tert-butyl, and cyclohexyl. In some other embodi is hydrogen or Caliphatic. In certain Such embodiments, ments, each of R and R' independently is hydrogen or each of R and e independently is selected from the group -(CH2), CO.H. In some such embodiments, p is 1. In consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, certain other embodiments, each of R and R' indepen 65 tert-butyl, and cyclohexyl. In certain other embodiments, dently is -(CH2) -CO2H. In certain such embodiments, p each of R and R' independently is -(CH2), CO.H, and is 1. p is 0 or 1. US 9, 175,018 B2 15 The variable p is 0, 1, or 2. In some embodiments, p is 0 or -continued 1. In certain embodiments, p is 0. In other certain embodi (IV) ments, p is 1. O In some embodiments, RandR'' are absent and R'' and R” taken together with the carbon atoms to which they are O Ral O attached, form a Substituted or unsubstituted phenyl ring. CO2H In Some embodiments, the beta-hydroxy carboxylic acid is P N N-1 no selected from the group consisting of malic acid, citric acid, 3-hydroxybutyric acid, beta-hydroxyisovaleric acid, and Sali Ra2 O Ra COH: cylic acid. In some other embodiments, the beta-hydroxy 10 carboxylic acid is selected from the group consisting of malic or a mixture thereof, wherein the variables P. A. R. R', acid, citric acid, 3-hydroxybutyric acid, beta-hydroxyisova and R' have the values described below. leric acid, tartaric acid, and Salicylic acid. In certain embodi In some other embodiments, wherein the alpha-hydroxy ments, the beta-hydroxy carboxylic acid is citric acid. Some 15 carboxylic acid or beta-hydroxy carboxylic acid is citric acid, other non-limiting examples of beta-hydroxy carboxylic a further bond can be formed between the carboxylic acid in acids include glucoheptonic acid, maltonic acid, lactobionic formula (III) or (IV) and the boron atom. Without being acid, and galactaric acid. Some other non-limiting examples limited by any chemical bonding theory, in Such embodi of beta-hydroxy carboxylic acids include embonic acid, 1-hy ments, the compound of general formula (I) may be repre droxy-2-naphthoic acid and 3-hydroxy-2-naphthoic acid. sented by formula (IIIa) or (IVa): In some embodiments, the alpha-hydroxy acid or beta hydroxy acid is selected from the group consisting of glycolic acid, malic acid, hexahydromandelic acid, 2-hydroxyisobu (IIIa) tyric acid, citric acid, mandelic acid, lactic acid, 3-hydroxy butyric acid, beta-hydroxyisovaleric acid, 2-hydroxy-3.3- 25 COH dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid, O Ral O 2 2-hydroxyisocaproic acid, tartaric acid, Salicylic acid, and S. l. benzilic acid. P N N-1 N In some embodiments, compounds of general formula (I) R.2 J. O R"5. ?o are characterized by formula (II): 30 -- O (IVa) (II) O O l R Rb2 35 O Ral O O Ral O pi H H Rb3 l COH : N N B P N N1 no R54 P N N-1 N a2 5. Ra2 O R 40 R A. O R. R. -- O wherein: or a mixture thereof, wherein the variables P. A. R. R', the variables P. A. R. R', R', and n have the values 45 and R' have the values described below. described below and the variables R', R. RandR It is recognized that, without being limited by any chemical have the values described above. bonding theory, there are other representations that could be In some embodiments, any one of R', R, R and R' used to depict this further bonding of the carboxylic acid with may contain a functional group that can form a further bond the boron atom in formulas (IIIa) and (IVa). with the boronatom. In certain embodiments, the functional 50 The following values are described for the variables in any group is a carboxylic acid. In other certain embodiments, the of formulas (I), (II), (III), (IIIa), (IV), or (IVa). functional group is a hydroxyl group. The variable P is hydrogen or an amino-group-blocking In some embodiments, wherein the alpha-hydroxy car moiety. Non-limiting examples of amino-group-blocking boxylic acid or beta-hydroxy carboxylic acid is citric acid, the moieties can be found in P. G. M. Wuts and T. W. Greene, compound of general formula (I) is characterized by formula 55 Greene's Protective Groups in Organic Synthesis (4" ed.), (III) or (IV): John Wiley & Sons, NJ (2007), and include, e.g., acyl, sulfo nyl, oxyacyl, and aminoacyl groups. In some embodiments, P is R C(O)—, R O C (III) (O) , R. N(R)-C(O) , R. S(O) , or R. N 9 CO.H. 60 (R')—S(O) , where R is selected from the group consist ing of Caliphatic, C. fluoroaliphatic, R. -T-RP, and O Ral O -T-R, and the variables T',R,R, and Rhave the values R. described below. P N N-'No The variable R is hydrogen, C. alkyl, C, a fluoroalkyl, H COH: 65 or Co ar(Calkyl), the aryl portion of which is Substituted Ra2 O i. A. or unsubstituted. In some embodiments, R is hydrogen or Cia alkyl. In certain embodiments, R is hydrogen. US 9, 175,018 B2 17 18 The variable T' is a C- alkylene chain substituted with 0-2 —N(Caliphatic). In some embodiments, each R" inde independently selected R' or R, wherein the alkylene pendently is Caliphatic, Ca fluoroaliphatic or halo. chain optionally is interrupted by C(R)=C(R) , In some embodiments, RP is a substituted or unsubstituted —C=C , or - O -. Each R" independently is selected mono- or bicyclic ring system. In some embodiments R’ is a from the group consisting of —F, -OH, -O(C. alkyl). Substituted or unsubstituted mono- or bicyclic ring system —CN, N(R), —C(O)—(C. alkyl). —CO.H. —CO, selected from the group consisting of furanyl, thienyl, pyrro (C. alkyl), —C(O)NH2, and —C(O)—NH(C. alkyl). lyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, imidazolyl, Each Rindependently is a Caliphatic optionally substi pyrazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridinyl, tuted with R" or R'; or two substituents R on the same pyridazinyl, pyrimidinyl, pyrazinyl, benzofuranyl, ben carbon atom, taken together with the carbon atom to which 10 Zothiophenyl, indolyl, benzoxazolyl, benzisoxazolyl, benz they are attached, form a 3- to 6-membered cycloaliphatic imidazolyl, indazolyl, purinyl, naphthyl, quinolinyl, iso ring. Each R is a substituted or unsubstituted aromatic group. quinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, In some embodiments, T is a C- alkylene chain. phthalazinyl, naphthyridinyl, tetrahydroquinolinyl, tetrahy The variable R is halo, -OR. -SR, S(O)R. droisoquinolinyl, tetrahydroquinoxalinyl, and dihydroben - SOR, SON(R), N(R), NRC(O)R, NRC 15 Zoxazinyl. In some embodiments, RP is a substituted or (O)N(R), NRCOR, N(R)SOR, N(R)SON unsubstituted mono- or bicyclic ring system selected from the (R), O C(O)R, OC(O)N(R), C(O)R, group consisting of phenyl, pyridinyl, pyrimidinyl, pyrazinyl, —COR, or C(O)N(R), where: naphthyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinox each Rindependently is hydrogen oran optionally substi alinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tet tuted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two rahydroquinoxalinyl, and dihydrobenzoxazinyl. R" on the same nitrogen atom, taken together with the nitro In some embodiments, the Substitutable ring carbon atoms gen atom, form an optionally Substituted 4- to 8-membered in RP are substituted on substitutable carbon atoms with 0-1 heterocyclyl ring having, in addition to the nitrogenatom,0-2 R" and 0-2 R'; wherein: ring heteroatoms independently selected from the group con 25 each R' independently is C, aliphatic, C. fluoro sisting of N, O, and S; aliphatic or halo; and each Rindependently is hydrogen oran optionally substi each R" independently is C, aliphatic, C, a fluoro tuted aliphatic, aryl, heteroaryl, or heterocyclyl group; and aliphatic or halo. each R' independently is an optionally substituted ali In some embodiments, the Substitutable ring carbon atoms phatic, aryl, or heteroaryl group. 30 in Rare substituted with 0-1 R and 0-2 R, wherein: The variable RP is a substituted or unsubstituted aromatic, T' is a C- alkylene chain that is unsubstituted or is sub heterocyclyl, or cycloaliphatic ring, any of which is option stituted with R or R: ally fused to a substituted or unsubstituted aromatic, hetero each R' independently is a substituted or unsubstituted cyclyl or cycloaliphatic ring. In some embodiments, R is aryl, heteroaryl, heterocyclyl, or cycloaliphatic ring; and substituted on substitutable ring carbon atoms with 0-2 R' 35 each R’ independently is —OR. -SR, S(O)R. and 0-2 R', and each substitutable ring nitrogen atom in R' - SOR, SON(R), N(R), NRC(O)R, NRC is unsubstituted or is substituted with C(O)R. —C(O)N (O)N(R), O C(O)R, OC(O)N(R), C(O)R, (R), —CO.R. —SO.R. —SO.N(R), Caliphatic, a —COR, or -C(O)N(R). The variables R. R. and R' Substituted or unsubstituted Co aryl, or a Co ar(C) have the values described above. alkyl, the aryl portion of which is substituted or unsubstituted. 40 In some embodiments, the variable R has the formula The variables R. R. and Rhave the values described above. -Q-R', where Q is - O -, -NH , or—CH2—, and R' is a Each R" independently is selected from the group consisting substituted or unsubstituted aryl, heteroaryl, heterocyclyl, or of Caliphatic, C, fluoroaliphatic, halo, R', R*, cycloaliphatic ring. In some embodiments, R is a substituted -T-R'', and -T-R, where the variables T, R'', R, and or unsubstituted phenyl, pyridinyl, pyrimidinyl, pyrazinyl, R" have the values described below. In some embodiments, 45 piperidinyl, piperazinyl, or morpholinyl ring. each Rindependently is selected from the group consisting In some embodiments, P has the formula R C(O)—, of Caliphatic, C. fluoroaliphatic and halo. where is C alkyl, C. fluoroalkyl, or Co ar(C) alkyl, T is a C- alkylene chain substituted with 0-2 indepen the aryl portion of which is substituted or unsubstituted. In dently selected R' or R, wherein the alkylene chain option certain Such embodiments, P is selected from the group con ally is interrupted by C(R)=C(R)-, -C=C- or 50 sisting of acetyl, trifluoroacetyl, and phenylacetyl. -O-. The variables R. R. and R have the values In some other embodiments, P has the formula RP C described above. (O) , where RP is a substituted or unsubstituted phenyl, Each R' independently is a substituted or unsubstituted pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl, or quinoxali aryl, heteroaryl, heterocyclyl, or cycloaliphatic ring. nyl. In yet some other embodiments, P has the formula Each Rindependently is NO, CN, C(R)=C(R), 55 RP C(O), where RP is a phenyl, pyridinyl, pyrazinyl, pyri midinyl, naphthyl, quinolinyl, quinoxalinyl, benzimidazolyl, or dihydrobenzoxazinyl substituted with 0-1 Rand 0-2 R. In certain embodiments, P has the formula RP C(O) , where R is 2-pyrazinyl. In other certain embodiments, Phas 60 the formula RP C(O)—, where RP is 2,5-dichlorophenyl. In yet other certain embodiments, P has the formula RP C (—NR) N(R), N(R)C(—NR) N(R) C(O)R, (O) , where R' is 6-phenyl-2-pyridinyl. or –C(=NR) N(R). The variables R. R. and R have In some other embodiments, P has the formula the values described above. R SO , where R is - RP or -T-RP, where T is Ca Each Rindependently is selected from the group consist 65 alkylene and R’ is a phenyl, pyridinyl, pyrazinyl, pyrimidi ing of C aliphatic, Ca fluoroaliphatic, halo, —OH, nyl, naphthyl, quinolinyl, quinoxalinyl, benzimidazolyl, or —O(C- aliphatic), —NH, -NH(C. aliphatic), and dihydrobenzoxazinyl substituted with 0-1 R and 0-2 R. US 9, 175,018 B2 19 20 The variable R is hydrogen, Caliphatic, C. fluoro some embodiments, R is hydrogen or a substituted or aliphatic, -(CH2), CH. R. —(CH), CH NHC unsubstituted aliphatic group. In some other embodiments, (—NR)NH Y, —(CH), CH CONCR), Rishydrogen or Caliphatic. In such embodiments, Ris -(CH), CH, N(R)CON(R), -(CH), CH(R)N selected from the group consisting of hydrogen, methyl, (R) -(CH), CH(R) OR, or -(CH), CH ethyl, isopropyl and isobutyl. In certain Such embodiments, (R) SR, where the variables R. R. and R have the val R’ is hydrogen. ues described above, and the variables R. R. R.Y. and m The variable m is 0, 1, or 2. In some embodiments, m is 0 have the values described below. or 1. In certain embodiments, m is 0. In other certain embodi In some embodiments, R is hydrogen, Caliphatic, C. ments, m is 1. fluoroaliphatic, or -(CH2), CH. R. In some other 10 embodiments, R is Caliphatic, or -(CH2), CH. R. The variable A is 0, 1, or 2. In some embodiments, A is 0 or In some further embodiments, R is Caliphatic. In yet other 1. In certain embodiments, A is 0. further embodiments, R is isobutyl, 1-naphthylmethyl, The variable n is 0, or 1. In certain embodiments, n is 0. In 2-naphthylmethyl, benzyl, 4-fluorobenzyl, 4-hydroxybenzyl, other certain embodiments, n is 1. 4-(benzyloxy)benzyl, benzylnapththylmethyl or phenethyl. 15 In some embodiments, A is 0; R is hydrogen, Cali In certain embodiments, R is isobutyl. phatic, C. fluoroaliphatic, or -(CH2), CH. R. R' is The variable R' is hydrogen, Caliphatic, C. fluoro hydrogen, Caliphatic, Ce fluoroaliphatic. —(CH2) - aliphatic, -(CH2), CH2 R', -(CH2), CH2 NHC CH, R, or -(CH), CH(R) OR: P is Re C(O)– (—NR)NH Y, —(CH), CH CONCR), or R S(O) : R is - RP; and m is 0 or 1. -(CH), CH, N(R)CON(R), -(CH), CH(R)N In some other embodiments, A is 0; R is Caliphatic or (R), -(CH), CH(R) OR, or -(CH), CH (CH), CH. R. R' is hydrogen, -(CH2), CH (R) SR, where the variables R. R. and R have the val R’, or -(CH), CH(R) OR; P is R C(O) or ues described above, and the variables R. R. R.Y. and m R S(O) : R is - RP; and m is 0 or 1. have the values described below. In some other embodiments, A is 0; R is Caliphatic; R' In some embodiments, R' is hydrogen, Caliphatic, C 25 is hydrogen, -(CH2), CH. R. or -(CH2), CH fluoroaliphatic, -(CH2), CH. R. or -(CH2), CH (R) OR; P is R. C(O); R is RP; and m is 0 or 1. (R) OR. In some other embodiments, R' is hydrogen, In some other embodiments, A is 0; R is isobutyl, R' is (CH), CH R', or -(CH), CH(R)-OR. In hydrogen, Caliphatic, Ce fluoroaliphatic. —(CH2) - yet some other embodiments, R' is isobutyl, 1-naphthylm CH, R, or -(CH), CH(R) OR; P is R. C ethyl 2-naphthylmethyl, benzyl, 4-fluorobenzyl, 4-hydroxy 30 (O) : R is - RP; and m is 0 or 1. benzyl, 4-(benzyloxy)benzyl, benzylnaphthylmethyl or In yet some other embodiments, A is 0; R is isobutyl; R' phenethyl. is hydrogen, Ce aliphatic, -(CH2), CH. R. or In certain embodiments, R' is —CH. R. In other cer -(CH), CH(R) OR; P is R. C(O) : R is RP: tain embodiments, R' is -CH(R)-OR. In yet other and m is 0 or 1. certain embodiments, R' is hydrogen. 35 The variable R' is hydrogen, Caliphatic, Ce fluoro In still yet some other embodiments, A is 0; R is isobutyl: aliphatic, -(CH2), CH2 R', -(CH2), CH2 NHC R" is hydrogen, —(CH), CH. R. or -(CH2), CH (—NR)NH Y, —(CH), CH CONCR), (R) OR; P is R. C(O) : R is RP; and m is 0 or 1. -(CH), CH, N(R)CON(R), -(CH), CH(R)N In certain embodiments, A is 0; R is isobutyl; R' is CH (R), -(CH), CH(R) OR, or -(CH), CH 40 R’, and R is phenyl: P is R C(O)–: Re is - RP; and RP (R) SR, where the variables R. R. and R have the val is 2-pyrazinyl. ues described above, and the variables R. R. R.Y. and m In other certain embodiments, A is 0; R is isobutyl; R' is have the values described below. hydrogen; P is R C(O)–: R is - RP; and RP is 2.5- In some embodiments, R is hydrogen, Caliphatic, C dichlorophenyl. fluoroaliphatic, -(CH2), CH. R. or -(CH2), CH 45 In yet other certain embodiments, A is 0; R is isobutyl; R' (R) OR. In some other embodiments, R' is isobutyl, is —CH(R)-OR: R is Caliphatic; R is hydrogen; 1-naphthylmethyl 2-naphthylmethyl, benzyl, 4-fluoroben P is R C(O)–: Re is - RP; and RP is 6-phenyl-2-pyridi Zyl, 4-hydroxybenzyl, 4-(benzyloxy)benzyl, benzylnaphth nyl-. ylmethyl or phenethyl. In some embodiments, the compound of formula (I) is Each R', independently, is a substituted or unsubstituted 50 characterized by formula (I-1): mono- or bicyclic ring system. In some embodiments, each R’ independently is a substituted or unsubstituted phenyl, pyridyl, indolyl, benzimidazolyl, naphthyl, quinolinyl, qui noxalinyl, or isoquinolinyl ring. In certain embodiments, R” (I-1) is a Substituted or unsubstituted phenyl ring. 55 The variable Y is hydrogen, —CN, or - NO. In some C O O embodiments, Y is NO. H COH : The variable R is hydrogen or a substituted or unsubsti N B tuted aliphatic, aryl, heteroaryl, or heterocyclyl group. In N 60 1. some embodiments, R is hydrogen or a substituted or O CO2H unsubstituted aliphatic group. In some other embodiments, R" is hydrogenor Caliphatic. In such embodiments, R is selected from the group consisting of hydrogen, methyl, C ethyl, isopropyl and isobutyl. In certain Such embodiments, R" is methyl. 65 or a crystalline form thereof. The variable R is hydrogen or a substituted or unsubsti In some other embodiments, the compound of formula (I) tuted aliphatic, aryl, heteroaryl, or heterocyclyl group. In is characterized by formula (I-15): US 9, 175, 018 B2 21 22 Coupling of compoundi with an N-protected amino acidii, (I-15) followed by N-terminal deprotection, provides compound iii or a salt thereof. Examples of suitable protecting groups (PG) O 5 include, without limitation, acyl protecting groups, e.g., formyl, acetyl (Ac). Succinyl (Suc), and methoxysuccinyl: O O and urethane protecting groups, e.g., tert-butoxycarbonyl H CO2H : (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycar N N n N n-No bonyl (Fmoc). Optionally, PG is hydrogen and deprotection is H 10 not necessary. The peptide coupling reaction can be con 2 O CO2H N ducted by prior conversion of the carboxylic acid moiety of compound ii to an activated ester or acid halide, e.g., an N O—(N-hydroxysuccinnimide) ester, followed by treatment 15 with compound i. Alternatively, the activated ester can be or a crystalline form thereof. generated in situ by contacting the carboxylic acid with a In yet some other embodiments, the compound of formula peptide coupling reagent. Examples of Suitable peptide cou (I) is characterized by formula (I-18): pling reagents include, without limitation, carbodiimide reagents, e.g., dicyclohexylcarbodiimide (DCC) or 1-(3-dim 2O (I-18) ethylaminopropyl)-3-ethylcarbodiimide (EDC); phospho nium reagents, e.g., (benzotriazol-1-yloxy)tris(dimethy O lamino)phosphonium hexafluorophosphate (BOP); and HO, O '' O uronium reagents, e.g., O-(1H-benzotriazol-1-yl)-N.N.N',N'- H CO2H ; 25 tetramethyluronium tetrafluoroborate (TBTU). N N B n N n-1 no Compound iii is then coupled with an amino-group block ing moiety, to afford compound iv. The peptide coupling 21 O S. CO2H conditions described above for the coupling of compounds i 30 and ii are also Suitable for coupling compound iii with the or a crystalline form thereof. amino-group blocking moiety. Deprotection of the boronic General Synthetic Methodology acid moiety then affords compound V. The deprotection step The compounds of formula (I) can be prepared by esteri- 35 preferably is accomplished by transesterificationina biphasic fication of corresponding boronic acids. Such boronic acid mixture comprising the boronic ester compound iv, an compounds can be prepared from methods known to one of organic boronic acid acceptor, a lower alkanol, a Css hydro ordinary skill in the art. See, e.g., Adams et al., U.S. Pat. No. carbon solvent, and aqueous mineral acid. Other reagents that 5,780,454; Pickersgill et al., International Patent Publication can be used for deprotection of the boronic acid moiety WO 2005/097809. An exemplary synthetic route is set forth 40 include, without limitation, BC1, lithium aluminium hydride in Scheme 1 below. and NaIO.

Scheme 1: CH3 CH3 ul-CH3 O Ral ul-CH3 H3C, 1. peptide coupling O Ral H3C, CF3CO2 HN-- B.9 -- PG a2 Hnot Heconditions NH N-B-H 9 Y O R A. O 2. deprotection H 2 N E O Ra ii R4 J O Ra i iii.

O Ral OH l i-BuB(OH)2, aq. HCl O n a P N n- OH MeOH hexane Ra2 O Ra rt P US 9, 175,018 B2

Scheme 2:

O Ral O Ral 1. peptide coupling amino blocking H N OPG conditions P N OH group moiety a2 H 2. deprotection a2 H R A. O R A. O vi vii.

-- CFCO HN

i peptide coupling conditions

CH3-CH O Ra pi 3C. III.ii.22.

Bal i-BuB(OH)2, aq. HCI O Ral O Y se P N N-1 OH MeOH hexane H I a2 H rt P N N- BN, R A. O Ra H E a2

iv

Alternatively, the order of coupling reactions can be The conversion of V to the compound of formula (I) can be reversed, as shown in Scheme 2. Thus, an O-protected amino accomplished under esterification conditions employing acid Vi is first coupled with an amino-group blocking moiety, approximately a molar equivalent of the alpha-hydroxy car followed by ester hydrolysis, to form compound vii. Option 40 boxylic acid or beta-hydroxy carboxylic acid in a solvent such ally, PG' is H and ester hydrolysis is not needed, leading as ethyl acetate at a temperature of between about 40°C. and about 80°C. The conversion of V to the compound of formula directly to compound vii. Coupling with compound i and (I) can also be accomplished as described above employing a boronic acid deprotection are then accomplished as described molar excess of the alpha-hydroxy carboxylic acid or beta above for Scheme 1 to afford compound V. hydroxy carboxylic acid. Examples of other suitable solvents Compound V is reacted with the appropriate alpha-hydroxy 45 for this conversion include, but are not limited to, methyl carboxylic acid or beta-hydroxy carboxylic acid to afford the isobutyl ketone, acetone, acetonitrile, 2-methyltetrahydrofu compound of formula (I) as shown in Scheme 3. ran, anisole, isopropyl acetate, dimethoxyethane, tetrahydro

Scheme 3:

O Ral OH alpha-hydroxy carboxylic acid or l beta-hydroxy carboxylic acid P N N1 No H E a2 R A. O Ra

w

O Ral H N P N n H Ra2 (I) US 9, 175,018 B2 25 26 furan, dioxane, dichloromethane, toluene, heptane, methyl In another aspect, therefore, the invention provides a cyclohexane, tert-butylmethyl ether, and mixtures thereof. method for inhibiting one or more peptidase activities of a The choice of the solvent will depend partly on the solubility proteasome in a cell, comprising contacting a cell in which of the alpha-hydroxy carboxylic acid or beta-hydroxy car proteasome inhibition is desired with a compound described boxylic acid used. The temperature selected for the conver herein, or a pharmaceutically acceptable salt, boronic ester, or sion of V to the compound of formula (I) will depend partly on boronic acid anhydride thereof. the boiling point of the solvent or solvent mixture used. The invention also provides a method for inhibiting cell The conversion of V to the compound of formula (I) may be proliferation, comprising contacting a cell in which Such catalyzed by an organic amine base Such as, but not limited to, inhibition is desired with a compound described herein. The triethylamine, triethylenediamine, pyridine, collidine, 2.6- 10 phrase “inhibiting cell proliferation' is used to denote the lutidine, 4-dimethylaminopyridine, di-tertbutylpyridine, ability of a compound of the invention to inhibit cell number N-methylmorpholine, N-methylpiperidine, tetrameth or cell growth in contacted cells as compared to cells not ylguanidine, diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-di contacted with the inhibitor. An assessment of cell prolifera azabicyclo[2.2.2]octane, 1,5-diazabicycle 4.3.0non-5-ene, tion can be made by counting cells using a cell counter or by N,N'diisopropylethylamine, or a mixture thereof. 15 an assay of cell viability, e.g., an MTT or WST assay. Where The compound of formula V and the alpha-hydroxy car the cells are in a Solid growth (e.g., a solid tumor or organ), boxylic acid or beta-hydroxy carboxylic acid are heated Such an assessment of cell proliferation can be made by together in the solvent of choice for a period of time. Follow measuring the growth, e.g., with calipers, and comparing the ing this period of time, the reaction mixture is allowed to cool size of the growth of contacted cells with non-contacted cells. for a period of time and the compound of formula (I) which Preferably, the growth of cells contacted with the inhibitor precipitates upon cooling is collected by filtration. The cool is retarded by at least about 50% as compared to growth of ing may be uncontrolled or may be controlled by the use of a non-contacted cells. In various embodiments, cell prolifera coolingapparatus. The reaction mixture may be stirred during tion of contacted cells is inhibited by at least about 75%, at this cooling period. Alternatively, the compound of formula least about 90%, or at least about 95% as compared to non (I) can also be isolated from the reaction mixture by cooling 25 contacted cells. In some embodiments, the phrase “inhibiting followed by evaporation of the solvent. The reaction mixture cell proliferation' includes a reduction in the number of con may be seeded with crystals of the compound of formula (I) in tacted cells, as compared to non-contacted cells. Thus, a order to effect precipitation. proteasome inhibitor that inhibits cell proliferation in a con A co-solvent such as, but not limited to, heptane, methyl tacted cell may induce the contacted cell to undergo growth cyclohexane, toluene, tert-butylmethyl ether, ethyl acetate, or 30 retardation, to undergo growth arrest, to undergo pro a mixture thereof, may be added during the cooling period. grammed cell death (i.e., apoptosis), or to undergo necrotic Following the addition of the co-solvent, the reaction mixture cell death. can be cooled further leading to the precipitation of the com In another aspect, the invention provides a pharmaceutical pound of formula (I). Alternatively, once the co-solvent is composition comprising a compound of formula (I), or a added, the reaction mixture can then be heated again to gen 35 pharmaceutically acceptable salt thereof, and a pharmaceuti erate a homogenous solution, which is then cooled leading to cally acceptable carrier. the precipitation of the compound of formula (I). The reaction In some embodiments, the composition also comprises the mixture may be seeded with crystals of the compound of free alpha-hydroxy carboxylic acid or a salt thereof or the formula (I) in order to effect precipitation. beta-hydroxy carboxylic acid or a salt thereof. In such In other embodiments, the compound of formula (I) is 40 embodiments, the alpha-hydroxy carboxylic acid or a salt isolated in Substantially pure form. In Such embodiments, the thereof or beta-hydroxy carboxylic acid or a salt thereof and purity is about 80%, about 85%, about 90%, about 91%, about the compound of formula (I) are present in a molar ratio 92%, about 93%, about 94%, about 95%, about 96%, about ranging from about 2:1 to about 200:1. In various embodi 97%, about 98%, about 99%, or about 99.5%. ments, the alpha hydroxy carboxylic acid or a salt thereof or In some embodiments, the compound of formula (I) is 45 beta-hydroxy carboxylic acid or a salt thereof and the com isolated in crystalline form. In some embodiments, the com pound of formula (I) are present in a ratio ranging from about pound of formula (I) is isolated in substantially crystalline 2:1 to about 200:1, from about 15:1 to about 80:1, or from form. In some other embodiments, the compound of formula about 20:1 to about 40:1. (I) is isolated in amorphous form. If a pharmaceutically acceptable salt of the compound of The compound of formula (I) can also be generated by the 50 the invention is utilized in these compositions, the salt pref co-lyophilization of compound V and the alpha-hydroxy car erably is derived from an inorganic or organic acid or base. boxylic acid or beta-hydroxy carboxylic acid. This is accom For reviews of suitable salts, see, e.g., Berge et al., J. Pharm. plished by Subjecting an aqueous solution comprising the Sci. 66:1-19 (1977) and Remington. The Science and Practice compound of formula V and a molar excess of the alpha of Pharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams hydroxy carboxylic acid or beta-hydroxy carboxylic acid to a 55 & Wilkins, 2000. lyophilization procedure. In some embodiments, the aqueous Nonlimiting examples of Suitable acid addition salts Solution additionally comprises a water-miscible co-solvent. include the following: acetate, adipate, alginate, aspartate, Examples of suitable co-solvents include, but are not limited benzoate, benzene Sulfonate, bisulfate, butyrate, citrate, cam to, tert-butyl alcohol, methanol, ethanol, and mixtures phorate, camphor Sulfonate, cyclopentanepropionate, diglu thereof. The co-lyophilization results in a composition that 60 conate, dodecylsulfate, ethanesulfonate, fumarate, lucohep contains the compound of formula (I) and the excess alpha tanoate, glycerophosphate, hemisulfate, heptanoate, hydroxy carboxylic acid or beta-hydroxy carboxylic acid. hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hy Uses, Formulation, and Administration droxyethanesulfonate, lactate, maleate, methanesulfonate, The present invention provides compounds that are potent 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pecti inhibitors of the proteasome. The compounds can be assayed 65 nate, persulfate, 3-phenyl-propionate, picrate, pivalate, pro in vitro or in vivo for their ability to inhibit proteasome pionate. Succinate, tartrate, thiocyanate, tosylate and unde mediated peptide hydrolysis or protein degradation. Canoate. US 9, 175,018 B2 27 28 Suitable base addition salts include, without limitation, The pharmaceutical compositions of the invention can be ammonium salts, alkali metal salts, such as lithium, Sodium manufactured by methods well known in the art such as and potassium salts; alkaline earth metal salts, such as cal conventional granulating, mixing, dissolving, encapsulating, cium and magnesium salts; other multivalent metal salts. Such lyophilizing, or emulsifying processes, among others. Com as Zinc salts; salts with organic bases, such as dicyclohexy positions may be produced in various forms, including gran lamine, N-methyl-D-glucamine, t-butylamine, ethylene ules, precipitates, or particulates, powders, including freeze diamine, ethanolamine, and choline; and salts with amino dried, rotary dried or spray dried powders, amorphous pow acids such as arginine, lysine, and so forth. ders, tablets, capsules, syrup, Suppositories, injections, emul The term “pharmaceutically acceptable carrier is used sions, elixirs, Suspensions or solutions. 10 According to a preferred embodiment, the compositions of herein to refer to a material that is compatible with a recipient this invention are formulated for pharmaceutical administra Subject, preferably a mammal, more preferably a human, and tion to a mammal, preferably a human being. Such pharma is Suitable for delivering an active agent to the target site ceutical compositions of the present invention may be admin without terminating the activity of the agent. The toxicity or istered orally, parenterally, by inhalation spray, topically, adverse effects, if any, associated with the carrier preferably 15 rectally, nasally, buccally, vaginally or via an implanted res are commensurate with a reasonable risk/benefit ratio for the ervoir. The term “parenteral' as used herein includes subcu intended use of the active agent. taneous, intravenous, intramuscular, intra-articular, intra The terms “carrier”, “excipient” or “vehicle' are used synovial, intrasternal, intrathecal, intrahepatic, intralesional interchangeably herein, and include any and all solvents, and intracranial injection or infusion techniques. Preferably, diluents, and other liquid vehicles, dispersion or Suspension the compositions are administered orally, intravenously, or aids, Surface active agents, pH modifiers, isotonic agents, subcutaneously. The formulations of the invention may be thickening or emulsifying agents, preservatives, Solid bind designed to be short-acting, fast-releasing, or long-acting. ers, lubricants and the like, as Suited to the particular dosage Still further, compounds can be administered in a local rather form desired. Remington. The Science and Practice of Phar than systemic means, such as administration (e.g., by injec macy, 20th Ed., ed. A. Gennaro, Lippincott Williams & 25 tion) at a tumor site. Wilkins, 2000 discloses various carriers used in formulating Liquid dosage forms for oral administration include, but pharmaceutically acceptable compositions and known tech are not limited to, pharmaceutically acceptable emulsions, niques for the preparation thereof. Strickley, Pharmaceutical microemulsions, Solutions, Suspensions, syrups and elixirs. Research, 21(2) 201-230 (2004) reviews pharmaceutically In addition to the active compounds, the liquid dosage forms acceptable excipients used in commercial products to solubi 30 may contain inert diluents commonly used in the art Such as, lize compounds for oral or parenteral administration. Except for example, water or other solvents, solubilizing agents and insofar as any conventional carrier medium is incompatible emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl with the compounds of the invention, such as by producing carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, any undesirable biological effect or otherwise interacting in a propylene glycol, 1,3-butylene glycol, cyclodextrins, dimeth deleterious manner with any other component(s) of the phar 35 ylformamide, oils (in particular, cottonseed, groundnut, corn, maceutically acceptable composition, its use is contemplated germ, olive, castor, and sesame oils), glycerol, tetrahydrofur to be within the scope of this invention. Some examples of furyl alcohol, polyethylene glycols and fatty acid esters of materials which can serve as pharmaceutically acceptable sorbitan, and mixtures thereof. Besides inert diluents, the oral carriers include, but are not limited to, ion exchangers, alu compositions can also include adjuvants such as wetting mina, aluminum Stearate, lecithin, serum proteins, such as 40 agents, emulsifying and Suspending agents, Sweetening, fla human serum albumin, buffer Substances such as phosphates, Voring, and perfuming agents. carbonates, magnesium hydroxide and aluminum hydroxide, Injectable preparations, for example, sterile injectable glycine, Sorbic acid, or potassium Sorbate, partial glyceride aqueous or oleaginous Suspensions may be formulated mixtures of Saturated vegetable fatty acids, water, pyrogen according to the known art using Suitable dispersing or wet free water, salts or electrolytes Such as protamine Sulfate, 45 ting agents and Suspending agents. The sterile injectable disodium hydrogen phosphate, potassium hydrogen phos preparation may also be a sterile injectable solution, Suspen phate, sodium chloride, and Zinc salts, colloidal silica, mag sion or emulsion in a parenterally acceptable diluent or Sol nesium trisilicate, polyvinyl pyrrolidone, polyacrylates, vent, for example, as a solution in 1,3-butanediol. Among the waxes, polyethylene-polyoxypropylene-block polymers, acceptable vehicles and solvents that may be employed are wool fat, Sugars Such as lactose, glucose. Sucrose, and man 50 water, Ringer's solution, U.S.P. and isotonic sodium chloride nitol, starches such as corn starch and potato starch, cellulose Solution. In addition, Sterile, fixed oils are conventionally and its derivatives Such as sodium carboxymethyl cellulose, employed as a solvent or Suspending medium. For this pur ethyl cellulose and cellulose acetate, powdered tragacanth; pose any bland fixed oil can be employed including synthetic malt, gelatin, talc, excipients such as cocoa butter and Sup mono- or diglycerides. In addition, fatty acids such as oleic pository waxes, oils such as peanut oil, cottonseed oil, saf 55 acid are used in the preparation of injectables. The injectable flower oil, sesame oil, olive oil, corn oil and Soybean oil, formulations can be sterilized, for example, by filtration glycols such as propylene glycol and polyethylene glycol, through a bacterial-retaining filter, or by incorporating steril esters such as ethyl oleate and ethyl laurate, agar, alginic acid, izing agents in the form of Sterile solid compositions which isotonic saline, Ringer's Solution, alcohols such as ethanol, can be dissolved or dispersed in sterile water or other sterile isopropyl alcohol, hexadecyl alcohol, and glycerol, cyclodex 60 injectable medium prior to use. Compositions formulated for trins such as hydroxypropyl B-cyclodextrin and Sulfobu parenteral administration may be injected by bolus injection tylether B-cyclodextrin, lubricants such as sodium lauryl Sul or by timed push, or may be administered by continuous fate and magnesium Stearate, petroleum hydrocarbons such infusion. as mineral oil and petrolatum. Coloring agents, releasing Solid dosage forms for oral administration include cap agents, coating agents, Sweetening, flavoring and perfuming 65 Sules, tablets, pills, powders, and granules. In Such solid dos agents, preservatives and antioxidants can also be present in age forms, the active compound is mixed with at least one the composition, according to the judgment of the formulator. inert, pharmaceutically acceptable excipient or carrier Such as US 9, 175,018 B2 29 30 Sodium citrate or dicalcium phosphate and/or a) fillers or transdermal patches, which have the added advantage of pro extenders such as starches, lactose, cellulose, Sucrose, glu viding controlled delivery of a compound to the body. Such cose, mannitol, and silicic acid, b) binders such as, for dosage forms can be made by dissolving or dispensing the example, carboxymethylcellulose, alginates, gelatin, polyvi compound in the proper medium. Absorption enhancers can nylpyrrolidinone. Sucrose, and acacia, c) humectants such as also be used to increase the flux of the compound across the glycerol, d) disintegrating agents such as agar-agar, calcium skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a carbonate, potato or tapioca Starch, alginic acid, certain sili polymer matrix or gel. cates, crospovidone, cellulose, croscaramellose Sodium, In some embodiments, the present invention provides phar Sodium starch glycolate, and Sodium carbonate, e) solution maceutical compositions comprising the compound of for retarding agents such as paraffin, f) absorption accelerators 10 mula (I), and additional excipients described herein. In some Such as quaternary ammonium compounds, g) Wetting agents other embodiments, the present invention provides pharma Such as, for example, cetyl alcohol and glycerol monostear ceutical compositions comprising the compound of formula ate, h) absorbents such as kaolin and bentonite clay, and i) (II), and additional excipients described herein. In yet some lubricants such as talc, calcium Stearate, magnesium Stearate, other embodiments, the present invention provides pharma Sodium Stearyl fumarate, Stearic acid, Solid polyethylene gly 15 ceutical compositions comprising the compound of formula cols, sodium lauryl Sulfate, glyceryl behenate, and mixtures (III) or (IV), and additional excipients described herein. thereof. In the case of capsules, tablets and pills, the dosage In further embodiments, the present invention provides form may also comprise buffering agents such as phosphates pharmaceutical compositions comprising the citrate ester of or carbonates. compound (VIII-1), and additional excipients described Solid compositions of a similar type may also be employed herein. In other further embodiments, the present invention as fillers in Soft and hard-filled gelatin capsules using Such provides pharmaceutical compositions comprising the citrate excipients as lactose or milk Sugar as well as high molecular ester of compound (VIII-15), and additional excipients weight polyethylene glycols and the like. The Solid dosage described herein. In further embodiments, the present inven forms of tablets, dragees, capsules, pills, and granules can be tion provides pharmaceutical compositions comprising the prepared with coatings and shells Such as enteric coatings and 25 citrate ester of (VIII-18) and additional excipients described other coatings well known in the pharmaceutical formulating herein. art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredi (VIII-1) ent(s) only, or preferentially, in a certain part of the intestinal C O OH tract, optionally, in a delayed manner. Examples of embed 30 ding compositions that can be used include polymeric Sub R. stances and waxes. Solid compositions of a similar type may H 1 N1 NOI also be employed as fillers in soft and hard-filled gelatin capsules using Such excipients as lactose or milk Sugar as well as high molecular weight polethylene glycols and the like. 35 O N The active compounds can also be in micro-encapsulated C form with one or more excipients as noted above. The solid (VIII-15) dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells Such as enteric coatings, release controlling coatings and other coatings well 40 known in the pharmaceutical formulating art. In such solid O OH dosage forms the active compound may be admixed with at least one inert diluent such as Sucrose, lactose or starch. Such N R. dosage forms may also comprise, as is normal practice, addi n N N1 No tional Substances other than inert diluents, e.g., tableting 45 2 O lubricants and other tableting aids such as magnesium Stear ate and microcrystalline cellulose. In the case of capsules, N N tablets and pills, the dosage forms may also comprise buffer ing agents. They may optionally contain opacifying agents (VIII-18) and can also be of a composition that they release the active 50 HO ingredient(s) only, or preferentially, in a certain part of the O ' OH intestinal tract, optionally, in a delayed manner. Examples of N R. n N N1 No embedding compositions that can be used include polymeric H Substances and waxes. In some embodiments, the excipients 21 O or carriers may include, but are not limited to Sodium Stearyl 55 fumarate, carboxymethylcellulose, magnesium Stearate, crospovidone, ethylcellulose, talc, and silicified microcrys talline cellulose. Dosage forms for topical or transdermal administration of In further embodiments, the present invention provides a compound of this invention include ointments, pastes, 60 pharmaceutical compositions comprising the compound creams, lotions, gels, powders, Solutions, sprays, inhalants or (I-1), or a crystalline form thereof. In still yet further embodi patches. The active component is admixed under sterile con ments, the present invention provides pharmaceutical com ditions with a pharmaceutically acceptable carrier and any positions comprising the compound (I-15), or a crystalline needed preservatives or buffers as may be required. Oph form thereof. In still yet some further embodiments, the thalmic formulation, ear drops, and eye drops are also con 65 present invention provides pharmaceutical compositions templated as being within the scope of this invention. Addi comprising the compound (I-18), or a crystalline form tionally, the present invention contemplates the use of thereof. US 9, 175,018 B2 31 32 The following description of pharmaceutical compositions Suitable fillers include, but are not limited to, powdered and methods for preparation of said pharmaceutical compo cellulose, microcrystalline cellulose, silicified microcrystal sitions are applicable to the compounds of formulas (I), (II), line cellulose, high density microcrystalline cellulose, low (III), (IIIa), (IV), or (IVa) and various embodiments of these moisture microcrystalline cellulose, pregeletanized starch, formulas as described herein. The following description of 5 Sodium starch glycolate, and mixtures thereof. In some other pharmaceutical compositions and methods for preparation of embodiments, the filler is selected from the group consisting said pharmaceutical compositions are also applicable to the of powdered cellulose, microcrystalline cellulose, silicified compounds (I-1), (I-15), or (I-18). microcrystalline cellulose, low-moisture microcrystalline In one embodiment, the pharmaceutical composition com cellulose, and mixtures thereof. In yet some other embodi prises the compound of formula (I), wherein the compound of 10 ments, the filler is low-moisture microcrystalline cellulose. In formula (I) is substantially crystalline. In another embodi some further embodiments, the filler is selected from the ment, the compound of formula (I) in the pharmaceutical group consisting of low-moisture microcrystalline cellulose, composition is at least about 95%, 96%, 97%, 98%, 99%, Sodium starch glycolate, pregeletanized starch, and mixtures 99.5%, 99.9% of a crystalline form. In yet another embodi thereof. ment, the compound of formula (I) in the pharmaceutical 15 composition is a crystalline form. In other embodiments, the filler is present in an amount of In Some embodiments, the pharmaceutical formulations of about 97% to about 99.8%, by weight as a percentage of total the invention provide stable solid oral dosage forms of active weight. In some other embodiments, the filler is present in an compound, by using excipients having low water or low amount from about 98% to about 99.75%, by weight as a moisture content, and manufactured using dry or non-aque percentage of total weight. In yet some other embodiments, ous formulation processes. when a lubricant is present, the amount of filler is reduced by In one embodiment, the pharmaceutical composition is an the corresponding percent amount of lubricant present. In oral pharmaceutical dosage form, selected from the group Some further embodiments, the filler is present in an amount consisting of capsules, tablets, pills, powders, and granules. of about 86.5% to about 99.8%, by weight as a percentage of In another embodiment, the oral pharmaceutical dosage form 25 total weight. is a capsule, wherein the capsule is a polymer-based capsule In some embodiments, the filler comprises a first filler and selected from the group consisting of gelatin, hydroxypropy a second filler. The first filler is present in an amount of 0% to lmethyl cellulose (HPMC), fish gelatin, and pullulan. In yet about 99.8%, by weight as a percentage of total weight, and another embodiment, the polymer-based capsule is selected the second filler is present in an amount of 0% to about 99.8% from the group consisting of gelatin, and hydroxypropylm 30 by weight as a percentage of total weight, as long as the total ethyl cellulose. In still yet another embodiment, the polymer based capsule is a hard gelatin capsule. amount of filler is no greater than about 99.8%. In some In one embodiment, the pharmaceutical composition com embodiments, the first filler is present in an amount of about prises the compound of formula (I), or a crystalline form 40% to about 60%, by weight as a percentage of total weight, thereof, a filler, and optionally a lubricant. In another embodi 35 and the second filler is present in an amount of about 40% to ment, the pharmaceutical composition comprises about 0.2% about 60% by weight as a percentage of total weight, as long to about 3% of the compound of formula (I), or a crystalline as the total amount offiller is not greater than about 99.8%, by form thereof about 97% to about 99.8% of a filler; and weight as a percentage of total weight. optionally up to about 1.5% of a lubricant. In yet another In some embodiments, the first filler is selected from the embodiment, the pharmaceutical composition comprises 40 group consisting of low-moisture microcrystalline cellulose, about 0.25% to about 2% of the compound of formula (I), or Sodium starch glycolate, pregeletanized starch, and mixtures a crystalline form thereof; and about 98% to about 99.75% of thereof. In some embodiments, the second filler is selected a filler. from the group consisting of low-moisture microcrystalline In another embodiment, the pharmaceutical composition cellulose, Sodium starch glycolate, pregeletanized starch, and further comprises an optional flow-aid, and an optional 45 mixtures thereof. buffer. In yet another embodiment, the pharmaceutical com Suitable lubricants include, but are not limited to, magne position comprises about 0.2% to about 3% of the compound sium Stearate, glyceryl behenate, hydrogenated vegetable oil, of formula (I), or a crystalline form thereof, about 86.5% to talc, Zinc Stearate, calcium Stearate, Sucrose Stearate, sodium about 99.8% of a filler, optionally up to about 1.5% of a Stearyl fumarate, and mixtures thereof. In some embodi lubricant, optionally up to about 5% of a flow-aid, and option 50 ments, the lubricant is magnesium Stearate. In other embodi ally up to about 5% of a buffer, by weight as a percentage of ments, the lubricant is present in an amount of up to about total weight. 1.5%, by weight as a percentage of total weight. In yet some In another embodiment, the pharmaceutical composition other embodiments, the lubricant is present in an amount of comprises about 0.2% to about 12% of the compound of about 1%, by weight as a percentage of total weight. formula (I), or a crystalline form thereof, about 76.5% to 55 Suitable flow-aids include, but are not limited to silicon about 99.8% of a filler, optionally up to about 1.5% of a dioxide, talc, and mixtures thereof. In some embodiments, the lubricant, optionally up to about 5% of a flow-aid, and option flow-aid is talc. In other embodiments, the flow-aid is present ally up to about 5% of a buffer, by weight as a percentage of in an amount of up to about 5%, by weight as a percentage of total weight. total weight. In some other embodiments, the flow-aid is In some embodiments, the compound of formula (I), or a 60 present in an amount of about 1%, by weight as a percentage crystalline form thereof, is present in the pharmaceutical of total weight. In yet some other embodiments, the flow-aid composition in an amount of about 0.2% to about 3%, by is present in an amount of about 2%, by weight as a percent weight as a percentage of total weight. In some other embodi age of total weight. ments, the compound of formula (I), or a crystalline form Suitable buffers include sodium citrate, citric acid, and thereof, is present in the pharmaceutical composition, in an 65 mixtures thereof. In some embodiments, the buffer is sodium amount of about 0.25% to about 2%, by weight as a percent citrate. In some other embodiments, the buffer is present in an age of total weight. amount of up to about 5%, by weight as a percentage of total US 9, 175,018 B2 33 34 weight. In yet some other embodiments, the buffer is present mula (I), or a crystalline form thereof, about 86.5% to about in an amount of about 2%, by weight as a percentage of total 99.8% of a filler, optionally up to about 1.5% of a lubricant, weight. optionally up to about 5% of a flow-aid, and optionally up to In some embodiments, the pharmaceutical composition about 5% of a buffer, by weight as a percentage of total comprises the compound of formula (I), or a crystalline form weight, wherein: thereof, a filler, and optionally a lubricant; wherein: the compound of formula (I) is (I-1), (I-15) or (I-18); the alpha-hydroxy carboxylic acid or beta-hydroxy car the filler is selected from the group consisting of low boxylic acid is citric acid; moisture microcrystalline cellulose, Sodium starch gly A is 0; colate, pregeletanized starch, and mixtures thereof. R" is isobutyl: 10 R" is hydrogen, Caliphatic, -(CH2), CH. R. or the lubricant, when present, is magnesium Stearate; -(CH), CH(R) OR; the flow-aid, when present, is talc, and P is R. C(O) ; the buffer, when present, is sodium citrate. R is RP; In some embodiments, the pharmaceutical composition M is 0 or 1; 15 comprises the compound of formula (I), or a crystalline form the filler is selected from the group consisting of low thereof, a filler, and optionally a lubricant; wherein the com moisture microcrystalline cellulose, Sodium starch gly pound of formula (I) is (I-1). In some other embodiments, the colate, pregeletanized starch, and mixtures thereof, and pharmaceutical composition comprises the compound of for the lubricant, when present, is magnesium Stearate. mula (I), or a crystalline form thereof, a filler, and optionally In some embodiments, the pharmaceutical composition a lubricant; wherein the compound of formula (I) is (I-1); the comprises the compound of formula (I), or a crystalline form filler is selected from the group consisting of low-moisture thereof, a filler, and optionally a lubricant; wherein: microcrystalline cellulose, Sodium starch glycolate, the compound of formula (I) is (I-1), (I-15) or (I-18): pregeletanized starch, and mixtures thereof, and the lubri the filler is selected from the group consisting of low cant, when present, is magnesium Stearate. moisture microcrystalline cellulose, Sodium starch gly 25 In some embodiments, the pharmaceutical composition colate, pregeletanized starch, and mixtures thereof, and comprises the compound of formula (I), or a crystalline form the lubricant, when present, is magnesium Stearate. thereof; wherein the compound of formula (I) is (I-1); and the In some embodiments, the pharmaceutical composition crystalline form is Form 2. comprises about 0.25% to about 2% of the compound of In some embodiments, the pharmaceutical composition formula (I), or a crystalline form thereof; and about 98% to 30 comprises the compound of formula (I-1) Form 2, and low about 99.75% of a filler; wherein: the compound of formula (I) is (I-1), (I-15) or (I-18); and moisture microcrystalline cellulose. In some other embodi the filler is selected from the group consisting of low ments, the pharmaceutical composition comprises the com moisture microcrystalline cellulose, Sodium starch gly pound of formula (I-1) Form 2, and silicified microcrystalline colate, pregeletanized starch, and mixtures thereof. 35 cellulose. In yet some other embodiments, the pharmaceuti In some embodiments, the pharmaceutical composition cal composition comprises the compound of formula (I-1) comprises the compound of formula (I), or a crystalline form Form 2, low-moisture microcrystalline cellulose, and magne thereof, a filler, optionally a lubricant; optionally a flow-aid: sium stearate. In still yet some further embodiments, the and optionally a buffer; wherein: pharmaceutical composition comprises the compound of for the alpha-hydroxy carboxylic acid or beta-hydroxy car 40 mula (I-1) Form 2, microcrystalline cellulose, and magne boxylic acid is citric acid; sium Stearate. A is 0; In some embodiments, the pharmaceutical composition R" is isobutyl: comprises the compound of formula (I-1) Form 2, low-mois R" is hydrogen, Caliphatic, -(CH2), CH. R. or ture microcrystalline cellulose, and talc. In some other -(CH), CH(R) OR; 45 embodiments, the pharmaceutical composition comprises the P is R. C(O) ; compound of formula (I-1) Form 2, and pregeletanized R is RP; starch. In yet some other embodiments, the pharmaceutical m is 0 or 1; composition comprises the compound of formula (I-1) Form the filler is selected from the group consisting of low 2, pregeletanized starch, talc, and magnesium Stearate. In still moisture microcrystalline cellulose, Sodium starch gly 50 yet some other embodiments, the pharmaceutical composi colate, pregeletanized starch, and mixtures thereof. tion comprises the compound of formula (I-1) Form 2, low the lubricant, when present, is magnesium Stearate; moisture microcrystalline cellulose, talc, and magnesium the flow-aid, when present, is talc, and Stearate. In some further embodiments, the pharmaceutical the buffer, when present, is sodium citrate. composition comprises the compound of formula (I-1) Form In some embodiments, the pharmaceutical composition 55 2, low-moisture microcrystalline cellulose, talc, magnesium comprises the compound of formula (I), or a crystalline form Stearate, and sodium citrate. In some other further embodi thereof, a filler, optionally a lubricant; optionally a flow-aid: ments, the pharmaceutical composition comprises the com and optionally a buffer, wherein: pound of formula (I-1) Form 2, low-moisture microcrystal the compound of formula (I) is (I-1), (I-15) or (I-18): line cellulose, talc, magnesium Stearate, and pregeletanized the filler is selected from the group consisting of low 60 starch. In still yet some further embodiments, the pharmaceu moisture microcrystalline cellulose, Sodium starch gly tical composition comprises the compound of formula (I-1) colate, pregeletanized starch, and mixtures thereof. Form 2, low-moisture microcrystalline cellulose, talc, mag the lubricant, when present, is magnesium Stearate; nesium Stearate, and Sodium starch glycolate. the flow-aid, when present, is talc, and When the compound of formula (I) is subjected to hydro the buffer, when present, is sodium citrate. 65 lytic conditions, the ester portion of the molecule is hydro In some embodiments, the pharmaceutical composition lyzed to give the compound of formula (VIII) in a 1:1 molecu comprises about 0.2% to about 3% of the compound of for lar ratio. US 9, 175,018 B2 35 36 maceutical composition comprises the compound of formula (VIII) (I-1), or a crystalline form thereof, wherein the compound of OH formula (I-1) is present in an amount equivalent to a molar weight basis of about 0.46 mg to about 0.54 mg of the com N1 B YOH pound of formula (VIII-1). In still some further embodiments, the unit dose pharmaceutical composition comprises the Ra2 O compound of formula (I-1), or a crystalline form thereof, wherein the compound of formula (I-1) is present in an amount equivalent to a molar weight basis of about 1.80 mg to Using an analytical method that involves hydrolytic con 10 about 2.20 mg of the compound of formula (VIII-1). ditions for sample preparation, the amount of the compound In some embodiments, the amount of the compound of of formula (VIII) presentinatest sample is measured (see e.g. formula (I-1), or a crystalline form thereof, present in a phar Analytical Test Method 1, below), by comparison to a refer maceutical composition is expressed as the equivalent ence standard of known purity. Using an analytical method amount of the compound of formula (VIII-1), based on the that does not subject the sample to hydrolytic conditions, the 15 relative molecular weights of the compound of formula (I-1) amount of the compound of formula (VIII) present in the and the compound of formula (VIII-1). sample of the compound of formula (I) is measured by com In some embodiments, the unit dose pharmaceutical com parison to a reference standard of known purity (see e.g. position comprises about 0.143 mg to about 4.3 mg of the Analytical Test Method 2 below). Therefore, the amount of compound of formula (I-1), or a crystalline form thereof, the compound of formula (VIII) measured in Analytical Test measured as about 0.1 mg to about 3.0 mg of the compound of Method 1 minus the amount of the compound of formula formula (VIII-1), on a weight for weight basis. (VIII) measured in Analytical Test Method 2, gives the In some other embodiments, the unit dose pharmaceutical amount of the compound of formula (VIII) in the sample that composition comprises about 0.214 mg to about 3.15 mg of is derived from hydrolysis of the compound of formula (I). the compound of formula (I-1), or a crystalline form thereof, Based on a 1:1 molecular ratio for the conversion of the 25 measured as about 0.15 mg to about 2.2 mg of the compound compound of formula (I) to the compound of formula (VIII), of formula (VIII-1), on a weight for weight basis. a molecular weight conversion gives the amount of com In yet some other embodiments, the unit dose pharmaceu pound of formula (I) present in the test sample. tical composition comprises about 0.258 mg to about 0.315 It will be recognized that such analytical methods as mg of the compound of formula (I-1), or a crystalline form described directly above, and in the Experimental section 30 thereof, measured as about 0.18 mg to about 0.22 mg of the below are applicable in a similar manner to any of the com compound of formula (VIII-1), on a weight for weight basis. pounds of formulas (I), (II), (III), (IIIa), (IV), or (IVa) and In still yet some other embodiments, the unit dose pharma various embodiments of these formula as described herein. ceutical composition comprises about 0.659 mg to about Such analytical methods as described directly above and in 0.773 mg of the compound of formula (I-1), or a crystalline the Experimental section below are applicable in a similar 35 form thereof, measured as about 0.46 mg to about 0.54 mg of manner to the compounds (I-1), (I-15), or (I-18). the compound of formula (VIII-1), on a weight for weight In some embodiments, the amount of the compound of basis. formula (VIII) present in a pharmaceutical composition is In some further embodiments, the unit dose pharmaceuti determined by measuring the amount of the compound of cal composition comprises about 2.58 mg to about 3.15 mg of formula (VIII) that is present after subjecting to the sample to 40 the compound of formula (I-1), or a crystalline form thereof, conditions under which the compound of formula (I) is measured as about 1.80 mg to about 2.20 mg of the compound hydrolyzed to the compound of formula (VIII). of formula (VIII-1), on a weight for weight basis. In some embodiments, the amount of the compound of In some embodiments, the invention provides a process for formula (I-1), or a crystalline form thereof, present in a phar the production of an oral pharmaceutical dosage form of the maceutical composition is expressed as the equivalent 45 compound of formula (I), or a crystalline form thereof, amount on a molar weight basis of the compound of formula wherein the oral pharmaceutical dosage form is a capsule, (VIII-1). comprising the steps of In some embodiments, the invention relates to a unit dose (a-1) mixing together screened filler and Screened com pharmaceutical composition comprising the compound of pound of formula (I), or a crystalline form thereof, in a formula (I-1), or a crystalline form thereof. 50 bag; In some other embodiments, the unit dose pharmaceutical (a-2) passing the resulting mixture from step (a-1) through composition comprises the compound of formula (I-1), or a a screen, then blending: crystalline form thereof, wherein the compound of formula (a-3) screening additional filler through the same screen, (I-1) is present in an amount equivalent to a molar weight passing it through the same bag, and blending in the basis of about 0.1 mg to about 3.0 mg of the compound of 55 same blending apparatus; formula (VIII-1). In yet some other embodiments, the unit (a-4) repeating step (a-3) up to two times; dose pharmaceutical composition comprises the compound (a-5) taking the resulting mixture from step (a-4), and of formula (I-1), or a crystalline form thereof, wherein the encapsulating it using a capsule-filling system; and compound of formula (I-1) is present in an amount equivalent (a-6) weight-sorting the resulting capsules from step (a-5). to a molar weight basis of about 0.15 mg to about 2.2 mg of 60 In some embodiments, step (a-3) may be repeated three or the compound of formula (VIII-1). In still yet some other more times. embodiments, the unit dose pharmaceutical composition When a lubricant is present in the pharmaceutical compo comprises the compound of formula (I-1), or a crystalline sition, the invention provides a process for the production of form thereof, wherein the compound of formula (I-1) is an oral pharmaceutical dosage form of the compound of present in an amount equivalent to a molar weight basis of 65 formula (I), or a crystalline form thereof, wherein the oral about 0.18 mg to about 0.22 mg of the compound of formula pharmaceutical dosage form is a capsule, comprising the (VIII-1). In some further embodiments, the unit dose phar steps of US 9, 175,018 B2 37 38 (b-1) mixing together screened filler, and screened com The mixing step using the high shear mixing apparatus can pound of formula (I), or a crystalline form thereof, in a take place in any conventional high shear mixing apparatus. bag; An example of such a high shear mixing apparatus is sold as (b-2) passing the resulting mixture from step (b-1) through Lab High Shear Granulator (Key International, Inc., English a screen, then blending: 5 town, N.J.). In some embodiments, the mixing is performed (b-3) screening additional filler through the same screen, for less than about 10 minutes. In some other embodiments, passing it through the same bag, and blending in the the mixing is performed for less than about 5 minutes. same blending apparatus; The capsule filling step outlined above can take place in (b-4) repeating step (b-3) up to two times; any conventional capsule filling system or apparatus. In some (b-5) blending together the mixture from step (b-4), and 10 embodiments, the capsule filling system is semi-automated, screened lubricant; and can handle Small batch sizes. An example of Such a (b-6) taking the resulting mixture from Step (b-5), and capsule filling system is sold as In-Cap (Isopak Limited, encapsulating it using a capsule-filling system; and Lincolnshire, Stamford, United Kingdom). In some embodi (b-7) weight-sorting the resulting capsules from step (b-6). ments, the capsule filling system is manual. An example of In some embodiments, step (b-3) may be repeated three or 15 such a capsule filling apparatus is sold as ProFill 100 (Torpac, more times. When additional components are present in the Inc., Fairfield, N.J., USA). pharmaceutical composition, such as buffer, second filler, or In some embodiments, the capsules are hard gelatin cap flow-aid, they may be added in any of steps (b-1) or (b-3). The sules, sold as Coni-Snap(R) (Capsugel, Peapack, N.J.). One of total amount of each component of the pharmaceutical com skill in the art will be able to select the appropriate capsule position may be added in one step or may be broken into size and color. In some embodiments, the capsules have a fill several amounts, which may or may not be of equal weight, weight of 85 mg, 120 mg. or 150 mg. and added in individual occurrences of steps (b-1) or (b-3). The weight-sorting step outlined above can take place In some embodiments, the invention provides a process for using any conventional weight-sorting apparatus or machine. the production of an oral pharmaceutical dosage form of the An example of a weight-sorting apparatus or machine is sold compound of formula (I), or a crystalline form thereof, 25 as the SADE SP Bench Top Tablet and Capsule Weight Sorter wherein the oral pharmaceutical dosage form is a capsule, (AC Compacting LLC, North Brunswick, N.J., USA). comprising the steps of In some embodiments, the capsules are packaged in (c-1) passing filler through a screen, then placing in a high bottles, foil pouches or blister packs. In some other embodi shear mixing apparatus; ments, the capsules are packaged in heat-induction sealed (c-2) passing the compound of formula (I), or a crystalline 30 high-density polyethylene (HDPE) bottles. In another form thereof, through a screen, then placing in the same embodiment, the capsules are packaged in air-tight sealed foil high shear mixing apparatus: pouches. In yet another embodiment, the capsules are pack (c-3) passing filler through a screen, then placing in the aged in foil-foil blister packs. In some other embodiments, same high shear mixing apparatus; the capsules are packaged with a desiccant. (c-4) mixing using the same high shear mixing apparatus 35 The physical and chemical stability of the oral pharmaceu for less than 10 minutes; tical dosage form may be tested in a conventional manner, for (c-5) taking the resulting mixture from step (c-4), and example, the measurement of dissolution, disintegration encapsulating it using a capsule-filling system; and time, assay for the compound of formula (I) degradation (c-6) weight-sorting the resulting capsules from Step (c-5). products, after storage at different temperatures for different In some embodiments, when using the high shear mixing 40 lengths of time. apparatus, additional components that are present in the phar In some other embodiments, the invention provides phar maceutical composition may be added by repeating either maceutical compositions for parenteral use. In yet some other step (c-1) or step (c-3). embodiments, the invention provides liquid pharmaceutical In some embodiments, the compound of formula (I) used in compositions for parenteral or oral use. the processes for preparation of Solid oral dosage forms 45 In some embodiments, the compound of formula (I) is described above is selected from the group consisting of (I-1), formulated as a lyophilized powder, in a manner analogous to (I-15), and (I-18). In some embodiments, the compound of that described in Plamondon et al., WO 02/059131, hereby formula (I) used in the processes for preparation of solid oral incorporated by reference in its entirety. In such embodi dosage forms described above is (I-1). ments, an aqueous mixture comprising a alpha-hydroxy car The process steps outlined above can take place using 50 boxylic acid or a beta-hydroxy carboxylic acid is lyophilized conventional apparatus and equipment. For a review, see e.g. to form the compound of formula (I). Remington. The Science and Practice of Pharmacy, 21 Ed., In some embodiments, the lyophilized powder also com Lippincott Williams & Wilkins, 2005. prises free alpha-hydroxy carboxylic acid, or beta hydroxy The blending steps outlined above can take place in any carboxylic acid. Preferably, the free alpha-hydroxy carboxy conventional blending apparatus. In some embodiments, the 55 lic acid or beta hydroxy carboxylic acid compound and the blending time for each individual blending step is between compound of formula (I) are present in the mixture in a molar about 1 minute and about 45 minutes. In some other embodi ratio ranging from about 0.5:1 to about 100:1, more prefer ments, the blending time for each individual blending step is ably from about 5:1 to about 100:1. In various embodiments between about 1 minute and about 20 minutes. In yet some wherein the alpha-hydroxy carboxylic acid or beta-hydroxy other embodiments, the blending time for each individual 60 carboxylic acid compound is citric acid, the lyophilized pow blending step is between about 2 minutes and about 15 min der comprises free citric acid and the corresponding boronate utes. ester in a molar ratio ranging from about 10:1 to about 100:1, The mixing step outlined above can take place in any from about 20:1 to about 100:1, or from about 40:1 to about conventional polyethylene bag. In some embodiments, the 100:1. mixing step takes between about 30 seconds and 5 minutes. In 65 In some embodiments, the lyophilized powder comprises Some embodiments, the mixing step outlined above can take citric acid and a compound of formula (I), Substantially free place in a stainless Steel container. of other components. However, the composition can further US 9, 175,018 B2 39 40 comprise one or more other pharmaceutically acceptable m is 0 or 1; excipients, carriers, diluents, fillers, salts, buffers, bulking the bulking agent is glycine; and agents, stabilizers, solubilizers, and other materials well the buffer is sodium citrate, and citric acid. known in the art. The preparation of pharmaceutically accept In some embodiments, the pharmaceutical composition able formulations containing these materials is described in, 5 comprises the compound of formula (I), a bulking agent, and e.g., Remington. The Science and Practice of Pharmacy, 20th a buffer; wherein: Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000, or the compound of formula (I) is represented by compounds latest edition, and Strickley, Pharmaceutical Research, 21(2) (I-1), (I-15) or (I-18): 201-230 (2004). the bulking agent is glycine; and Upon dissolution in aqueous medium, equilibrium is estab 10 the buffer is sodium citrate, and citric acid. lished between the boronate ester compound of formula (I) In some embodiments, the pharmaceutical composition and the corresponding free boronic acid compound. In some comprises the compound of formula (I), a bulking agent, and embodiments, equilibrium is reached quickly, e.g., within a buffer in a lyophilized powder; wherein: 1-15 minutes, after the addition of aqueous medium. The the alpha-hydroxy carboxylic acid or beta-hydroxy car relative concentrations of boronate ester, boronic acid, and 15 boxylic acid is citric acid; any intermediate species present at equilibrium is dependent A is 0; upon parameters such as, e.g., the pH of the Solution, tem R" is isobutyl: perature, the nature of the alpha-hydroxy carboxylic acid or R" is hydrogen, Caliphatic, -(CH2), CH. R. or beta-hydroxy carboxylic acid, and the ratio of the alpha -(CH), CH(R) OR; hydroxy carboxylic acid or beta-hydroxy carboxylic acid to P is R. C(O) ; boronate ester compound of formula (I) present in the lyo R is RP; philized powder. m is 0 or 1; In some embodiments, the pharmaceutical composition the bulking agent is glycine; and comprises the compound of formula (I), a bulking agent, and the buffer is sodium citrate and citric acid. a buffer. In some other embodiments, the pharmaceutical 25 In some embodiments, the pharmaceutical composition composition comprises the compound of formula (I), a bulk comprises the compound of formula (I), a bulking agent, and ing agent, and a buffer in a lyophilized powder. a buffer in a lyophilized powder; wherein: In some embodiments, the compound of formula (I) is the compound of formula (I) is represented by compounds pre-formed. In some other embodiments, the compound of (I-1), (I-15) or (I-18): formula (I) is formed in situ, from the corresponding boronic 30 the bulking agent is glycine; and acid of formula (VIII). In yet some other embodiments, the the buffer is sodium citrate and citric acid. compound (I-1) is pre-formed. In still yet some other embodi In some embodiments, the pharmaceutical composition ments, the compound (I-15) is formed in situ from compound comprises the compound (I-1) in a lyophilized powder. In (VIII-15). Some other embodiments, the pharmaceutical composition Suitable bulking agents include glycine. In some embodi 35 comprises the compound (I-1), glycine, Sodium citrate, and ments, the amount of the bulking agent present is about 1% citric acid in a lyophilized powder. In yet some other embodi weight/volume (w/v) to about 5% w/v. In some other embodi ments, the pharmaceutical composition comprises the com ments, the amount of the bulking agent present is about 3% pound (I-15) in a lyophilized powder. In still yet some other w/v. embodiments, the pharmaceutical composition comprises the Suitable buffers include sodium citrate, citric acid, and 40 compound (I-15), glycine, sodium citrate, and citric acid in a mixtures thereof. In some embodiments, the buffer is sodium lyophilized powder. citrate, and citric acid. In some embodiments, the invention provides a unit dose In some embodiments, the buffer is present in a concentra pharmaceutical composition comprising the compound of tion of about 45 mM to about 65 mM. In some other embodi formula (I-1), a bulking agent, and a buffer in a lyophilized ments, the buffer is present in a concentration of about 50 mM 45 powder. In some embodiments, the unit dose pharmaceutical to about 60 mM. composition comprises the compound of formula (I-1), gly In some embodiments, the ratio of the buffer to the com cine, sodium citrate, and citric acid in a lyophilized powder. pound of formula (I) is from about 50:1 to about 10:1. In some In some embodiments, the compound of formula (I-1) is other embodiments, the ratio of the buffer to the compound of present in the unit dose pharmaceutical composition in an formula (I) is from about 30:1 to about 10:1. In yet some other 50 amount equivalent on a molar weight basis of about 1 mg to embodiments, the ratio of the buffer to the compound of about 10 mg of the compound of formula (VIII-1). In some formula (I) is about 20:1. embodiments, the compound of formula (I-1) is present in the In some embodiments, the pH of the pharmaceutical com unit dose pharmaceutical composition in an amount equiva position is between about pH 4.7 and pH 6.1. The pH of the lent on a molar weight basis of about 1 mg to about 5 mg of the pharmaceutical composition can be adjusted using any Suit 55 compound of formula (VIII-1). In some embodiments, the able inorganic acid or organic acid. compound of formula (I-1) is present in the unit dose phar In some embodiments, the pharmaceutical composition maceutical composition in an amount equivalent on a molar comprises the compound of formula (I), a bulking agent, and weight basis of about 1.0 mg, about 1.5 mg, about 2.0 mg. a buffer; wherein: about 2.5 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or the alpha-hydroxy carboxylic acid or beta-hydroxy car 60 about 5.0 mg of the compound of formula (VIII-1). In some boxylic acid is citric acid; embodiments, the compound of formula (I-1) is present in the A is 0; unit dose pharmaceutical composition in an amount equiva R" is isobutyl: lent on a molar weight basis of about 3.5 mg of the compound R" is hydrogen, Caliphatic, -(CH2), CH. R. or of formula (VIII-1). -(CH), CH(R) OR; 65 In Some embodiments, the amount of glycine present in the P is R. C(O) ; unit dose pharmaceutical composition is about 0.01 g to about R is RP: 0.50 g. In some embodiments, the amount of glycine present US 9, 175,018 B2 41 42 in the unit dose pharmaceutical composition is about 0.03 g to iv. an alpha-hydroxy carboxylic acid, or a salt thereof; or about 0.250 g. In some embodiments, the amount of glycine a beta-hydroxy carboxylic acid, or a salt thereof, or a present in the unit dose pharmaceutical composition is about combination thereof; to form a mixture; and 0.06 g to about 0.125 g. (e-2) lyophilizing the mixture. In some embodiments, the sodium citrate and citric acid is 5 In some embodiments, the aqueous solvent mixture com present in the unit dose pharmaceutical composition in an prises one or more co-solvents in addition to water. In some amount equivalent to about 0.005 g to about 0.250 g of citrate embodiments, the co-solvent is miscible with water. In some ion. In some embodiments, the Sodium citrate and citric acid other embodiments, the co-solvent is an alcohol including, is present in the unit dose pharmaceutical composition in an but not limited to, ethanol, tert-butyl alcohol and mixtures 10 thereof. In some other embodiments, the co-solvent is tert amount equivalent to about 0.025 g to about 0.125 g of citrate butyl alcohol. 1O. In some embodiments, the aqueous solvent mixture com In some embodiments, the invention provides a unit dose prises about 1% V/v to about 40% V/v alcohol. In some other pharmaceutical composition comprising the compound of embodiments, the aqueous solvent mixture comprises about formula (I-15), a bulking agent, and a buffer in a lyophilized 15 3% V/v to about 10% V/v alcohol. In some other embodi powder. In some embodiments, the unit dose pharmaceutical ments, the aqueous solvent mixture comprises about 3% V/v composition comprises the compound of formula (I-15), gly to about 6% v/v alcohol. In yet some other embodiments, the cine, sodium citrate, and citric acid in a lyophilized powder. solvent mixture comprises about 3% V/v to about 6% V/v In some embodiments, the compound of formula (I-15) is tert-butyl alcohol. In still yet some other embodiments, the present in the unit dose pharmaceutical composition in an solvent mixture comprises about 5% V/v tert-butyl alcohol. amount equivalent on a molar weight basis of about 1 mg to In some embodiments, a method is provided for preparing about 10 mg of the compound of formula (VIII-15). In some the compound (I-1) as a lyophilized powder, the method embodiments, the compound of formula (I-15) is present in comprising the steps: the unit dose pharmaceutical composition in an amount (f1) combining: equivalent on a molar weight basis of about 1 mg to about 5 25 i. water; mg of the compound of formula (VIII-15). In some embodi ii. the compound (I-1); ments, the compound of formula (I-15) is present in the unit iii. glycine; dose pharmaceutical composition in an amount equivalent on iv. Sodium citrate; and a molar weight basis of about 1.0 mg, about 1.5 mg, about 2.0 V. citric acid; to form a mixture; and mg, about 2.5 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg. 30 (f-2) lyophilizing the mixture. or about 5.0 mg of the compound of formula (VIII-15). In In some embodiments, a method is provided for preparing some embodiments, the compound of formula (I-15) is the compound (I-15) as a lyophilized powder, the method present in the unit dose pharmaceutical composition in an comprising the steps: amount equivalent on a molar weight basis of about 3.5 mg of (g-1) combining: the compound of formula (VIII-15). 35 i. an aqueous solvent mixture comprising water and In some embodiments, the amount of glycine present in the tert-butyl alcohol: unit dose pharmaceutical composition is about 0.01 g to about ii. the compound (VIII-15); 0.50 g. In some embodiments, the amount of glycine present iii. glycine; in the unit dose pharmaceutical composition is about 0.03 g to iv. Sodium citrate; and about 0.250 g. In some embodiments, the amount of glycine 40 V. citric acid; to form a mixture; and present in the unit dose pharmaceutical composition is about (g-2) lyophilizing the mixture. 0.06 g to about 0.125 g. In some other embodiments, for the method described In some embodiments, the sodium citrate and citric acid is directly above, the amount of tert-butyl alcohol present in the present in the unit dose pharmaceutical composition in an aqueous solvent mixture is about 3% V/v to about 6% V/v. amount equivalent to about 0.005 g to about 0.250 g of citrate 45 The lyophilization or freeze-drying can be performed ion. In some embodiments, the Sodium citrate and citric acid using any conventionallyophilizers or freeze-dryers. In some is present in the unit dose pharmaceutical composition in an embodiments, the lyophilization comprises the steps: (i) amount equivalent to about 0.025 g to about 0.125 g of citrate loading the liquid mixture as prepared above, and freezing; 1O. (ii) annealing; (iii) second freeze cycle; (iv) drying under In another aspect, the invention provides a method for 50 vacuum; and (v) secondary drying. The temperatures and preparing the compound of formula (I) as a lyophilized pow times for each step will depend on the lyophilizer or freeze der; the method comprising the steps: dryer that is employed. (d-1) combining: In some embodiments, the resulting lyophilized powder i. an aqueous solvent mixture; has a residual moisture content of less than about 2%. In some ii. the compound of formula (I); 55 other embodiments, the resulting lyophilized powder has a iii. a bulking agent; and residual moisture content of less than about 1%. iv. a buffer; to form a mixture; and In another aspect, the invention provides a method for the (d-2) lyophilizing the mixture. preparation of a pharmaceutical composition of the com In some embodiments, the compound of formula (I) is pound of formula (I) as a liquid pharmaceutical dosage form, formed in situ from the corresponding compound of formula 60 said method comprising the step of reconstituting a lyo (VIII). Thus, the invention also provides a method for prepar philized powder of the compound of formula (I) with an ing the compound of formula (I) as a lyophilized powder; the aqueous solvent Suitable for pharmaceutical administration. method comprising the steps: Suitable reconstitution solvents include, but are not limited (e-1) combining: to, water, saline, phosphate buffered saline (PBS), and mix i. an aqueous solvent mixture; 65 tures thereof. In some embodiments, the reconstitution sol ii. the compound of formula (VIII); vent is water, water for injection, Saline, and mixtures thereof. iii. a bulking agent; and In some other embodiments, the reconstitution solvent is US 9, 175,018 B2 43 44 water for injection. Following reconstitution, the liquid phar Suitable tonicity modifiers include, but are not limited to, maceutical dosage form can contain concentrations of the amino acids such as arginine, histidine, and glycine; salts compound of formula (I) as described herein. Such as Sodium chloride, potassium chloride, Sodium citrate, In some embodiments, a method is provided for the prepa propylene glycol; and mixtures thereof. In some embodi ration of a pharmaceutical composition of the compound (I-1) ments, the tonicity modifier is propylene glycol. In some as a liquid pharmaceutical dosage form, said method com other embodiments, the tonicity modifier is sodium chloride. prising the step of reconstituting a lyophilized powder of the Upon dissolution in a aqueous solvent mixture, an equilib compound (I-1) as described herein with an aqueous solvent rium is established between the compound of formula (I) and Suitable for pharmaceutical administration. In some embodi the corresponding boronic acid of formula (VIII). Thus, ments, a method is provided for the preparation of a pharma 10 either a compound of formula (I) or a compound of formula ceutical composition of the compound (I-1) as a liquid phar (VIII) may be used in the preparation of the liquid pharma maceutical dosage form, said method comprising the step of ceutical composition. Typically, equilibrium is reached reconstituting a lyophilized powder of the compound (I-1) as quickly within about 10-15 minutes after the addition of the described herein with water for injection, or normal saline. In aqueous solvent mixture. The relative concentrations of the Some embodiments, a method is provided for the preparation 15 boronate ester and boronic acid present at equilibrium is of a pharmaceutical composition of the compound (I-1) as a dependent upon the pH of the Solution, temperature, and the liquid pharmaceutical dosage form, said method comprising ratio of the alpha-hydroxy or beta-hydroxy acid compound to the step of reconstituting a lyophilized powder of the com boronic acid compound. In some embodiments, the excess pound (I-1) as described herein with water for injection. alpha-hydroxy or beta-hydroxy acid may act as a stabilizer, In some embodiments, a method is provided for the prepa which pushes the equilibrium towards the boronate ester. In ration of a pharmaceutical composition of the compound Some embodiments, the tonicity modifier may also act as a (I-15) as a liquid pharmaceutical dosage form, said method stabilizer. comprising the step of reconstituting a lyophilized powder of In some embodiments, the liquid pharmaceutical compo the compound (I-15) as described herein with an aqueous sition optionally further comprises a preservative. Solvent Suitable for pharmaceutical administration. In some 25 In some embodiments, the liquid pharmaceutical compo embodiments, a method is provided for the preparation of a sition comprises the compound of formula (I), a buffer, and pharmaceutical composition of the compound (I-15) as a optionally a tonicity modifier; wherein: liquid pharmaceutical dosage form, said method comprising the alpha-hydroxy carboxylic acid or beta-hydroxy car the step of reconstituting a lyophilized powder of the com boxylic acid is citric acid; pound (I-15) as described herein with water for injection, or 30 A is 0; normal saline. In some embodiments, a method is provided R" is isobutyl: for the preparation of a pharmaceutical composition of the R" is hydrogen, Caliphatic, -(CH2), CH. R. or compound (I-15) as a liquid pharmaceutical dosage form, -(CH), CH(R) OR; said method comprising the step of reconstituting a lyo P is R. C(O) ; philized powder of the compound (I-15) as described herein 35 R is RP; with water for injection. m is 0 or 1; Upon reconstitution in a reconstitution solvent, an equilib the buffer is sodium citrate and citric acid; and rium is established between the compound of formula (I) and the tonicity modifier, when present, is sodium chloride. the corresponding boronic acid of formula (VIII). Typically, In some embodiments, the liquid pharmaceutical compo equilibrium is reached quickly within about 10-15 minutes 40 sition comprises the compound of formula (I), a buffer, and after the addition of the reconstitution solvent. The relative optionally a tonicity modifier; wherein: concentrations of the boronate ester and boronic acid present the compound of formula (I) is represented by compounds at equilibrium is dependent upon the pH of the solution, (I-1), (I-15) or (I-18): temperature, and the ratio of the alpha-hydroxy or beta-hy the buffer is sodium citrate and citric acid; and droxy acid compound to boronic acid compound. 45 the tonicity modifier, when present, is sodium chloride. In another aspect, the invention provides a liquid pharma In some embodiments, wherein the alpha-hydroxy car ceutical composition comprising the compound of formula boxylic acidorbeta-hydroxy carboxylic acid is citric acid, the (I), and additional excipients described herein. In some liquid pharmaceutical composition of the compound of for embodiments, the liquid pharmaceutical composition is Suit mula (I) comprises the compound of formula (I), water, citric able for parenteral use. In some other embodiments, the liquid 50 acid, Sodium citrate, and Sodium chloride. In some other pharmaceutical composition is Suitable for oral use. embodiments, wherein the alpha-hydroxy carboxylic acid or In Such embodiments, the liquid pharmaceutical composi beta-hydroxy carboxylic acid is citric acid, the liquid phar tion comprises the compound of formula (I), a buffer, and maceutical composition comprises the compound of formula optionally a tonicity modifier. (I), water, citric acid, and propylene glycol. In yet some other In some embodiments, the ratio of the buffer to the com 55 embodiments, the liquid pharmaceutical composition com pound of formula (I) is from about 50:1 to about 10:1. In some prises the compound of formula (I), wherein the compound of other embodiments, the ratio of the buffer to the compound of formula (I) is the compound (I-1), water, citric acid, sodium formula (I) is from about 30:1 to about 10:1. In yet some other citrate and sodium chloride. embodiments, the ratio of the buffer to the compound of In Such embodiments, wherein the alpha-hydroxy car formula (I) is about 20:1. 60 boxylic acidorbeta-hydroxy carboxylic acid is citric acid, the In some embodiments, the buffer is present in a concentra liquid pharmaceutical dosage form of the compound of for tion of about 45 mM to about 65 mM. In some other embodi mula (I) has a pH of between about pH 3 and about pH 7. In ments, the buffer is present in a concentration of about 50 mM certain such embodiments, the pH is between about pH 4.9 to about 60 mM. and about pH 6.7. In other certain such embodiments, the pH Suitable buffers include sodium citrate, citric acid, and 65 is between about pH 5.5 and about pH 6.5. mixtures thereof. In some embodiments, the buffer is sodium In some embodiments, wherein the alpha-hydroxy car citrate, and citric acid. boxylic acidorbeta-hydroxy carboxylic acid is citric acid, the US 9, 175,018 B2 45 46 liquid pharmaceutical composition of the compound of for mixture prior to step (h-5). In yet some other embodiments, mula (I) is prepared in situ from a stock vehicle Solution and after step (h-5), the liquid in the vials can be overlayed with the compound of formula (VIII). In some embodiments, the nitrogen prior to capping. stock vehicle solution comprises water, citric acid, sodium In some embodiments, the compound of formula (I) is citrate and propylene glycol. In such embodiments, the result formed in situ from the compound of formula (VIII). In such ing solution can be further diluted with stock vehicle solution embodiments, in step (h-2), the compound of formula (VIII), or with a sodium chloride solution to generate liquid pharma or a crystalline form thereof, is added to the mixture. In some ceutical compositions of the compound of formula (I) of embodiments, the alpha-hydroxy acid or beta-hydroxy acid is desired concentrations. added in step (h-2). In some other embodiments, the alpha 10 hydroxy acid or beta-hydroxy acid is present in step (h-1) as In another aspect the invention provides a unit dose liquid the buffer. pharmaceutical composition, comprising the compound of The pharmaceutical compositions of the invention prefer formula (I), a buffer, and optionally a tonicity modifier. In ably are formulated for administration to a patient having, or Some embodiments, the unit dose liquid pharmaceutical com at risk of developing or experiencing a recurrence of a pro position comprises the compound of formula (I), a buffer, and 15 teasome-mediated disorder. The term “patient’, as used optionally a tonicity modifier, wherein the compound of for herein, means an animal, preferably a mammal, more prefer mula (I) is the compound (I-1). In some embodiments, the ably a human. Preferred pharmaceutical compositions of the compound of formula (I) is present in the unit dose liquid invention are those formulated for oral, intravenous, or Sub pharmaceutical composition at a concentration of about 0.5 cutaneous administration. However, any of the above dosage mg/ml to about 3 mg/ml of the compound of formula (VIII). forms containing atherapeutically effective amount of a com In some other embodiments, the compound of formula (I) is pound of the invention are well within the bounds of routine present in the unit dose liquid pharmaceutical composition at experimentation and therefore, well within the scope of the a concentration of about 1 mg/ml of the compound of formula instant invention. In some embodiments, the pharmaceutical (VIII). In some other embodiments, wherein the compound of composition of the invention may further comprise another formula (I) is compound (I-1), the compound (I-1) in the unit 25 therapeutic agent. In some embodiments, such other thera dose liquid pharmaceutical composition is present at a con peutic agent is one that is normally administered to patients centration of about 0.5 mg/ml to about 3 mg/ml of the com with the disease or condition being treated. pound of formula (VIII-1). In yet some other embodiments, By “therapeutically effective amount' is meant an amount wherein the compound of formula (I) is compound (I-1), the Sufficient to cause a detectable decrease in proteasome activ compound (I-1) in the unit dose liquid pharmaceutical com 30 ity or the severity of a proteasome-mediated disorder. The position is present at a concentration of about 1 mg/ml of the amount of proteasome inhibitor needed will depend on the compound of formula (VIII-1). In still yet some other effectiveness of the inhibitor for the given cell type and the embodiments, wherein the compound of formula (I) is the length of time required to treat the disorder. It should also be compound (I-15), the compound (I-15) in the unit dose liquid understood that a specific dosage and treatment regimen for pharmaceutical composition is present at a concentration of 35 any particular patient will depend upon a variety of factors, about 1 mg/ml of the compound of formula (VIII-15). including the activity of the specific compound employed, the In some embodiments in the unit dose liquid pharmaceu age, body weight, general health, sex, and diet of the patient, tical composition, the Sodium citrate and citric acid are time of administration, rate of excretion, drug combinations, present in an amount equivalent to about 0.005 g to about the judgment of the treating physician, and the severity of the 0.250 g of citrate ion. In some embodiments in the unit dose 40 particular disease being treated. The amount of additional liquid pharmaceutical composition, the Sodium citrate and therapeutic agent present in a composition of this invention citric acid are present in an amount equivalent to about 0.025 typically will be no more than the amount that would nor g to about 0.125 g of citrate ion. mally be administered in a composition comprising that In some embodiments in the unit dose liquid pharmaceu therapeutic agent as the only active agent. Preferably, the tical composition, the Sodium chloride is present in an amount 45 amount of additional therapeutic agent will range from about of about 0.0045 g to about 0.09g. In some embodiments in the 50% to about 100% of the amount normally present in a unit dose liquid pharmaceutical composition, the sodium composition comprising that agent as the only therapeutically chloride is present in an amount of about 0.01 g to about 0.04 active agent. 9. In another aspect, the invention provides a method for In some embodiments in the unit dose liquid pharmaceu 50 treating a patient having, or at risk of developing or experi tical composition, the pharmaceutical composition is stored encing a recurrence of a proteasome-mediated disorder. As frozen until use. used herein, the term “proteasome-mediated disorder In another aspect the invention provides a method for pre includes any disorder, disease or condition which is caused or paring the compound of formula (I), as a unit dose liquid characterized by an increase in proteasome expression or pharmaceutical composition; the method comprising the 55 activity, or which requires proteasome activity. The term steps: “proteasome-mediated disorder also includes any disorder, (h-1) dissolving the buffer in an aqueous solvent; disease or condition in which inhibition of proteasome activ (h-2) dissolving the compound of formula (I), or a crystal ity is beneficial. line form thereof, in the mixture obtained in step (h-1); For example, compounds and pharmaceutical composi (h-3) dissolving the toncity modifier in the mixture 60 tions of the invention are useful in treatment of disorders obtained in step (h-2); mediated via proteins (e.g., NFkB, p27*P, p21 '''', p53) (h-4) adding further aqueous solvent to the required batch which are regulated by proteasome activity. Relevant disor Volume; and ders include inflammatory disorders (e.g., rheumatoid arthri (h-5) filling vials with an amount of the mixture obtained in tis, inflammatory bowel disease, asthma, chronic obstructive step (h-4). 65 pulmonary disease (COPD), osteoarthritis, dermatosis (e.g., In some embodiments, the vials are capped after step (h-5). atopic dermatitis, psoriasis)), Vascular proliferative disorders In some other embodiments, nitrogen is bubbled through the (e.g., atherosclerosis, restenosis), proliferative ocular disor US 9, 175,018 B2 47 48 ders (e.g., diabetic retinopathy), benign proliferative disor teasome inhibitor of the invention may be administered with ders (e.g., hemangiomas), autoimmune diseases (e.g., mul the other therapeutic agent in a single dosage form or as a tiple Sclerosis, tissue and organ rejection), as well as separate dosage form. When administered as a separate dos inflammation associated with infection (e.g., immune age form, the other therapeutic agent may be administered responses), neurodegenerative disorders (e.g., Alzheimer's prior to, at the same time as, or following administration of disease, Parkinson's disease, motor neurone disease, neuro the proteasome inhibitor of the invention. pathic pain, triplet repeat disorders, astrocytoma, and neuro In some embodiments, a proteasome inhibitor of formula degeneration as result of alcoholic liver disease), ischemic (I), or pharmaceutical composition of the compound of for injury (e.g., stroke), and cachexia (e.g., accelerated muscle mula (I) is administered in conjunction with an anticancer 10 agent. As used herein, the term “anticancer agent” refers to protein breakdown that accompanies various physiological any agent that is administered to a subject with cancer for and pathological states, (e.g., nerve injury, fasting, fever, aci purposes of treating the cancer. dosis, HIV infection, cancer affliction, and certain endocrino Non-limiting examples of DNA damaging chemothera pathies)). peutic agents include topoisomerase I inhibitors (e.g., irino The compounds and pharmaceutical compositions of the 15 tecan, topotecan, camptothecin and analogs or metabolites invention are particularly useful for the treatment of cancer. thereof, and doxorubicin); topoisomerase II inhibitors (e.g., As used herein, the term "cancer refers to a cellular disorder etoposide, teniposide, and daunorubicin); alkylating agents characterized by uncontrolled or disregulated cell prolifera (e.g., melphalan, chlorambucil, buSulfan, thiotepa, ifosfa tion, decreased cellular differentiation, inappropriate ability mide, carmustine, lomustine, Semustine, streptozocin, decar to invade surrounding tissue, and/or ability to establish new bazine, methotrexate, mitomycin C, and cyclophosphamide); growth at ectopic sites. The term “cancer includes, but is not DNA intercalators (e.g., cisplatin, oxaliplatin, and carbopl limited to, solid tumors and bloodborne tumors. The term atin); DNA intercalators and free radical generators such as "cancer encompasses diseases of skin, tissues, organs, bone, bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, cartilage, blood, and vessels. The term “cancer further capecitibine, gemcitabine, fludarabine, cytarabine, mercap encompasses primary and metastatic cancers. topurine, thioguanine, pentostatin, and hydroxyurea). Non-limiting examples of solid tumors that can be treated 25 Chemotherapeutic agents that disrupt cell replication with the disclosed proteasome inhibitors or pharmaceutical include: paclitaxel, docetaxel, and related analogs; Vincris compositions include pancreatic cancer, bladder cancer; col tine, vinblastin, and related analogs; thalidomide, lenalido orectal cancer, breast cancer, including metastatic breast can mide, and related analogs (e.g., CC-5013 and CC-4047); cer; prostate cancer, including androgen-dependent and protein tyrosine kinase inhibitors (e.g., imatinib mesylate and androgen-independent prostate cancer, renal cancer, includ 30 gefitinib); proteasome inhibitors (e.g., bortezomib); NF-kB ing, e.g., metastatic renal cell carcinoma; hepatocellular can inhibitors, including inhibitors of IKB kinase; antibodies cer, lung cancer, including, e.g., non-Small cell lung cancer which bind to proteins overexpressed in cancers and thereby (NSCLC), bronchioloalveolar carcinoma (BAC), and adeno downregulate cell replication (e.g., trastuzumab, rituximab, carcinoma of the lung; ovarian cancer, including, e.g., pro cetuximab, and bevacizumab); and other inhibitors of pro gressive epithelial or primary peritoneal cancer, cervical can teins or enzymes known to be upregulated, over-expressed or cer; gastric cancer, esophageal cancer, head and neck cancer, 35 activated in cancers, the inhibition of which downregulates including, e.g., squamous cell carcinoma of the head and cell replication. neck; melanoma; neuroendocrine cancer, including meta In order that this invention be more fully understood, the static neuroendocrine tumors; brain tumors, including, e.g., following preparative and testing examples are set forth. glioma, anaplastic oligodendroglioma, adult glioblastoma These examples illustrate how to make or test specific com multiforme, and adult anaplastic astrocytoma; bone cancer, 40 pounds, and are not to be construed as limiting the scope of and soft tissue sarcoma. the invention in any way. Non-limiting examples of hematologic malignancies that can be treated with the disclosed proteasome inhibitors or EXAMPLES pharmaceutical compositions include acute myeloid leuke mia (AML); chronic myelogenous leukemia (CML), includ 45 Abbreviations ing accelerated CML and CML blast phase (CML-BP); acute lymphoblastic leukemia (ALL); chronic lymphocytic leuke DCM methylene chloride mia (CLL); Hodgkin’s disease (HD); non-Hodgkin’s lym DIPEAN,N'-diisopropylethylamine phoma (NHL), including follicular lymphoma and mantle DMF N,N'-dimethylformamide cell lymphoma; B-cell lymphoma; T-cell lymphoma; mul 50 EDCI N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide tiple myeloma (MM); Waldenstrom's macroglobulinemia; hydrochloride myelodysplastic syndromes (MDS), including refractory EtOAc ethyl acetate anemia (RA), refractory anemia with ringed siderblasts h hours (RARS), (refractory anemia with excess blasts (RAEB), and HPLC high performance liquid chromatography RAEB in transformation (RAEB-T); and myeloproliferative syndromes. 55 MIBK methyl isobutyl ketone In some embodiments, the compound or pharmaceutical PES polyethersulfone compositions of the invention are used to treat a patient hav TBTU 0-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium ing or at risk of developing or experiencing a recurrence in a tetrafluoroborate cancer selected from the group consisting of multiple TFA trifluoroacetic acid myeloma and mantle cell lymphoma. 60 THF tetrahydrofuran In some embodiments, the proteasome inhibitor or phar HOBt 1-hydroxybenztriazole maceutical compositions of the invention is administered in LCMS liquid chromatography mass spectrum conjunction with another therapeutic agent. The other thera min minutes peutic agent may also inhibit the proteasome, or may operate General Methods by a different mechanism. In some embodiments, the other 65 therapeutic agent is one that is normally administered to The spectra are acquired at ambient temperature on a JOEL patients with the disease or condition being treated. The pro ECX-400 NMR spectrometer operating at 400 MHz for H US 9, 175,018 B2 49 50 NMR. The resulting FIDs are transferred to a PC and pro C. per minute. A nitrogen purge flowing at 60 mL per minute cessed using NUTS NMR processing software from Acorn is maintained over the sample during measurements. Typi NMR Inc. Chemical shifts are referenced to DMSO solvent, cally 5 mg to 15 mg of sample is loaded onto a pre-tared 2.50 ppm. A solvent blank is prepared by adding 0.75 mL of platinum crucible. DMSO-d6 to the NMR tube. After a "H spectrum is acquired 5 on the solvent blank, the sample is added and completely Example 1 dissolved. Mass Spectrometry: Synthesis of 4-(R.S)-(carboxymethyl)-2-((R)-1-(2- Mass spectrometry studies are run on a Thermo-Finnigan (2,5-dichlorobenzamido)acetamido)-3-methylbutyl)- LCQ Deca-XP ion trap mass spectrometer. The electrospray 10 6-oxo-1,3,2-dioxaborinane-4-carboxylic acid (I-1) ion source was used in both positive and negative modes with a high voltage of 5 Icy, sheathgas flow rate of 35 arb, capillary temperature of 275°C., capillary voltage of 9 V and tube lens offset of 35 V. An analyte was dissolved in acetonitrile to C O OH generate a 0.5 mg/ml solution. An Agilent 1100 HPLC system 15 was used for LC-Mass spectrometry flow analysis. The pump C O flow rate was 1.0 ml/minute. 10 ul of each sample solution Her was injected from the autosampler into a T-joint. About 2% of NaOH the Solution from the T-joint was infused into the mass spec trometer. X-Ray Powder Diffractometry (XRPD): C X-ray powder diffraction patterns are acquired on either: i) a Bruker AXS D8 Advance diffractometer. The data is I () collected over an angular range of 2.9° to 29.6° 20 in con CFCOOH.H.N.N-B- C? tinuous scan mode using a step size of 0.0520 and a step time 25 C O R of 2 seconds. The sample is run under ambient conditions and prepared as a flat plate specimen using powder as received OH N without grinding; or N1 O TBTU ii) a PANalytical XPert Pro diffractometer. Each specimen is analyzed using Cu radiation produced using an Optix long 30 C fine-focus Source. An elliptically graded multilayer mirror is used to focus the Cu K C. X-rays of the source through the specimen and onto the detector. The specimen is sandwiched C O between 3-micron thick films, analyzed in transmission geometry, and rotated to optimize orientation statistics. A 35 beam-stop is used to minimize the background generated by air scattering. Helium and the anti-scatter extension are not used. Soller slits are used for the incident and diffracted C beams to minimize axial divergence. The diffraction patterns are collected using a scanning position-minimize axial diver 40 C O O gence. The diffraction patterns are collected using a scanning R. I. position-sensitive detector (X Celerator) located 240 mm N ~ N1 3 citric acid from the specimen. Prior to the analyses, a silicon specimen (NIST standard reference material 640c) is analyzed to verify the position of the silicon 111 peak. 45 O s Differential Scanning Calorimetry (DSC): C Differential scanning calorimetry (DSC) data are collected O on either: i) a TA Instruments Q100 differential scanning calorimeter C O O equipped with a 50 position auto-sampler. The energy and 50 CO2H temperature calibration standard is indium. Samples are heated at a rate of 10° C. perminute between 25°C. and 300° C. A nitrogen purge flowing at 50 mL per minute is main CO2H tained over the sample during a scan. Between 1 mg and 3 mg of sample is analyzed. All samples are crimped in a hermeti 55 cally sealed aluminum pan with a pinhole to alleviate the C pressure accumulated from the solvent vapor; or ii) a TA Instruments differential scanning calorimeter 2920. The sample is placed into an aluminum DSC pan and the Step 1: 2.5-(dichlorobenzoyl)aminoacetic acid weight is accurately recorded. The open pan is covered with a 60 lid and then crimped. The sample cell is equilibrated at 25°C., To a mixture of NaOH (12 g,300 mmol) and glycine (18 g. and heated under a nitrogen purge at a rate of 10° C./min. 239 mmol) in water (120 mL) was added dropwise over 45 Indium metal was used as the calibration standard. min a solution of 2,5-dichlorobenzoyl chloride (10 g, 48 Thermal Gravimetric Analysis (TGA): mmol) in THF (15 mL) keeping the internal temperature Thermal gravimetric analysis (TGA) data are collected on 65 below about 25°C. After 1 h, the mixture was acidified with a TA Instruments Q500 thermal gravimetric analyzer, cali 2.0 M HCl (125 mL) keeping the internal temperature below brated with Nickel/Alumel and running at a scan rate of 10° about 5°C. The resulting precipitate was collected by vacuum US 9, 175,018 B2 51 52 filtration. The crude product was recrystallized from water to diluted with DCM (40 mL), the layers were separated, and the give 2.5-(dichlorobenzoyl)aminoacetic acid as a white, resulting aqueous layer was washed three times with DCM crystalline solid (6.1 g, 52%). mp 173.3° C. "H NMR (300 (3x10 mL). The combined DCM extracts were dried over MHz, DMSO-de 8): 12.72 (bs, 1H), 8.89 (t, J=6.0 Hz, 1H), MgSO4 (25 g) and evaporated to a thick oil. The product was 7.54 (m, 2H), 7.48 (m, 1H), 3.93 (d. 0.1=6.0 Hz). 'C NMR precipitated with heptane (50 mL) and collected by filtration (75 MHz, DMSO-de 8): 41.6, 129.3, 129.6, 1314, 132.2, to yield N,N',N'-boroxin-2,4,6-triyltris(1R)-3-methylbu 138.2, 171.4, 165.9. MS (m/z): M+H calculated for tane-1,1-diyl)imino(2-oxoethane-2,1-diyl)tris(2,5-dichlo CHC1NO. 248.0. found, 248.0: M+Na calculated for robenzamide) as a white solid (6.6 g., 74%). "H NMR (300 CHC1NNaO 270.0. found 270.2. MHz, DMSO-d, 8): 8.93 (t, J=6.0 Hz, 1H), 8.68 (bs, 1H), 2.5-(dichlorobenzoyl)aminoacetic acid was also be pre 10 7.63 (m. 1H), 7.52 (m, 2H), 4.00 (d. J=6.0 Hz, 2H), 2.62 (m, pared via the following procedure: To a mixture of glycine 1H), 1.59 (m, 1H), 1.33 (m, 1H), 1.24 (m, 1H), 0.81 (d. J=5.9 (21.5 g. 286 mmol) in water (437 mL), was added 2.0 M NaOH (130 mL) and the resulting solution was cooled to 0° Hz, 6H). 'C NMR (125 MHz, DMSO-de 8): 23.2, 25.8, C. A solution of 2,5-dichlorobenzoyl chloride (50.0 g, 239 40.1, 40.7, 43.0, 129.0, 130.0, 131.0, 137.5, 165.0, 172.5. MS mmol) in THF (75 mL) was added dropwise at such a rate that 15 (m/z) in CHCN: M+H calculated for CHBC1.N.O. the internal temperature was maintained at 0+1° C. During 1027.2. found, 1027.3; M+Na calculated for the addition, the pH was controlled at 11.0+0.2 using a pH CHSBC1NNaO. 1049.2. found 1049.5. controller titrated with 2.0MNaOH. After complete addition, Step 4: 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5- the mixture was stirred at 0+1° C. for an additional 2 h. The dichlorobenzamido)acetamido)-3-methylbutyl)-6- mixture was then acidified with 2.0 MHCl (176 mL) to a final pH of 2.5. The resulting precipitate was collected by filtra OXO-1,3,2-dioxaborinane-4-carboxylic acid (I-1) tion, washed with cold water (125 mL), and dried at 45°C. in a vacuum oven to afford 2.5-(dichlorobenzoyl)aminoacetic Form 1: acid as a white solid (57.6 g. 97.3%). To a solution of citric acid (2.75 g, 14.3 mmol) in EtOAc 25 (85 mL) with an internal temperature of about 74°C. was Step 2: 2,5-dichloro-N-2-((1R)-3-methyl-1-(3aS, added N,N',N'-boroxin-2,4,6-triyltris(1R)-3-methylbu 4S,6S,7aR)-3a,5.5-trimethylhexahydro-4,6-methano tane-1,1-diyl)imino(2-oxoethane-2,1-diyl))}tris(2.5- 1,3,2-benzodioxaborol-2-ylbutylamino)-2-oxoet dichlorobenzamide) (5.00 g, 4.87 mmol) as a solid. The solu hylbenzamide tion was cooled uncontrolled until the internal temperature 30 was about 25°C. and the mixture was stirred overnight. The To a solution of 2.5-(dichlorobenzoyl)aminoacetic acid resulting precipitate was collected by filtration to yield 2,2'- (6.10g, 24.6 mmol) and TBTU (8.34g, 26.0 mmol) in DMF {2-(1R)-1-((2,5-dichlorobenzoyl)aminoacetylamino)- (40 mL) with an internal temperature below about 5°C. was 3-methylbutyl-5-oxo-1,3,2-dioxaborolane-4.4- added (1R)-3-methyl-1-(3aS.4S,6S,7aR)-3a,5.5-trimethyl diyldiacetic acid Form 1 as a crystalline solid (6.65 g, 88%). hexahydro-4,6-methano-1,3,2-benzodioxaborol-2-ylbutan 35 "H NMR (500 MHz, DMSO-d 8 110° C.): 10.08 (s, 1H), 1-amine.TFA (9.35g, 24.7 mmol). DIPEA (13 mL, 75 mmol) 8.69 (s, 1H), 7.61 (s, 1H), 7.52 (d. J–1.3 Hz, 2H), 4.26 (d. was then added dropwise over 2 hkeeping the internal tem J=5.5 Hz, 2H), 2.70 (q, J=14.5 Hz, 4H), 2.70 (bs, 1H), 1.72 perature below about 5° C. After 40 min, the mixture was (sept, J=6.5 Hz, 1H), 1.42 (ddd, J=5.2 Hz, J=8.6 Hz, J=13.9 diluted with EtOAc (90 mL), washed with 5% NaCl (150 HZ, 1H), 1.28 (ddd, J=5.3, J=9.4 Hz, J=14.3 Hz, 1H), 0.91 mL), twice with 10% NaCl (2x40 mL), once with 2% KCO, 40 (dd, J–3.3 Hz, J=6.6 Hz, 6H). MS (m/z) in CHCN: M+Na (1x40 mL), once with 1% HPO (1x40 mL), and once with calculated for CHBCNNaO, 539.1. found, 539.1. 10% NaCl (1x40 mL). The resulting organic layer was con The XRPD data for I-1 Form 1 is shown in FIG. 1 and in centrated to a thick oil, diluted with heptane (40 mL) and Table 1. evaporated to yield 2,5-dichloro-N-2-((1R)-3-methyl-1- (3aS.4S,6S,7aR)-3a,5.5-trimethylhexahydro-4,6-methano 45 TABLE 1 1,3,2-benzodioxaborol-2-ylbutylamino)-2-oxoethylben Zamide as a white Solid which was used in the next step XRPD Data I-1 Form 1 without purification. Angle 20 o Intensity % 50 Step 3: N,N',N'-boroxin-2,4,6-triyltris(1R)-3- 6.441 100 methylbutane-1,1-diylimino(2-oxoethane-2,1-diyl) 8.304 29.5 tris(2,5-dichlorobenzamide) 10.35 19 11619 S.1 12.695 13.6 To a solution of 2,5-dichloro-N-2-({(1R)-3-methyl-1- 15.077 28.2 (3aS.4S,6S,7aR)-3a,5.5-trimethylhexahydro-4,6-methano 55 16.352 28.7 1,3,2-benzodioxaborol-2-ylbutylamino)-2-oxoethylben 17.504 16.3 18231 6 Zamide (12.2g, 24.6 mmol) in methanol/hexane (1:1) (250 19.086 21.4 mL) were added 1N HCl (30 mL, 30 mmol) and (2-methyl 20.405 11.7 propyl)boronic acid (6.5 g. 64 mmol). The reaction mixture 21.231 7.6 was allowed to stir overnight. The phases were separated and 60 21.916 7.6 the methanol layer was washed twice with additional heptane 25.371 15.2 (2x55 mL). The resulting organic layer was concentrated to 27.588 6.2 about 10 mL and partitioned between 2.0M NaOH (30 mL) and DCM (25 mL). The DCM layer was washed once with The Differential Scanning calorimetry (DSC) data for I-1 additional 2.0M NaOH (5 mL). The basic aqueous layers 65 Form 1 is shown in FIG. 2. The profile is characterized by an were then combined, washed twice with DCM (2x25 mL) and endothermic transition with anonset temperature of 191.8° C. acidified with 1M HCl (60 mL). The resulting mixture was with a melt of 198.8° C. A second endothermic transition US 9, 175,018 B2 53 54 corresponding to decomposition has an onset temperature of nane-4-carboxylic acid Form 2 was also prepared by dissolv 225°C. These temperatures have an error oft 5°C. ing in acetone followed by addition of EtOAc as an The Thermal Gravimetric Analysis (TGA) data for I-1 antisolvent. Form 1 is shown in FIG. 2. The profile graphs the percent loss The XRPD data for I-1 Form 2 is shown in FIG. 3 and in of weight of the sample as a function of temperature, the 5 Table 3. temperature rate change being about 10°C/min. The weight loss represents a loss of about 0.72% of the weight of the TABLE 3 sample as the temperature is changed from 50° C. to 200°C. These temperatures have an error of +5°C. XRPD Data I-1 Form 2 Form 2: To a solution of citric acid (10.1 g, 52.6 mmol) in 10 Angle EtOAc (300 mL) with an internal temperature of about 74°C. 2-Go Intensity % was added a solution of N,N',N'-boroxin-2,4,6-triyltris 5.817 100 (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- 7.614 93.4 11.575 71.1 diyl)tris(2,5-dichlorobenzamide) (20.0 g, 19.5 mmol) in 15 11.896 67.1 EtOAc (60 mL). The solution was cooled slowly (about 0.33° 12.571 24.3 C./min) until the internal temperature was about 60° C. and 14.43 32.2 16.689 65.8 the mixture was stirred for 3 h. The resulting slurry was 17.362 17.8 cooled slowly (rate of about 0.12°C./min) until the internal 18.232 53.9 temperature was about 25° C. and the mixture was stirred 19.596 77.6 overnight. The resulting precipitate was collected by filtration 19.959 63.8 20.376 36.2 to yield 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlo 20.998 32.2 robenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-diox 21.5 40.1 aborinane-4-carboxylic acid Form 2 as a crystalline Solid 21764 43.4 25 22.407 77.6 (26.7 g., 98%). "H NMR (500 MHz, DMSO-d 8 110° C.): 23.12 33.6 10.08 (s, 1H), 8.69 (s, 1H), 7.61 (s, 1H), 7.52 (d. J=1.3 Hz, 23.901 26.3 2H), 4.26 (d. J=5.5 Hz, 2H), 2.70 (q, J=14.5 Hz, 4H), 2.70 (bs, 24-402 20.4 24.882 19.7 1H), 1.72 (sept, J=6.5 Hz, 1H), 1.42(ddd, J=5.2 Hz, J=8.6 Hz, 25.764 19.1 J=13.9 Hz, 1H), 1.28 (ddd, J=5.3, J=9.4 Hz, J=14.3 Hz, 1H), 26.464 39.5 0.91 (dd, J=3.3 Hz, J=6.6 Hz, 6H). 'C NMR (100 MHz, 30 27.347 21.7 27.65 17.1 DMSO-d 8 100° C.): 21.65, 23.34, 25.09, 38.39, 38.98, 27.979 16.4 42.07, 76.25, 128.97, 129.14, 130.94, 131.48, 131.73, 29.41 20.4 137.05, 165.44, 170.23, 175.74, 17743. MS (m/z) in CHCN:M+Na calculated for CHBC1NNaO,539.1. found, 539.1. 35 The Differential Scanning calorimetry (DSC) data for I-1 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichloroben Form 2 is shown in FIG. 4. The profile is characterized by an Zamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori endothermic transition with anonset temperature of 206.5°C. nane-4-carboxylic acid Form 2 was also prepared by adding a with a melt of 219.9° C. A second endothermic transition solution of citric acid (21 g, 0.11 mmol) in THF (80 mL) to a corresponding to decomposition has an onset temperature of solution of N,N',N'-boroxin-2,4,6-triyltris(1R)-3-meth 40 225°C. These temperatures have an error oft 5°C. ylbutane-1,1-diyl)imino(2-oxoethane-2,1-diyl)tris(2.5- The Thermal Gravimetric Analysis (TGA) data for I-1 dichlorobenzamide) (40 g, 0.11 mmol) in THF (80 mL) at 60° Form 2 is shown in FIG. 4. The profile graphs the percent loss C. The solution was then seeded with Form 2 crystals (400 of weight of the sample as a function of temperature, the mg). After stirring for 30 min at 60°C., EtOAc (400 mL) was temperature rate change being about 10°C/min. The weight added over a period of 9 h. After complete addition of the 45 loss represents a loss of about 1.1% of the weight of the EtOAc, the temperature was lowered to 20°C. over 5h. The sample as the temperature is changed from 50° C. to 200° C. resulting Suspension was filtered to collect 4-(R.S)-(car These temperatures have an error of +5°C. boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-car Example 1A boxylic acid Form 2 as a crystalline solid (40 g, 70%). 50 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichloroben Alternate synthesis of 4-(R.S)-(carboxymethyl)-2- Zamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori ((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3- nane-4-carboxylic acid Form 2 was also prepared in the same methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxy general manner using the conditions described in Table 2. lic acid (I-1) Form 2 55 A 50-L glass reactor equipped with mechanical stirrer, TABLE 2 dropping funnel, temperature indicator, and heating/cooling Additional conditions for preparation of I-1 Form 2 control unit (under nitrogen) was charged with 1.2 micron filtered EtOAc (18.9 kg) and anhydrous citric acid (0.561 kg, Initial Seed Isolated Yield 60 2.9 mol). The mixture was heated to 71° C. and a solution Solvent Temperature Temperature I-1 Form 2 resulted. N,N',N'-boroxin-2,4,6-triyltris(1R)-3-meth acetonitrile 80° C. No seeding 779, ylbutane-1,1-diyl)imino(2-oxoethane-2,1-diyl)tris(2.5- MIBK 80° C. No seeding 80% dichlorobenzamide) (1.109 kg, 3.1 mol) dissolved in EtOAc 2-methyltetrahydrofuran 80° C. 60° C. 729% (4.0 kg) was clarified using an in-line filter (1.2 micron), and 65 the Solution was added to the reaction mixture under stirring 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichloroben (193 rpm) over a period of 20 min while maintaining a tem Zamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori perature of 73° C. to 75°C. The stirring was reduced to 96 rpm US 9, 175,018 B2 55 56 and the mixture was cooled as follows: (1) The mixture was EtOAc (1.0 mL). The solution was cooled uncontrolled until kept at 73° C.75° C. for 25 min; (2)The mixture was stepwise the internal temperature was about 25°C. and the solvent was cooled to 40°C. at the rate of approximately 5°C/30 min; (3) removed by evaporation to yield 2,5-dichloro-N-(2-(1R)- The mixture was allowed to cool uncontrolled overnight to 3-methyl-1-(4-oxo-1,3,2-dioxaborolan-2-yl)butylamino ambient temperature with stirring. The product was then iso 2-oxoethyl)benzamide as a white solid (0.215 g, 95%). MS lated by filtration, washed on the filter with 1.2 micron filtered (m/z) in CHCN: M--EtN+H calculated for EtOAc (2x1.2 kg), and dried under vacuum at 40-41° C. CHBC1.N.O.s, 502.2. found,502.0. MS (m/z) in CHCN: overnight (22 hours) to give 1.458 kg (92%) of the title M-H calculated for CHBC1NO,399.1. found,399.0. compound. "H NMR (400 MHz, DMSO-de 8): 12.13 (s. 2H), 10.69 (s, 1H), 9.11 (t, J=5.6 Hz, 1H), 7.66 (t, J=1.2 Hz, 1H), 10 Example 3 7.56 (d. J=1.2 Hz, 2H), 4.27 (bs, 2H), 2.9-2.55 (m, 5H), 1.67 Synthesis of{(4S)-2-[(1R)-14{(2,5-dichloroben (bs, 1H), 1.4-1.15 (bs, 2H), 0.86 (d. J=6.4 Hz, 6H). Zoyl)amino-acetylaminol-3-methylbutyl-5-oxo-1, The XRPD data for the compound (I-1) Form 2 is shown in 3.2-dioxaborolan-4-yl)acetic acid (I-3) FIG. 7 and in Table 6. 15 To a solution of L-malic acid (0.0958 g., 0.714 mmol) in TABLE 6 EtOAc (2.0 mL) with an internal temperature of about 60° C. Angle was added a solution of N,N',N'-boroxin-2,4,6-triyltris 2-Go Intensity % (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- S.69 100 diyl)tris(2,5-dichlorobenzamide) (0.239 g, 0.233 mmol) in 7.64 66 EtOAc (1.0 mL). The solution was cooled uncontrolled until 9.66 4 the internal temperature was about 25°C. and the solvent was 1122 23 removed by evaporation to yield {(4S)-2-[(1R)-1-({(2.5- 11.42 51 11.79 37 dichlorobenzoyl)amino-acetylamino)-3-methylbutyl-5- 25 12.41 15 OXO-1,3,2-dioxaborolan-4-yl)acetic acid as a white solid 1423 15 (0.307 g. 96%). MS (m/z) in CHCN: M+EtN+H calcu 15.60 6 16.53 32 lated for CHBC1NO7,560.1. found, 560.1. MS (m/z) in 17.15 4 CHCN: M-H calculated for CHBC1NO7, 457.1. 18.07 31 found, 457.1. 1939 55 30 19.79 41 Example 4 20.24 21 20.79 15 21.36 2O Synthesis of 2,5-dichloro-N-2-({(1R)-1-(4S)-4- 21.61 22 cyclohexyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3- 22.23 63 35 methylbutylamino)-2-oxoethylbenzamide (I-4) 22.55 14 22.97 2O 23.22 7 To a solution of (S)-hexahydromandelic acid (0.0881 g, 23.67 10 0.557 mmol) in EtOAc (2.0 mL) with an internal temperature 23.90 7 of about 60°C. was added a solution of N,N',N'-boroxin-2, 24.19 10 40 4,6-triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoet 24.74 7 24.97 3 hane-2,1-diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 25.64 8 mmol) in EtOAc (1.0 mL). The solution was cooled uncon 26.31 24 trolled until the internal temperature was about 25°C. and the 26.64 10 solvent was removed by evaporation to yield 2,5-dichloro-N- 27.21 7 45 27.40 7 2-({(1R)-1-(4S)-4-cyclohexyl-5-oxo-1,3,2-dioxaborolan 27.88 5 2-yl)-3-methylbutylamino)-2-oxoethylbenzamide as a 28.25 4 white solid (0.251 g, 93%). MS (m/z) in CHCN: M+EtN+ 29.27 11 H calculated for CHSBC1NOs 584.3. found, 584.1. MS 29.72 10 (m/z) in CHCN: M-H calculated for CHBC1NOs, 50 481.1. found 481.1. The Differential Scanning calorimetry (DSC) data for the compound (I-1) Form 2 is shown in FIG. 8. The profile is Example 5 characterized by two endothermic transitions; the first with a melt at about 231.3°C., and the second with a melt at about Synthesis of 2,5-dichloro-N-(2-(1R)-1-(4,4-dim 239.9°C. These temperatures have an error off 5° C. 55 ethyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3-methylbutyl amino-2-oxoethyl)benzamide (I-5) Example 2 To a solution of 2-hydroxyisobutyric acid (0.0567 g., 0.545 Synthesis of 2,5-dichloro-N-(2-(1R)-3-methyl-1- mmol) in EtOAc (2.0 mL) with an internal temperature of (4-oxo-1,3,2-dioxaborolan-2-yl)butylamino-2- 60 about 60°C. was added a solution of N,N',N'-boroxin-2,4, oxoethyl)benzamide (I-2) 6-triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoet hane-2,1-diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 To a solution of glycolic acid (0.041 g, 0.54 mmol) in mmol) in EtOAc (1.0 mL). The solution was cooled uncon EtOAc (2.0 mL) with an internal temperature of about 60° C. trolled until the internal temperature was about 25°C. and the was added a solution of N,N',N'-boroxin-2,4,6-triyltris 65 solvent was removed by evaporation to yield 2,5-dichloro-N- (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- (2-(1R)-1-(4,4-dimethyl-5-oxo-1,3,2-dioxaborolan-2-yl)- diyl)tris(2,5-dichlorobenzamide) (0.199 g, 0.19 mmol) in 3-methylbutylamino-2-oxoethyl)benzamide as a white US 9, 175,018 B2 57 58 solid (0.225 g, 96%). MS (m/z) in CHCN: M+EtN+H TABLE 4-continued calculated for CHBC1.N.O.s, 530.2. found, 530.0. MS (m/z) in CHCN: M-H calculated for CHBC1NOs, XRPD Data I-7 427.1. found, 427.0. Angle 2-Go Intensity % Example 6 12.195 100 13.71 7.3 Synthesis of 2,5-dichloro-N-2-({(1R)-3-methyl-1- 14.594 26.5 (5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl) 15.3O2 8.3 butylamino)-2-oxoethylbenzamide (I-6) 10 15.772 31 17.299 26.8 17.859 25.8 To a solution of (R)-mandelic acid (0.168 g, 1.10 mmol) in 18.549 22.7 EtOAc (2.0 mL) with an internal temperature of about 60° C. 19.943 55.5 was added a solution of N,N',N'-boroxin-2,4,6-triyltris 20.214 33.9 15 20.606 50 (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- 2148 15.6 diyl)tris(2,5-dichlorobenzamide) (0.382 g, 0.37 mmol) in 21.887 23 EtOAc (1.0 mL). The solution was cooled uncontrolled until 22.75 30.1 the internal temperature was about 25° C. and the resulting 23.028 53.1 23.334 28.9 precipitate was collected by filtration to yield 2,5-dichloro 24.243 18.2 N-2-({(1R)-3-methyl-1-(5R)-4-oxo-5-phenyl-1,3,2-diox 25.2 13.3 aborolan-2-ylbutylamino)-2-oxoethylbenzamide as a 2S.S66 37.7 27.221 10 white solid (0.343 g, 65%). "H NMR (300 MHz, DMSO-d 29.103 9.2 8): 10.88 (s, 1H), 9.22 (m, 1H), 7.68-7.27 (m, 8H), 5.15 (s, 29.383 12.6 1H), 4.33 (d. J=6.0 Hz, 2H), 2.8-2.76 (m, 1H), 1.71-1.62 (m, 1H), 1.50-1.28 (m, 2H), 0.89 (m, 6H). MS (m/z) in CHCN: 25 IM+EtN+H calculated for CHBCINOs 578.2. found, 578.1. MS (m/z) in CHCN: M-H calculated for Example 8 CHBC1.N.O.s, 475.1. found 475.1. Synthesis of 2,5-dichloro-N-2-({(1R)-3-methyl-1- Example 7 30 (4S)-4-methyl-6-oxo-1,3,2-dioxaborinan-2-yl) butylamino)-2-oxoethylbenzamide (I-8) Synthesis of 2,5-dichloro-N-2-({(1R)-3-methyl-1- (4S)-4-methyl-5-oxo-1.32-dioxaborolan-2-yl) To a solution of (S)-3-hydroxybutyric acid (0.0598 g. butylamino)-2-oxoethylbenzamide (I-7) 0.566 mmol) in EtOAc (2.0 mL) with an internal temperature 35 of about 60°C. was added a solution of N,N',N'-boroxin-2, To a solution of L-lactic acid (0.675 g, 7.34 mmol) in 4,6-triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoet EtOAc (3.0 mL) with an internal temperature of about 70° C. hane-2,1-diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 was added a solution of N,N',N'-boroxin-2,4,6-triyltris mmol) in EtOAc (1.0 mL). The solution was cooled uncon (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- trolled until the internal temperature was about 25°C. and the diyl)tris(2,5-dichlorobenzamide) (2.50 g, 2.43 mmol) in 40 solvent was removed by evaporation to yield 2,5-dichloro-N- EtOAc (7.5 mL). The solution was cooled uncontrolled until 2-({(1R)-3-methyl-1-(4S)-4-methyl-6-oxo-1,3,2-diox the internal temperature was about 60° C. After 30 min, aborinan-2-ylbutylamino)-2-oxoethylbenzamide as a heptane (11.5 mL) was added until the solution became tur white solid (0.225 g, 95%). H NMR (300 MHz, DMSO-d bid. The suspension was heated until the internal temperature 8): 10.45 (s, 1H), 9.11 (t, J=6.0 Hz, 1H), 7.65 (m, 1H), 7.55 was at or about 70° C., at which point a homogenous solution 45 (m. 2H), 4.21 (d. J=6.0 Hz, 2H), 3.98-3.90 (m, 1H), 2.51 (m, resulted. The solution was cooled at a rate of 0.17° C./min 1H), 2.33 (dd, J =19.2 Hz, J=2.7 Hz, 1H), 2.24-2.21 (m. 1H), until the internal temperature was about 30°C., then cooled 1.61-1.52 (m, 1H), 1.33-1.19 (m, 2H), 1.07-104 (m, 3H), uncontrolled until until the internal temperature was about 0° 0.84 (m, 6H). MS (m/z) in CHCN: M+EtN+H calculated C. The resulting precipitate was collected by filtration to yield for CH3BC1.N.O.s, 530.2. found, 530.0. MS (m/z) in 2.5-dichloro-N-2-({(1R)-3-methyl-1-(4S)-4-methyl-5- 50 CHCN: M-H calculated for CHBC1NO. 427.1. oxo-1,3,2-dioxaborolan-2-ylbutylamino)-2-oxoethylben found, 427.1. Zamide as a white, crystalline solid (2.32 g, 81%). MS (m/z) in CHCN: M+EtN+H calculated for CH,BC1NOs, Example 9 515.9, found, 516.0. MS (m/z) in CHCN: M-H calculated for C.H.BC1NOs, 413.1. found 413.0. 55 Synthesis of 2,5-dichloro-N-(2-(1R)-1-(4,4-dim The XRPD data for I-7 is shown in FIG. 5 and in Table 4. ethyl-6-oxo-1,3,2-dioxaborinan-2-yl)-3-methylbutyl amino-2-oxoethyl)benzamide (I-9) TABLE 4 To a solution f3-hydroxyisovaleric acid (0.0841 g, 0.712 XRPD Data I-7 60 mmol) in EtOAc (2.0 mL) with an internal temperature of Angle about 60°C. was added a solution of N,N',N'-boroxin-2,4, 2-Go Intensity % 6-triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoet hane-2,1-diyl)tris(2,5-dichlorobenzamide) (0.260 g, 0.253 7.404 46 8.783 63.5 mmol) in EtOAc (1.0 mL). The solution was cooled uncon 9.402 16.1 65 trolled until the internal temperature was about 25°C. and the 11.9 20.6 solvent was removed by evaporation to yield 2,5-dichloro-N- (2-(1R)-1-(4,4-dimethyl-6-oxo-1,3,2-dioxaborinan-2-yl)- US 9, 175,018 B2 59 60 3-methylbutylamino-2-oxoethyl)benzamide as a white lated for CH,BC1NNaOs, 479.1. found, 479.1. MS (m/z) solid (0.296 g. 95%). MS (m/z) in CHCN: M+EtN+H in CHCN: M-H calculated for CHBC1.N.O.s, 455.1. calculated for CHBC1NOs, 544.3. found, 544.0. MS found 455.1. (m/z) in CHCN: M-H calculated for CHBC1NOs, 44.1.1. found, 441.0. 5 Example 13 Example 10 Synthesis of 2,5-dichloro-N-(2-(1R)-3-methyl-1- (4-oxo-4H-1,3,2-benzodioxaborinin-2-yl)butyl Synthesis of 2,5-dichloro-N-2-({(1R)-1-(4S)-4-tert amino-2-oxoethyl)benzamide (I-13) butyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3- 10 methylbutylamino)-2-oxoethyl-2,5-dichlorobenza To a solution of salicylic acid (0.075.8 g., 0.549 mmol) in EtOAc (2.0 mL) with an internal temperature of about 60° C. mide (I-10) was added a solution of N,N',N'-boroxin-2,4,6-triyltris To a solution of (S)-2-hydroxy-3,3-dimethylbutyric acid (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- 15 diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 mmol) in (0.0712g, 0.553 mmol) in EtOAc (2.0 mL) with an internal EtOAc (1.0 mL). The solution was cooled uncontrolled until temperature of about 60°C. was added a solution of N,N',N'- the internal temperature was about 25° C. and the resulting {boroxin-2,4,6-triyltris(1R)-3-methylbutane-1,1-diyl precipitate was collected by filtration to yield 2,5-dichloro imino(2-oxoethane-2,1-diyl)tris(2,5-dichlorobenzamide) N-(2-(1R)-3-methyl-1-(4-oxo-4H-1,3,2-benzodioxabori (0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was nin-2-yl)butylamino-2-oxoethyl)benzamide as a white cooled uncontrolled until the internal temperature was about solid (0.198g, 78%). MS (m/z) in CHCN: M+Na calcu 25° C. and the solvent was removed by evaporation to yield lated for CHBC1NNaOs, 485.1. found, 485.1. MS (m/z) 2.5-dichloro-N-2-({(1R)-1-(4S)-4-tert-butyl-5-oxo-1,3,2- in CHCN: M-H calculated for CHBC1NOs, 461.1. dioxaborolan-2-yl)-3-methylbutylamino)-2-oxoethyl-2,5- found, 461.0. dichlorobenzamide as a white solid (0.245 g, 97%). MS (m/z) 25 The XRPD data for 1-13 is shown in FIG. 6 and in Table 5. in CHCN: M+EtN+H calculated for CHBC1NOs, 558.3. found, 558.0. MS (m/z) in CHCN: M-H calculated TABLE 5 for CHBC1NOs, 455.1. found, 455.0. XRPD Data I-13 Example 11 30 Angle 2-Go Intensity %

Synthesis of 2,5-dichloro-N-2-({(1R)-1-(4S)-4- 6.784 88.1 isopropyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3- 8.372 100 methylbutylamino)-2-oxoethylbenzamide (I-11) 11.855 66.6 35 13.18 85.2 To a solution of (S)-2-hydroxy-3-methylbutyric acid 14.118 7.7 14.546 19.3 (0.0659 g, 0.558 mmol) in EtOAc (2.0 mL) with an internal 15.614 9.6 temperature of about 60°C. was added a solution of N,N',N'- 16.123 19.3 {boroxin-2,4,6-triyltris(1R)-3-methylbutane-1,1-diyl 16.417 14.1 imino(2-oxoethane-2,1-diyl)tris(2,5-dichlorobenzamide) 40 16.738 7.7 17.29 43.7 (0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was 19. OS 17.4 cooled uncontrolled until the internal temperature was about 19.28 28.9 25° C. and the solvent was removed by evaporation to yield 19.726 52.1 2.5-dichloro-N-2-({(1R)-1-(4S)-4-isopropyl-5-oxo-1,3,2- 20.401 60.8 45 20.591 37.6 dioxaborolan-2-yl)-3-methylbutylamino)-2-oxoethylben 21.233 43.7 Zamide as a white solid (0.246g, 99%). MS (m/z) in CHCN: 21.658 16.7 IM+Na calculated for CHBCNNaOs, 465.1. found, 22.029 18.6 22.718 30.9 465.1. MS (m/z) in CHCN: M-H calculated for 23.557 41.5 CHBC1NOs, 441.1. found, 441.0. 24.236 22.2 50 24.717 62.1 Example 12 25.309 26 25648 13.5 26.1.86 69.1 Synthesis of 2,5-dichloro-N-2-({(1R)-1-(4S)-4- 26.653 17.4 isobutyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3- 26.995 36.3 methylbutylamino)-2-oxoethylbenzamide (I-12) 55 27.956 25.4 28.898 8.4 To a solution of 2-hydroxyisocaproic acid (0.0752g, 0.569 29.47 8.7 mmol) in EtOAc (2.0 mL) with an internal temperature of about 60° C. was added a solution of N,N',N'-boroxin-2,4, 6-triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoet 60 Example 14 hane-2,1-diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooled uncon Synthesis of 2,5-dichloro-N-(2-(1R)-3-methyl-1- trolled until the internal temperature was about 25°C. and the (5-oxo-4,4-diphenyl-1,3,2-dioxaborolan-2-yl)butyl solvent was removed by evaporation to yield 2,5-dichloro-N- amino-2-oxoethyl)benzamide (I-14) 2-({(1R)-1-(4S)-4-isobutyl-5-oxo-1,3,2-dioxaborolan-2- 65 yl)-3-methylbutylamino)-2-oxoethylbenzamide as a white To a solution of benzilic acid (0.126 g., 0.552 mmol) in solid (0.253 g, 95%). MS (m/z) in CHCN: M+Na calcu EtOAc (2.0 mL) with an internal temperature of about 60° C. US 9, 175,018 B2 61 62 was added a solution of N,N',N'-boroxin-2,4,6-triyltris {(1R)-3-methylbutane-1,1-diyl)imino (2S)-1-oxo-3-phe (1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane-2.1- nylpropane-1,2-diyl)tripyrazine-2-carboxamide (0.179 g, diyl)tris(2,5-dichlorobenzamide) (0.200 g, 0.195 mmol) in 0.163 mmol) as a solid. The solution was cooled uncontrolled EtOAc (1.0 mL). The solution was cooled uncontrolled until until the internal temperature was about 25° C. and the sol the internal temperature was about 25°C. and the solvent was vent was removed by evaporation to yield N-(1S)-1-benzyl removed by evaporation to yield 2,5-dichloro-N-(2-(1R)- 2-({(1R)-3-methyl-1-(4S)-4-methyl-6-oxo-1,3,2-diox 3-methyl-1-(5-oxo-4,4-diphenyl-1,3,2-dioxaborolan-2-yl) aborinan-2-ylbutylamino)-2-oxoethylpyrazine-2- butylamino-2-oxoethyl)benzamide as a white solid (0.291 carboxamide as a white solid (0.213 g, 96%). MS (m/z) in g, 95%). MS (m/z) in CHCN: M+Na calculated for CHCN: M+Na calculated for CHBNNaOs, 475.2. CHBC1NNaOs, 575..1. found, 575.2. MS (m/z) in 10 found, 475.2. MS (m/z) in CHCN: M-H calculated for CHCN: M-H calculated for CHBC1NOs, 551.1. CHBNOs, 451.2. found, 451.1. found, 551.1. Example 18 Example 15 15 Preparation of formulations of 4-(R.S)-(carboxym Synthesis of 2,2'-(2-(1R)-3-methyl-1-((2S)-3-phe ethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta nyl-2-(pyrazin-2-ylcarbonyl)amino mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4- propanoyl)amino)butyl-5-oxo-1,3,2-dioxaborolane carboxylic acid (I-1) for parenteral or oral 4.4-diy1}diacetic acid (I-15) administration To a solution of citric acid (0.257g, 1.34 mmol) in EtOAc Formulation A: A vessel was charged with 90 mL water and (7.4 mL) with an internal temperature of about 74°C. was citric acid monohydrate (0.08 g) and sodium citrate dihydrate added N,N',N'-(boroxin-2,4,6-triyltris (1R)-3-methylbu (1.5 g) were added and stirred until dissolved. To this solu tane-1,1-diylimino (2S)-1-oxo-3-phenylpropane-1,2-diyl tion, 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlo })tripyrazine-2-carboxamide (0.500 g, 0.455 mmol) as a 25 robenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-diox solid. The resulting solution was cooled uncontrolled until the aborinane-4-carboxylic acid (I-1) Form 2 (0.142 g) was internal temperature was about 25°C. and was evaporated to added, and the mixture was stirred until a solution was yield 2,2'-(2-(1R)-3-methyl-1-((2S)-3-phenyl-2- obtained. To this solution, sodium chloride (0.45 g) was (pyrazin-2-ylcarbonyl)aminolpropanoyl)amino)butyl-5- added, and the pH was adjusted to pH 5.45 using 2NHC1. The oxo-1,3,2-dioxaborolane-4,4-diyl diacetic acid as a white 30 final volume of the resulting solution was adjusted to 100 mL solid (0.730 g. 99%). MS (m/z) in CHCN: M+EtN+H with water and filtered through a 0.2 Lum PES membrane to calcd for CHSBNO, 642.3. found, 642.2. MS (m/z) in yield Formulation A which was stored at -20°C. CHCN: M-H calcd for CHBNO. 539.2. found, Formulation B was prepared as for Formulation A, except 539.2. that the pH was adjusted to pH 6.2 using 2N NaOH. 35 Formulation C: A vessel was charged with 90 mL water and Example 16 citric acid monohydrate (0.08 g), sodium citrate dihydrate (1.5 g), and propylene glycol (1.0 g) were added and stirred Synthesis of N-(1S)-1-benzyl-2-({(1R)-3-methyl-1- until dissolved. To this solution, 4-(R.S)-(carboxymethyl)-2- (5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl) ((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbu butylamino)-2-oxoethylpyrazine-2-carboxamide 40 tyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylic acid (I-1) Form (I-16) 2 (0.142 g) was added, and the mixture was stirred until a solution was obtained. The pH was adjusted to 6.2 using 2N To a solution of (R)-mandelic acid (0.0738g, 0.485 mmol) NaOH, and the final volume of the resulting solution was in EtOAc (2.0 mL) with an internal temperature of about 60° adjusted to 100 mL with water and filtered through a 0.2 um C. was added N,N',N'-(boroxin-2,4,6-triyltris (1R)-3-me 45 PES membrane to yield Formulation C which was stored at thylbutane-1,1-diyl)imino (2S)-1-oxo-3-phenylpropane-1, -20° C. 2-diyl)tripyrazine-2-carboxamide (0.178 g., 0.162 mmol) as a solid. The solution was cooled uncontrolled until the Example 19 internal temperature was about 25°C. and the resulting pre cipitate was collected by filtration to yield N-(1S)-1-benzyl 50 In situ preparation of formulations of 4-(R.S)-(car 2-({(1R)-3-methyl-1-(5R)-4-oxo-5-phenyl-1,3,2-diox boxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido) aborolan-2-ylbutylamino)-2-oxoethylpyrazine-2- acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxabori carboxamide as a white solid (0.195 g, 80%). MS (m/z) in nane-4-carboxylic acid (I-1) for parenteral or oral CHCN: M+Na calculated for C-HBNNaOs, 523.2. administration found, 523.2. MS (m/z) in CHCN: M-H calculated for 55 C.H.BNOs 499.2. found, 499.2. Stock Formulation Vehicle: A vessel was charged with approximately 160 mL water and citric acid monohydrate Example 17 (0.714 g) and sodium citrate dihydrate (2.24 g) were added and stirred until dissolved. To this solution, propylene glycol Synthesis of N-(1S)-1-benzyl-2-({(1R)-3-methyl-1- 60 (2.0 g) was added, and the mixture was stirred until a homo (5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl) geneous solution was obtained. The final pH was pH 5.14. butylamino)-2-oxoethylpyrazine-2-carboxamide The final weight of the resulting solution was adjusted to 200 (I-17) g (assuming density of 1 g/mL) with water and filtered through a 0.2 Lum PES membrane filter unit and stored at a To a solution of (S)-3-hydroxybutyric acid (0.0509 g, 65 temperature between about 2°C. and about 8°C. 0.489 mmol) in EtOAc (2.0 mL) with an internal temperature Formulation Stock (1 mg/mL): To a vessel, 0.105 grams of about 60° C. was added N,N',N'-(boroxin-2,4,6-triyltris (approximately 95.4% purity) of N,N',N'-boroxin-2,4,6- US 9, 175,018 B2 63 64 triyltris(1R)-3-methylbutane-1,1-diyl)imino(2-oxoethane cases, Small fragments of tumor tissue (approximately 1 2,1-diyl)tris(2,5-dichlorobenzamide) was added. To this mm) are implanted Subcutaneously in the right dorsal flank was added approximately 90 g of the Stock Formulation of anesthetized (3-5% isoflouraneoxygen mixture) C.B-17/ SCID mice (age 5-8 weeks, Charles River) via a 13-gatrocar Vehicle, and the resulting mixture was stirred for 48 hours (Popper & Sons 7927). Beginning at day 7 after inoculation protected from light. The final pH was pH 5.12. The final tumors are measured twice weekly using a vernier caliper. weight of the resulting solution was adjusted to 100 g (assum Tumor Volumes are calculated using standard procedures ing density of 1 g/mL) with Stock Formulation Vehicle and (0.5x(lengthxwidth)). When the tumors reach a volume of filtered through a 0.2 Lum PES membrane filter unit and stored approximately 200 mm mice are randomized into treatment protected from light at a temperature between about 2°C. and groups and begin receiving drug treatment. Dosing and about 8° C. 10 schedules are determined for each experiment based on pre Formulation D: The Formulation Stock was diluted to con vious results obtained from pharmacokinetic/pharmacody centrations of 0.05 mg/mL and 0.1 mg/mL with Stock For namic and maximum tolerated dose studies. The control mulation Vehicle prior to use. group will receive vehicle without any drug. Typically, test Formulation E: The Formulation Stock was diluted to con compound (100-200LL) is administered via intravenous (27 centrations of 0.05 mg/mL and 0.1 mg/mL with a 0.9% 15 ga needle), oral (20-ga gavage needle) or Subcutaneous (27 Sodium chloride Solution prior to use. ga needle) routes at various doses and Schedules. Tumor size and body weight are measured twice a week and the study is Example 20 terminated when the control tumors reach approximately 2000 mm. 20S Proteasome Assay Example 23 To 1 uL oftest compound dissolved in DMSO in a 384-well Synthesis of N—((S)-1-((R)-3-methyl-1-(4-oxo-4H black microtiter plate is added 25uL of assay buffer at 37°C. benzod 1.3.2dioxaborinin-2-yl)butylamino)-1- containing human PA28 activator (Boston Biochem, 12 nM oXo-3-phenylpropan-2-yl)pyrazine-2-carboxamide final) with Ac-WLA-AMC (B5 selective substrate) (15 uM 25 (I-19) final), followed by 25uL of assay buffer at 37° C. containing human 20S proteasome (Boston Biochem, 0.25 nM final). A mixture of N,N',N'-(boroxin-2,4,6-triyltris (1R)-3- Assay buffer is composed of 20 mM HEPES, 0.5 mM EDTA methylbutane-1,1-diylimino (2S)-1-oxo-3-phenylpropane and 0.01% BSA, pH7.4. The reaction is followed on a BMG 1,2-diyl)tripyrazine-2-carboxamide (0.250 g, 0.228 mmol) Galaxy plate reader (37°C., excitation 380 nm, emission 460 30 and salicyclic acid (269.6 mg, 0.68 mmol) were mixed in EtOAc (10 mL). The mixture was heated to form a solution. nm, gain 20). Percent inhibition is calculated relative to 0% The solution was cooled uncontrolled until the internal tem inhibition (DMSO) and 100% inhibition (10Mbortezomib) perature was about 25°C. Heptane (16 mL) was added. White controls. Solid precipitated out and the resultant slurry was agitated at ambient temperature for 3 h. The slurry was filtered to collect Example 21 35 solid N-4S)-1-((R)-3-methyl-1-(4-oxo-4H-benzod 1.3.2 dioxaborinin-2-yl)butylamino)-1-oxo-3-phenylpropan-2-yl) Antiproliferation Assay pyrazine-2-carboxamide (0.249 g, 75%). MS (m/z) in CHCN: M+H calculated for CHBN.O.s, 487.2153. HCT-116 (1000) or other tumor cells in 100 uL of appro found, 487.3. priate cell culture medium (McCoy's 5A for HCT-116. Invit 40 rogen) supplemented with 10% fetal bovine serum (Invitro Example 24 gen) are seeded in wells of a 96-well cell culture plate and Synthesis of 2-((S)-2-((R)-3-methyl-1-((S)-3-phenyl incubated overnight at 37°C. Test compounds are added to 2-(pyrazine-2-carboxamido)propanamido)butyl)-5- the wells and the plates are incubated for 96 hours at 37° C. oxo-1,3,2-dioxaborolan-4-yl)acetic acid (I-20) MTT or WST reagent (10 uL. Roche) are added to each well 45 and incubated for 4 hours at 37° C. as described by the A mixture of N,N',N'-(boroxin-2,4,6-triyltris (1R)-3- manufacturer. For MTT the metabolized dye is solubilized methylbutane-1,1-diylimino (2S)-1-oxo-3-phenylpropane overnight according to manufacturers instructions (Roche). 1,2-diyl)tripyrazine-2-carboxamide (0.500 g, 0.455 mmol) The optical density for each well is read at 595 nm (primary) and L-malic acid (213.6 mg, 0.55 mmol) were mixed in THF and 690 nm (reference) for the MTT and 450 nm for the WST 50 (5 mL). The mixture was heated to form a solution. The using a spectrophotometer (Molecular Devices). For the Solution was cooled uncontrolled until the internal tempera MTT the reference optical density values are subtracted from ture was about 25° C. White solid precipitated out and the the values of the primary wavelength. Percent inhibition is resultant slurry was agitated at ambient temperature for 1 h. calculated using the values from a DMSO control set to The slurry was filtered to collect solid 2-((S)-2-((R)-3-me 100%. 55 thyl-1-(S)-3-phenyl-2-(pyrazine-2-carboxamido)propana Example 22 mido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)acetic acid (0.625 g, 95%). MS (m/z) in CHCN: M+H calculated for In Vivo Tumor Efficacy Model CHBNO, 483.2051. found, 483.2. 60 Example 25 Freshly dissociated HCT-116 (2-5x10) or other tumor cells in 100 uL of RPMI-1640 media (Sigma-Aldrich) are Synthesis of 2-((R)-2-((R)-3-methyl-1-(S)-3-phe aseptically injected into the Subcutaneous space in the right nyl-2-(pyrazine-2-carboxamido)propanamido)butyl)- dorsal flank of female CD-1 nude mice (age 5-8 weeks, 5-oxo-1,3,2-dioxaborolan-4-yl)acetic acid (I-21) Charles River) using a 1 mL 263/8-ga needle (Becton Dick 65 inson Ref#309.625). Alternatively, some xenograft models A mixture of N,N',N'-(boroxin-2,4,6-triyltris (1R)-3- require the serial passaging of tumor fragments. In these methylbutane-1,1-diylimino (2S)-1-oxo-3-phenylpropane US 9, 175,018 B2 65 66 1,2-diyl)tripyrazine-2-carboxamide (0.305 g, 0.278 mmol) TABLE 7 and D-malic acid (130.3 mg, 0.33 mmol) were mixed in acetone (3 mL). The mixture was heated to form a solution. Capsule composition The solution was cooled uncontrolled until the internal tem Component Function mg Capsule perature was about 25°C. White solid precipitated out and the resultant slurry was agitated at ambient temperature for 3 h. Compound of formula (I-1) Form 2 O.29 The slurry was filtered to collect solid 2-((R)-2((R)-3-methyl Microcrystalline cellulose (low moisture) Filler 89.71 1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido) Total capsule content weight, mg 90.00 butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)acetic acid (0.410 g, Size 4 white opaque gelatin capsules 100%). M+H calculated for CHBNO, 483.2051. 10 found, 483.2. Example 29 Example 26 15 Pharmaceutical Composition 2 Synthesis of (R)-2-hydroxy-2-((R)-2-((R)-3-methyl 1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)pro The composition of the capsule is shown in Table 8 below. panamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl) TABLE 8 acetic acid (I-22) Capsule composition A mixture of N,N',N'-(boroxin-2,4,6-triyltris (1R)-3- Component Function mg Capsule methylbutane-1,1-diylimino2S)-1-oxo-3-phenylpropane Compound of formula (I-1) Form 2 O.29 1,2-diyl)tripyrazine-2-carboxamide (0.270 g, 0.246 mmol) Silicified microcrystalline cellulose Filler 109.71 and L-tartaric acid (149.5 mg, 0.33 mmol) were mixed in 25 acetone (3 mL). The mixture was heated to form a solution. Total capsule content weight, mg 110.00 The solution was cooled uncontrolled until the internal tem Size 4 white opaque gelatin capsules perature was about 25° C. Heptane (2.5 mL) was added. White solid precipitated out and the resultant slurry was agi 30 tated at ambient temperature for 1.5h. The slurry was filtered Example 30 to collect solid (R)-2-hydroxy-2-((R)-2-((R)-3-methyl-1- ((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)bu tyl)-5-oxo-1,3,2-dioxaborolan-4-yl)acetic acid (0.388 g) Pharmaceutical Composition 3 which also contained a dimeric species. MS (m/z) in CHCN: 35 The composition of the capsule is shown in Table 9 below. M+H calculated for CHBNOs 499.2000. found, 499.2. TABLE 9 Capsule composition Example 27 40 Component Function mg Capsule Synthesis of (S)-2-hydroxy-2-((S)-2-((R)-3-methyl Compound of formula (I-1) Form 2 O.29 1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)pro Microcrystalline cellulose (low moisture) Filler 88.81 panamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl) Magnesium stearate Lubricant O.90 acetic acid (I-23) 45 Total capsule content weight, mg 90.00 Size 4 white opaque gelatin capsule A mixture of N,N',N'-(boroxin-2,4,6-triyltris (1R)-3- methylbutane-1,1-diylimino (2S)-1-oxo-3-phenylpropane 1,2-diyl)tripyrazine-2-carboxamide (0.180g, 0.164 mmol) Example 31 and D-tartaric acid (147.5 mg, 0.33 mmol) were mixed in 50 acetone (4 mL). The mixture was heated to form a solution. The solution was cooled uncontrolled until the internal tem Pharmaceutical Composition 4 perature was about 25° C. Heptane (8 mL) was added. The mixture was evaporated to yield (S)-2-hydroxy-2-((S)-2- The composition of the capsule is shown in Table 10 below. ((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido) 55 propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)acetic TABLE 10 acid (0.447 g) which also contained a dimeric species. MS (m/z) in CHCN: M-H calculated for CHBNOs, Capsule composition 499.2000. found, 499.2. 60 Component Function mg Capsule Compound of formula (I-1) Form 2 O.29 Example 28 Microcrystalline cellulose Filler 78.91 Magnesium stearate Lubricant O.80 Total capsule content weight, mg 8O.OO Pharmaceutical Composition 1 65 Size 4 white opaque gelatin capsule The composition of the capsule is shown in Table 7 below. US 9, 175,018 B2 67 68 Example 32 TABLE 14-continued Pharmaceutical Composition 5 Batch composition Item The composition of the capsule is shown in Table 11 below. 5 Number Component g/Batch mg Capsule (Emcocel (R) XLM90; low moisture) TABLE 11 3 Microcrystalline cellulose, NF 192.9 8.2O (Emcocel (R) XLM90; low moisture) Capsule composition 4 Microcrystalline cellulose, NF 300 12.75 10 (Emcocel (R) XLM90; low moisture) Component Function mg Capsule 5 Microcrystalline cellulose, NF 500 21.25 (Emcocel (R) XLM90; low moisture) Compound of formula (I-1) Form 2 O.29 6 Microcrystalline cellulose, NF 900 38.25 Microcrystalline cellulose (low moisture) Filler 84.71 (Emcocel (R) XLM90; low moisture) Total capsule contents weight, mg 8S.OO Total weight 2OOO.O 85.OO Size 4 white opaque gelatin capsule 15 Size 4 white opaque gelatin capsules

Example 33 The batch was prepared according to the following pro CSS Pharmaceutical Composition 6 1) Microcrystalline cellulose, NF (Emcocel(RXLM90; low moisture) (Item #2) was screened through a 40 micron The composition of the capsule is shown in Table 12 below. mesh screen. 2) The screened material from step 1) was added to the PK TABLE 12 25 blender and was blended for 2 minutes. 3) Compound of formula (I-1) Form 2 that had been Capsule composition Screened through a 60 micron mesh screen, was weighed Component Function mg Capsule out (Item #1). Compound of formula (I-1) Form 2 0.72 4) The compound of formula (I-1) Form 2 from step 3), and Microcrystalline cellulose (low moisture) Filler 119.28 30 the microcrystalline cellulose, NF (Emcocel(RXLM90; low moisture) (Item #3) were combined in a polyethyl Total capsule content weight, mg 12O.OO ene bag, and the polyethylene bag was shaken; then the Size 3 darkgreen gelatin capsule contents of the polyethylene bag were passed through the same 40 micron screen as was used in step 1). 35 5) The material from step 4) was added to the PK blender Example 34 and blended for 15 minutes. Pharmaceutical Composition 7 6) Microcrystalline cellulose, NF (Emcocel(RXLM90; low moisture) (Item #4) was screened through the same 40 40 micron mesh screen, transferred to the same polyethyl The composition of the capsule is shown in Table 13 below. enebag used in step 4) and shaken in the polyethylene TABLE 13 bag. 7) The material from step 6) was added to the PK blender, Capsule composition which still contained the material from step 5) and 45 Component Function mg Capsule blended for 10 minutes. 8) Microcrystalline cellulose, NF (Emcocel(RXLM90; low Compound of formula (I-1) Form 2 2.89 moisture) (Item #5) was screened through the same 40 Microcrystalline cellulose (low moisture) Filler 147.11 micron mesh screen, transferred to the same polyethyl Total capsule content weight, mg 1SO.OO ene bag used in steps 4) and 6), and shaken in the poly Size 2 Swedish orange gelatin capsule 50 ethylene bag. 9) The material from step 8) was added to the PK blender, which still contained the material from steps 5) and 7), Example 35 and blended for 10 minutes. 55 10) Microcrystalline cellulose, NF (Emcocel(R) XLM90; Pharmaceutical Composition 8 low moisture) (Item #6) was screened through the same 40 micron mesh screen, transferred to the same polyeth The composition is shown in Table 14 below. ylene bag used in steps 4), 6), and 8), and shaken in the polyethylene bag. TABLE 1.4 60 11) The material from step 10) was added to the PK Batch composition blender, which still contained the material from steps 5), Item 7), and 9), and blended for 10 minutes. Number Component gBatch mg/Capsule 12) The material from the blender was encapsulated in size 4 white opaque gelatin capsules using the In-Cap sys 1 Compound of formula (I-1) Form 2 7.06 O.30 65 2 Microcrystalline cellulose, NF 1OO 4.25 tem. 13) The capsules were de-dusted, and weight-sorted. US 9, 175,018 B2 69 70 Example 36 TABLE 1.8 Capsule composition Pharmaceutical Composition 9 Component Function mg Capsule 5 The composition of the capsules is shown in Table 15 Compound of formula (I-1) Form 2 O.3 below. Microcrystalline cellulose (Emcocel (R) Filler 89.25 XLM90; low moisture) Magnesium Stearate Lubricant O45 TABLE 1.5 10 Total capsule content weight, mg 90.00 Capsule composition Size 4 white opaque gelatin capsule Component Function mg Capsule Compound of formula (I-1) Form 2 O.3 Pregeletanized Starch (Starch 1500) Filler 122.82S Example 40 Talc Flow-aid 1.25 15 Magnesium Stearate Lubricant O.625 Pharmaceutical Composition 13 Total capsule content weight, mg 125.00 Size 4 white opaque gelatin capsule The composition of the capsule is shown in Table 19 below. TABLE 19 Example 37 Capsule composition Component Function mg Capsule

Pharmaceutical Composition 10 25 Compound of formula (I-1) Form 2 O.3 Microcrystalline cellulose (Emcocel (R) Filler 88.35 XLM90; low moisture) The composition of the capsules is shown in Table 16 Talc Flow-aid O.9 below. Magnesium Stearate Lubricant O45 Total capsule content weight, mg 90.00 TABLE 16 30 Size 4 white opaque gelatin capsule Capsule composition Component Function mg Capsule Example 41 Compound of formula (I-1) Form 2 O.3 35 Pregeletanized Starch (Starch 1500) Filler 124.7 Pharmaceutical Composition 14 Total capsule content weight, mg 125.00 Size 4 white opaque gelatin capsule The composition of the capsule is shown in Table 20 below. TABLE 20 40 Capsule composition Example 38 Component Function mg Capsule Pharmaceutical Composition 11 Compound of formula (I-1) Form 2 O.3 45 Microcrystalline cellulose (Emcocel (R) Filler 51.15 XLM90; low moisture) The composition of the capsule is shown in Table 17 below. Talc Flow-aid O.98 Magnesium Stearate Lubricant O49 Pregeletanized Starch (Starcap) 45.08 TABLE 17 50 Total capsule content weight, mg 98.00 Capsule composition Size 4 white opaque gelatin capsule Component Function mg Capsule Compound of formula (I-1) Form 2 O.3 Microcrystalline cellulose (Emcocel (R) Filler 124.7 Example 42 XLM90; low moisture) 55 Talc Flow-aid 1.25 Pharmaceutical Composition 15 Total capsule content weight, mg 12S.OO Size 4 white opaque gelatin capsule The composition of the capsule is shown in Table 21 below.

60 TABLE 21 Capsule composition Example 39 Component Function mg Capsule Compound of formula (I-1) Form 2 O.3 Pharmaceutical Composition 12 65 Microcrystalline cellulose (Emcocel (R) Filler 61.65 XLM90; low moisture) The composition of the capsule is shown in Table 18 below. US 9, 175,018 B2 71 72 TABLE 21-continued 7) Talc (Item #7), and Sodium Citrate (Item #8) were Capsule composition Screened through the same 40 micron mesh screen. 8) The materials from step 6) and 7) were added to the PK Component Function mg Capsule blender, which still contained the material from step 5) Talc Flow-aid 1.18 and blended for 10 minutes. Magnesium Stearate Lubricant O.S9 Sodium Starch Glycolate (Explotab) 54.28 9) Microcrystalline cellulose, NF (Emcocel(RXLM90; low moisture) (Item #5) was screened through the same 40 Total capsule content weight, mg 118.00 Size 4 white opaque gelatin capsule 10 micron mesh screen, transferred to the same polyethyl ene bag used in steps 4) and 6), and shaken in the poly ethylene bag. Example 43 10) The material from step 9) was added to the PK blender,

15 which still contained the material from steps 5) and 8), Pharmaceutical Composition 16 and blended for 10 minutes. 11) Microcrystalline cellulose, NF (Emcocel(R) XLM90; The composition of the batch is shown in Table 22 below. low moisture) (Item #6) was screened through the same TABLE 22 Batch composition

Item Number Component g/Batch mg Capsule 1 Compound of formula (I-1) Form 2 O.33 O.30 2 Microcrystalline cellulose, NF (Emcocel (R) XLM90; S.OO 4...SO low moisture) 3 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 8.17 7.35 low moisture) 4 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 14.00 1260 low moisture) 5 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 2S.OO 22.50 low moisture) 6 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 44.OO 39.60 low moisture) 7 Talc 1.OO 8 Sodium Citrate 2.OO 9 Magnesium Stearate O.SO

Total weight 100.00 90.00 Size 4 white opaque gelatin capsules

40 The batch was prepared according to the following pro 40 micron mesh screen, transferred to the same polyeth CSS ylene bag used in steps 4), 6), and 9), and shaken in the 1) Microcrystalline cellulose, NF (Emcocel(RXLM90; low polyethylene bag. moisture) (Item #2) was screened through a 40 micron 45 mesh screen. 12) The material from step 11) was added to the PK 2) The screened material from step 1) was added to the PK blender, which still contained the material from steps 5), blender and was blended for 2 minutes. 8), and 10), and blended for 10 minutes. 3) Compound of formula (I-1) Form 2 that had been 13) Magnesium Stearate (Item #9) was screened through Screened through a 60 micron mesh screen, was weighed 50 the same 40 micron mesh screen. out (Item #1). 14) The material from step 13) was added to the PK 4) The compound of formula (I-1) Form 2 from step 3), and blender, which still contained the material from steps 5), the microcrystalline cellulose, NF (Emcocel(RXLM90; 8), 10), and 12) and blended for 5 minutes. low moisture) (Item #3) were combined in a polyethyl 55 ene bag, and the polyethylene bag was shaken; then the 15) The material from the blender was encapsulated in size contents of the polyethylene bag were passed through 4 white opaque gelatin capsules using the Profill system. the same 40 micron screen as was used in step 1). 16) The capsules were de-dusted, and weight-sorted. 5) The material from step 4) was added to the PK blender 60 and blended for 15 minutes. Example 44 6) Microcrystalline cellulose, NF (Emcocel(RXLM90; low moisture) (Item #4) was screened through the same 40 Pharmaceutical Composition 17 micron mesh screen, transferred to the same polyethyl 65 enebag used in step 4) and shaken in the polyethylene bag. The composition of the batch is shown in Table 23 below. US 9, 175,018 B2 73 74 TABLE 23 Batch composition

Item Number Component g/Batch mg Capsule 1 Compound of formula (I-1) Form 2 7.06 O.30 2 Microcrystalline cellulose, NF (Emcocel(R) XLM90; 4.94 O.21 ow moisture) 3 Microcrystalline cellulose, NF (Emcocel(R) XLM90; 25.00 1.06 ow moisture) 4 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 53.00 2.25 ow moisture) 5 Talc 10.00 O43 6 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 90 3.83 ow moisture) 7 Talc 30 1.28 8 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 170 7.23 ow moisture) 9 Microcrystalline cellulose, NF (Emcocel(R) XLM90; 3OO 12.75 ow moisture) 10 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 500 21.25 ow moisture) 11 Microcrystalline cellulose, NF (Emcocel (R) XLM90; 800 34 ow moisture) 12 Magnesium Stearate 10 O43 Total weigh 2OOO 85 Size 4 white opaque gelatin capsules

The batch was prepared according to the following pro 13) The second half of material from step 10) was added to CSS the small PK blender from steps 5) and 7), and 11), 1) Microcrystalline cellulose, NF (Emcocel(RXLM90; low 30 blended for 3 minutes, transferred to the same polyeth moisture) (Item #2) was screened through a 40 micron ylene bag used in steps 9) and 11), and shaken in the mesh screen. polyethylene bag. 14) The material from step 13) was added to the larger PK 2) The screened material from step 1) was added to the blender, which still contained the material from steps 8), Small PK blender and blended for 2 minutes. 9), and 12), and blended for 10 minutes. 3) Compound of formula (I-1) Form 2 that had been 35 15) Microcrystalline cellulose, NF (Emcocel(R) XLM90; Screened through a 60 micron mesh screen was weighed low moisture) (Item #9) was screened through the same out (Item #1). 40 micron mesh screen, transferred to the same polyeth 4) The compound of formula (I-1) Form 2 from step 3), and ylene bag used in steps 9), 11), and 13), and shaken in the the microcrystalline cellulose, NF (Emcocel(RXLM90; polyethylene bag. low moisture) (Item #3) were combined, and then passed 40 16) The material from step 15) was added to the same larger through the same 40 micron screen as was used in step PKblender, which still contained material from steps 8), 1). 9), 12), and 14), and blended for 10 minutes. 5) The material from step 4) was added to the small PK 17) Microcrystalline cellulose, NF (Emcocel(R) XLM90; blender and blended for 30 minutes. low moisture) (Item #10) was screened through the same 6) Microcrystalline cellulose, NF (Emcocel(RXLM90; low 45 40 micron mesh screen. 18) The material from step 17) was added to the same larger moisture) (Item #4) and Talc (Item #5) were screened PKblender, which still contained material from steps 8), through the same 40 micron mesh screen. 9), 12), 14), and 16), and blended for 10 minutes. 7) The material from step 6) was added to the small PK 19) Microcrystalline cellulose, NF (Emcocel(R) XLM90; blender, which still contained the material from step 5) low moisture) (Item #11) was screened through the same and blended for 15 minutes. 50 40 micron mesh screen. 8) Microcrystalline cellulose, NF (Emcocel(RXLM90; low 20) The material from step 19) was added to the same larger moisture) (Item #6) was screened through the same 40 PKblender, which still contained material from steps 8), micron mesh screen, transferred to a second larger PK 9), 12), 14), 16), and 18), and blended for 10 minutes. blender, and blended for 2 minutes. 21) Magnesium Stearate (Item #12) was screened through 9) The contents of the small PK blender from steps 5) and 55 the same 40 micron mesh screen. 7) were emptied into a polyethylene bag, and then trans 22) The material from step 21) was added to the same larger ferred to the larger PK blender from step 8). PKblender, which still contained material from steps 8), 10) Talc (Item #7) and Microcrystalline cellulose, NF (Em 9), 12), 14), 16), 18), and 20), and blended for 5 minutes. cocel(R) XLM90; low moisture) (Item #8) were screened 23) The material from the blender was encapsulated in size through the same 40 micron mesh screen. 60 4 white opaque gelatin capsules using the Incap system. 11) Half the material from step 10) was added to the small 24) The capsules were de-dusted, and weight-sorted. PK blender from steps 5) and 7), blended for 3 minutes, transferred to the same polyethylene bag used in step 9). Example 45 and shaken in the polyethylene bag. 12) The material from step 11) was added to the larger PK 65 Pharmaceutical Composition 18 blender, which still contained the material from steps 8) and 9). The composition of the batch is shown in Table 24 below. US 9, 175,018 B2 75 76 TABLE 24 the batch amount) was added to the pre-compounding con tainer with stirring. The container was rinsed with the Batch composition reserved tert-butyl alcohol/water solution, and the rinses were added to the pre-compounding container. The mixture was Item stirred until the sodium citrate was completely dissolved. Number Component gBatch mg/Capsule 5 N-(2-pyrazine)carbonyl-L-phenyl-L-leucine boronic acid 1 Compound of formula (I-1) Form 2 3.53 O.30 (VIII-15) was added to the pre-compounding container with 2 Microcrystalline cellulose, NF 496.5 4SO stirring. The container was rinsed with the reserved tert-butyl (Emcocel (R) XLM90; low moisture) alcohol/water solution, and the rinses were added to the pre 3 Microcrystalline cellulose, NF 500 7.35 compounding container. The mixture was stirred until the (Emcocel (R) XLM90; low moisture) 10 boronic acid was completely dissolved. The citric acid, Total weight 1OOO 85 Sodium citrate, and boronic acid mixture from the pre-com Size 4 white opaque gelatin capsules pounding container was transferred to the main compounding vessel. The pre-compounding container was rinsed with water for injection and the rinses were added to the main The batch is prepared according to the following process: 15 compounding vessel. Citric acid (70% of the batch amount) 1) Microcrystalline cellulose, NF (Emcocel(RXLM90; low was added to the main vessel with stirring. The container was moisture) (Item #2) is screened through a 40 micron rinsed with the water, and the rinses were added to the main mesh screen and added to a high shear mixer. vessel. The mixture was stirred until the citric acid was com 2) Compound of formula (I-1) Form 2 is screened through pletely dissolved. Sodium citrate (70% of the batch amount) a 60 micron mesh screen and weighed out (Item #1) and was added to the main vessel with stirring. The container was added to the same high shear mixer from step 1). rinsed with water, and the rinses were added to the pre 3) Microcrystalline cellulose, NF (Emcocel(RXLM90; low compounding container. The mixture was stirred until the moisture) (Item #3) is screened through the same 40 Sodium citrate was completely dissolved. Glycine was added micron mesh screen, and added to the same high shear to the main vessel and the residual glycine was rinsed with mixer from steps 1) and 2). water, and the rinses were added to the main vessel. The 4) The high shear mixer from steps 1), 2), and 3) is run for 25 mixture was stirred until the glycine was completely dis 4 minutes. solved. Sufficient water was added to reduce the total alcohol 5) The material from the high shear mixer is encapsulated content to 4.7% V/v. The mixture was filtered through a 0.22 in size 4 white opaque gelatin capsules using the Incap um filter. Aliquots of the filtered solution were placed into system. vials. The vials were sealed with lyophilization stoppers and 6) The capsules are de-dusted, and weight-sorted. 30 were placed on lyophilizer chamber shelves maintained at 20° C. The lyophilization chamber shelves were cooled to Example 46 -45° C. using the appropriate ramp rate and held at that temperature for 200 minutes. The shelf was warmed to -20° Lyophilized Powder 1 C. using an appropriate ramp rate and maintained at that 35 temperature for 480 minutes. The shelf was re-cooled to -45° In a clean container, a solution of 40% tert-butyl alcohol C. using an appropriate ramp rate and maintained at that 60% water for injection was prepared by warming the temperature. After 200 minutes, the lyophililization chamber required amount of tert-butyl alcohol to 35° C. and adding was evacuated, and the chamber pressure was adjusted to 150 water for injection. The solution was cooled to 15-30°C. A microns with nitrogen. The chamber shelves were warmed up portion of the required amount (60% of the total batch) of to -25° C. using an appropriate ramp rate, and held at that tert-butyl alcohol/water solution was added to a pre-com 40 temperature for 3000 minutes. After each of the product ther pounding container. Approximately 40% of the solution was mocouples read -25°C. or warmer, the shelf was warmed to reserved for use in rinsing. Citric acid (30% of the batch 27°C. and maintained at that temperature for 600 minutes. At amount) was added to the pre-compounding container with the end of the terminal drying phase, the chamber pressure stirring. The container was rinsed with the reserved tert-butyl was restored using nitrogen, and vials were sealed and alcohol/water solution, and the rinses were added to the pre 45 removed. Prelyophilized solution contained: 52 mM citrate, compounding container. The mixture was stirred until the 3% glycine, 4.7% tert-butyl alcohol (as shown in Table 25 citric acid was completely dissolved. Sodium citrate (30% of below). TABLE 25 Batch composition

Amount No. Component Amount/mL mM Batch per vial Compound (VIII-15) 0.001 g 2.6 0.300 g 3.5 mg Citric Acid Monohydrate, 0.00382 g 18.2 1.147 g 13.37 mg USPEP Sodium Citrate Dihydrate, 0.00994g 33.8 2.982 g 34.79 mg USPEP Glycine, USP/EP 0.03g 399.6 9.0 g 105 mg Tert-butyl alcohol, ACS na na 14.1 mL 0.1645 mL. grade Water for Injection, na na Fill to na USPEP batch volume Total volume na na 300 mL. 3.5 mL. Final measured pH na na S.O8 na US 9, 175,018 B2 Example 47 Lyophilized Powder 2 Prepared as described in Example 46. Pre-lyophilized 5 solution contained: 52 mM citrate; 3% glycine; and 4.7% tert-butyl alcohol (as shown in Table 26 below). TABLE 26 Batch composition

Amount No. Component Amount mL mM Batch per vial 1. Compound (VIII-15) 0.001 g 2.6 0.300 g 3.5 mg 2. Citric Acid Monohydrate, 0.00168 g 8.0 0.504 g 5.88 mg USPEP 3. Sodium Citrate Dihydrate, 0.0129 g 44.0 3.882 g 45.15 mg USPEP 4. Glycine, USP/EP 0.03g 399.6 9.0 g 105 mg 5. Tert-butyl alcohol, ACS na na 14.1 mL 0.1645 mL. grade 6. Water for Injection, na na Fill to na USPEP batch volume 7. Total volume na na 300 mL. 3.5 mL. 8. Final measured pH na na 5.84 na

Example 48 Example 49

Lvophilizedyop Powder 3 Lyophilized- Powder 4 The formulation was prepared as described in Example 46, 30 except that the lyophilization cycle was modified. The vials A clean vessel was charged with water for injection. Citric were sealed with lyophilization stoppers and placed on lyo acid and sodium citrate were added and stirred until dis philizer chambershelves maintained at 20°C. The lyophiliza tion chamber shelves were cooled to -45° C. using the appro solved. To this solution, N-(2-pyrazine)carbonyl-L-phenyl priate ramp rate and held at that temperature for 200 minutes. 35 L-leucine boronic acid (VIII-15) was added and stirred until The shelf was warmed to -20°C. using an appropriate ramp dissolved. Glycine was added to the vessel and the residual rate and maintained at that temperature for 480 minutes. The shelf was re-cooled to -45° C. using an appropriate ramp rate glycine was rinsed with water, and the rinses were added to and maintained at that temperature. After 200 minutes, the the main vessel. The mixture was stirred until the glycine was lyophilization chamber was evacuated, and the chamber pres 40 completely dissolved. Sufficient water was added to batch sure was adjusted to 150 microns with nitrogen. The chamber volume. The mixture was filtered through a 0.22 Lum filter. shelves was warmed up to -15°C. using an appropriate ramp rate, and held at that temperature for 2700 minutes. After each Aliquots of the filtered solution were placed into vials. The of the product thermocouples read -15° C. or warmer, the vials were sealed with lyophilization stoppers and were shelf was warmed to 37° C. and maintained at that tempera 45 placed on lyophilizer chamber shelves maintained at 20° C. ture for 300 minutes. At the end of the terminal drying phase, The lyophilization chamber shelves were cooled to -45° C. the chamber pressure was restored using nitrogen, and the vials were sealed and removed. Pre-lyophilized solution con using the appropriate ramp rate and held at that temperature tained: 52 mM citrate; 3% glycine; and 4.7% tert-butyl alco for 200 minutes. The shelf was warmed to -20°C. using an hol (as shown in Table 27 below). appropriate ramp rate and maintained at that temperature for TABLE 27 Batch composition

Amount No. Component Amount mL mM Batch per vial 1. Compound (VIII-15) 0.001 g 2.6 1.0 g 3.5 mg 2. Citric Acid Monohydrate, 0.00382 g 18.2 3.82 g 13.37 mg USPEP 3. Sodium Citrate Dihydrate, 0.00994g 33.8 9.94 g 34.79 mg USPEP 4. Glycine, USP/EP 0.03 g 399.6 30.0 g 105 mg 5. Tert-butyl alcohol, ACS grade na na 47.0 mL. O.1645 mL. 6. Water for Injection, USP/EP na nia Fill to batch na volume 7. Total volume na na 1000 mL. 3.5 mL. 8. Final measured pH na na 5.05 na US 9, 175,018 B2 79 80 480 minutes. The shelf was re-cooled to -45° C. using an To this solution, 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5- appropriate ramp rate and maintained at that temperature. dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3, After 200 minutes, the lyophilization chamber was evacuated 2-dioxaborinane-4-carboxylic acid (I-1) is added and stirred with and the chamber pressure was adjusted to 150 microns until dissolved. Glycine is added to the vessel and the residual with nitrogen. The chamber shelves were warmed up to -25° glycine is rinsed with water, and the rinses are added to the C. using an appropriate ramp rate, and held at that tempera main vessel. The mixture is stirred until the glycine is com ture for 3000 minutes. After each of the product thermo pletely dissolved. Sufficient water is added to batch volume. couples read -25°C. or warmer, the shelf was warmed to 27° The mixture is filtered through a 0.22um filter. Aliquots of the C. and maintained at that temperature for 600 minutes. At the 10 filtered solution are placed into sterilized vials. The vials are end of the terminal drying phase, the chamber pressure was sealed with lyophilization stoppers and are placed on lyo restored using nitrogen, and vials were sealed and removed. philizer chamber shelves maintained at 20°C. The lyophiliza Pre-lyophilized solution contained: 52 mM citrate; and 3% tion chamber shelves are cooled to -45° C. using an appro glycine (as shown in Table 28 below). priate ramp rate, and then are held at that temperature for 200 TABLE 28 Batch composition

Amount Component Amount mL mM Batch per vial 1. Compound (VIII-15) 0.001 g 2.6 0.30 g 3.5 mg 2. Citric Acid Monohydrate, 0.0040.97g 19.5 1.229 g 14.34 mg USPEP 3. Sodium Citrate Dihydrate, 0.009557g 32.5 2.867 g 33.45 mg USPEP 4. Glycine, USP/EP 0.03 g 399.6 9.0 g 105 mg 5. Water for Injection, USP/EP na na Fill to batch na volume 6. Total volume na na 300 mL. 3.5 mL. 7. Final measured pH na na 4.90 na

Example 50 minutes. The shelf is warmed to -20°C. using an appropriate ramp rate and then is maintained at that temperature for 480 Lyophilized Powder 5 35 minutes. The shelf is re-cooled to -45° C. using an appropri ate ramp rate and is maintained at that temperature. After 200 Prepared as described in Example 49. Pre-lyophilized minutes, the lyophilization chamber is evacuated, and the solution contained: 52 mM citrate; and 3% glycine (as shown chamber pressure is adjusted to 150 microns with nitrogen. in Table 29 below). In this example, the pH of the pre-lyo 40 The chamber shelves are warmed up to -25° C. using an philized solution was adjusted to the final measured pH by the appropriate ramp rate, and held at that temperature for 3000 addition of 2N HC1. minutes. After each of the product thermocouples reads -25° TABLE 29 Batch composition

Amount No. Component Amount mL mM Batch per vial 1. Compound (VIII-15) 0.001 g 2.6 0.30 g 3.5 mg 2. Citric Acid Monohydrate, 0.00168 g 8.0 0.504 g 5.88 mg USPEP 3. Sodium Citrate Dihydrate, 0.01294g 44.0 3.882 g 45.29 mg USPEP 4. Glycine, USP/EP 0.03 g 399.6 9.0 g 105 mg 5. Water for Injection, USP/EP na na Fill to batch na volume 6. Total batch volume na na 300 mL. 3.5 mL. 7. Final measured pH na na 5.84 na

Example 50 60 C. or warmer, the shelf is warmed to 27°C. and is maintained at that temperature for 600 minutes. At the end of the terminal Lyophilized Powder 6 drying phase, the chamber pressure is restored using nitrogen, and vials are sealed and removed. The composition of the 65 A clean vessel is charged with water for injection. Citric pre-lyophilized solution is 55 mM citrate; and 3% glycine (as acid and sodium citrate are added and stirred until dissolved. shown in Table 30 below). US 9, 175,018 B2

TABLE 30 Batch composition Amount per No. Component Amount mL mM Batch vial 1. Compound (I-1) expressed as 0.001 g 2.75 0.50 g 3.5 mg amount of compound (VIII 1) 2. Citric Acid Monohydrate, 0.0012g 5.5 0.578 g 4.2 mg USPEP 3. Sodium Citrate Dihydrate, 0.0147g 49.5 7.279 g 51.45 mg USPEP 4. Glycine, USP/EP 0.03 g 399.6 15.0 g 105 mg 5. Water for Injection, USP/EP na nia Fill to batch na volume 6. Total volume na na 500 mL. 3.5 mL.

Example 51 Example 53 Reconstitution of Lyophilized Powders 2O Analytical Test Method 1 The lyophilized powders (e.g. as prepared in Examples Reversed-phase HPLC using a C8 column at 25°C. with 46-50) are analyzed using XRPD, DSC, gas chromatography, ultraviolet (UV) detection at 225 nm. and Karl Fisher for cake structure, cake stability, residual Mobile phase: The gradient system starts at 85% mobile solvent, and residual moisture, respectively. The lyophilized phase A (0.01% trifluoroacetic acid in water) and 15% mobile powders are reconstituted with the appropriate amount of 25 phase B (0.01% trifluoroacetic acid in acetonitrile) and ends sterile water for injection or with sterile 0.9% sodium chlo at 75% mobile phase B after 40 minutes. ride solution for injection. The reconstituted solutions are The test sample is prepared by dissolving the content of the analyzed using HPLC, and NMR, for purity, and percentage capsules in diluent which is 15:85 (v/v) acetonitrile:20 mM ester. citrate buffer. Under these aqueous conditions, the compound of formula (I-1) completely hydrolyzes the citrate ester por Example 52 30 tion of the molecule to give the compound of formula (VIII-1) in a 1:1 molecular ratio. The presence of the compound of Preparation of formulation of 4-(R.S)-(carboxym formula in the test sample is confirmed by comparison of the ethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)aceta sample retention time to that of the reference standard. The mido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4- amount of the compound of formula (VIII-1) present in a sample is calculated from the area under the peak, on a carboxylic acid (I-1) Form 2 for parenteral or oral 35 weight-to-weight comparison including molecular weight administration conversion, with the area under the peak of the reference standard. The reference standard employed is a known A vessel was charged with water and citric acid monohy amount of the compound of formula (I-1), of known purity, drate and sodium citrate dihydrate were added and stirred which is prepared under the same hydrolyzing conditions as until dissolved. To this solution, 4-(R.S)-(carboxymethyl)-2- 40 the test sample. The limit of quantitation for the method is ((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbu 0.05% and the calculated limit of detection is 0.02%. tyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylic acid (I-1) Form 2 was added, and the mixture was stirred until a solution was Example 54 obtained. To this solution, sodium chloride was added and stirred until dissolved. Sufficient water was added to batch Analytical Test Method 2 volume and the solution was filtered through a 0.2 um PES membrane. Aliquots of the filtered solution were placed into Normal-phase HPLC using isocratic elution with a mobile vials. The vials were sealed with stoppers and stored at -20° phase of 40/60/0.1 (v/v/v) THF/n-Hexane/TFA on a cyano C. The batch and vial composition is as described below in HPLC column at 25°C. for 8 minutes, with UV detection at Table 31. 230 nm. TABLE 31 Batch composition Amount per No. Component Amount mL mM Batch vial 1. Compound (I-1) expressed 0.001 g 2.75 20 g 3.3 mg as amount of compound (VIII-1)) 2. Citric Acid Monohydrate, 0.0012g 5.5 23.198 g 3.282 mg USPEP 3. Sodium Citrate Dihydrate, 0.014.7 g 49.5 291.183 g 48.05 mg USPEP 4. Sodium Chloride, USP/EP 0.0045 g 77 89.991 g 14.85 mg 5. Water for Injection, USP/EP na nia Fill to batch na volume 6. Total volume na na 2O L. 3.3 mL 7. Final measured pH na na 5.72 na US 9, 175,018 B2 83 84 The test sample is prepared by dissolving the content of the group consisting of phenyl, pyridinyl, pyrimidinyl, capsules in 40/60 (v/v) THF/n-Hexane. Under these condi naphthyl, benzimidazolyl, quinolinyl, isoquinolinyl, tions, the compound of formula (I-1) is not hydrolyzed to the quinoxalinyl, tetrahydroquinolinyl, tetrahydroisoquino compound of formula (VIII-1). The amount of the compound linyl, tetrahydroquinoxalinyl, and dihydrobenzoxazi of formula (VIII-1) present in the test sample is calculated 5 nyl: from the area under the peak, on a weight to weight compari R" is hydrogen, Caliphatic, son, with the area under the peak of the reference standard. C1-6 fluoroaliphatic, -(CH2), CH. R. The reference standard employed is a known amount of the -(CH), CH NHC(=NR)NH Y, compound of formula (VIII-1), of known purity, which is —(CH), CH CONCR), —(CH), CH N prepared under the same conditions as the test sample. The 10 limit of quantitation for detection of the compound of formula —(CH),(RCON(R) CH(R ) OR(SH.), s CH(R')N(R'). (I-1) is 0.2%. or -(CH), CH(R) SR; To calculate the amount of compound of formula (I-1) R" is hydrogen, Caliphatic, present in a test sample, both Analytical Test Method 1 and C1-6 fluoroaliphatic, -(CH2), CH. R. Analytical Test Method 2 are used. Analytical Test Method 1 15 -(CH), CH NHC(=NR)NH Y, is used to calculate the amount on a weight basis of the —(CH), CH CONCR), —(CH), CH N compound of formula (VIII-1) that is present in a test sample, containing the compound of formula (I-1). Analytical Test —(CH),(RCON(R) CH(R ) OR(SH.), s CH(R')N(R'). Method 2 is also used to calculate the amount of the com or -(CH), CH(R) SR; pound of formula (VIII-1) present in the sample of the com each R* independently is hydrogen, Caliphatic, pound of formula (I-1) obtained without induced hydrolysis. C1-6 fluoroaliphatic, -(CH2), CH. R. The amount of the compound of formula (VIII-1) obtained -(CH), CH NHC(=NR)NH Y, from Analytical Test Method 1 minus the amount of the —(CH), CH CONCR), —(CH), CH N compound of formula (VIII-1) obtained from Analytical Test Method 2 gives the measured amount of the compound of 25 —(CH),(RCON(R) CH(R ) OR(SH.), s CH(R')N(R'). formula (VIII-1) that is produced by the induced hydrolysis of or -(CH), CH(R) SR; the compound of formula (I-1) present in the test sample. each R” independently is a substituted or unsubstituted Based on a 1:1 molecular ratio, a molecular weight calcula mono- or bicyclic ring system; tion gives the amount of the compound of formula (I-1) each R' independently is hydrogen or a substituted or present in the test sample. 30 unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl While the foregoing invention has been described in some group; or two R on the same nitrogen atom, taken detail for purposes of clarity and understanding, these par together with the nitrogen atom, form a substituted or ticular embodiments are to be considered as illustrative and unsubstituted 4- to 8-membered heterocyclyl ring hav not restrictive. It will be appreciated by one skilled in the art ing, in addition to the nitrogen atom, 0-2 ring heteroat from a reading of this disclosure that various changes inform 35 oms independently selected from N, O, and S; and detail can be made without departing from the true scope each R independently is hydrogen or a substituted or of the invention, which is to be defined by the appended unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl claims rather than by the specific embodiments. group; The patent and scientific literature referred to herein estab each R" independently is hydrogen or a substituted or lishes knowledge that is available to those with skill in the art. 40 unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl Unless otherwise defined, all technical and scientific terms group; used herein have the same meaning as commonly understood each R independently is hydrogen or a substituted or by one of ordinary skill in the art to which this invention unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl belongs. The issued patents, applications, and references that group; are cited herein are hereby incorporated by reference to the 45 each R independently is a substituted or unsubstituted same extent as if each was specifically and individually indi aliphatic, aryl, or heteroaryl group; cated to be incorporated by reference. In the case of incon Y is hydrogen, —CN, or - NO; sistencies, the present disclosure, including definitions, will m is 0, 1, or 2: control. each of R'' and R' independently is hydrogen, What is claimed is: 50 —COH, -OH, or a substituted or unsubstituted ali phatic, aryl, heteroaryl, or heterocyclyl group; 1. A process for generating a compound of Formula (II) each of R and R' independently is hydrogen, —COH, or a substituted or unsubstituted aliphatic, (II) aryl, heteroaryl, or heterocyclyl group; 55 or R'' and R'' are each independently hydrogen, and l R Rb2 R'' and R', taken together with the carbon atoms to O Ral O pi which they are attached, form an unsubstituted or 3 substituted fused 4- to 8-membered non-aromatic R. R ring having 0-3 ring heteroatoms selected from the P N N1 No R54 60 group consisting of O, N, and S, wherein said ring 2 may be optionally fused to an unsubstituted or substi Ra A. O tuted 4- to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroat or a pharmaceutically acceptable salt thereof, wherein oms selected from the group consisting of O, N, and S. A is 0, 1, or 2: 65 or R'' and R'' are absent, and R'' and R', taken P is R. C(O)–: R is RP: RP is substituted or unsubsti together with the carbon atoms to which they are tuted mono- or bicyclic ring system selected from the attached, form an unsubstituted or substituted fused US 9, 175,018 B2 85 5- to 6-membered aromatic ring having 0-3 ring het -continued eroatoms selected from the group consisting of O. N. CH and S, wherein said ring may be optionally fused to an H3C will-CH unsubstituted or substituted 4- to 8-membered non aromatic ring, or 5- to 6-membered aromatic ring X O having 0-3 ring heteroatoms selected from the group HN l He consisting of O, N, and S; and 3 N-1 No. n is 0 or 1; R the process comprising: 10 (1a) coupling a compound of Formula (vi) with a com pound, P-LG, to form a compound of Formula (vii)

O Ra 15 H O Ral N H P N N OPG H H N + P-LG -> Ra2 A. O Ra2 A. O iv vi wherein X is a counter anion; (4) deprotecting the compound of Formula (iv) to form a O Ral compound of Formula (v) H 25 N OPG P N s H Ra2 A. O vii 30 O Ral

wherein PG is hydrogen or a protecting group; and LG is a P N n1 No leaving group; Ra2 A. O Ra (2a) deprotecting the compound of Formula (vii) to form a 35 compound of Formula (viia) iv O Ral OH (, i? O Ral P N Y YOH : H 40 N OPG Ra2 O Ra P N -e-

Ra2 A. O vii and 45 (5) reacting the compound of Formula (v) with an alpha hydroxy carboxylic acid or a beta hydroxy carboxylic O Ral acid to form a compound of Formula (II). H N OH 2. The process of claim 1, wherein the reaction of step (1a) P N or step (3a) or both is conducted in the presence of a peptide H 50 coupling reagent. Ra2 A. O 3. The process of claim 2, wherein the peptide coupling via reagent is selected from the group consisting of a carbodiim ide reagent, phosphonium reagent, and uronium reagent. 4. The process of claim 3, wherein the peptide coupling (3.a) coupling the compound of Formula (viia) with a com 55 reagent is selected from one or more of the group consisting pound of Formula (i) to form a compound of Formula of dicyclohexylcarbodiimide (DCC), 1-(3-dimethylamino propyl)-3-ethylcarbodiimide (EDC), benzotriazol-1-yloxy (iv) tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetram 60 ethyluronium tetrafluoroborate (TBTU). 5. The process of claim 1, further comprising converting the carboxylic acid moiety of compound (via) to an activated ester or acid halide prior to the reaction of step (3a). 6. The process of claim 5, wherein said activated ester or 65 acid halide is an O—(N-hydroxysuccinnimide) ester. 7. The process of claim 1, wherein the reaction of step (1a) is conducted in the presence of a solvent. US 9, 175,018 B2 87 88 8. The process of claim 7, wherein the solvent is tetrahy 27. The process of claim 1, wherein drofuran. A is 0; 9. The process of claim 1, wherein the reaction of step (4) is conducted in the presence of an aqueous mineral acid. R’ is 2,5-dichlorophenyl: 10. The process of claim 9, wherein the mineral acid is R" is isobutyl: hydrochloric acid. R' is hydrogen; 11. The process of claim 1, wherein the reaction of step (4) each R'' and R' independently is hydrogen; is conducted in the presence of an organic boronic acid accep tOr. each RandR independently is -(CH2), CO.H; p is 12. The process of claim 11, wherein the organic boronic 10 0 or 1; and n is 0 or 1. acid acceptor is i-BuB(OH). 28. The process of claim 1, wherein the alpha hydroxy 13. The process of claim 1, wherein the reaction of step (5) carboxylic acid or a beta hydroxy carboxylic acid is citric is conducted in the presence of a solvent selected from the acid. group consisting of ethyl acetate, methyl isobutyl ketone, 15 29. The process of claim 1, wherein the compound of acetone, acetonitrile, 2-methyltetrahydrofuran, anisole, iso Formula (II) is 4-(R.S)-(carboxymethyl)-2-((R)-1-(2-(2,5- propyl acetate, dimethoxyethane, tetrahydrofuran, dioxane, dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3, dichloromethane, toluene, heptane, methyl-cyclohexane, 2-dioxaborinane-4-carboxylic acid. tert-butylmethyl ether, and any combination thereof. 14. The process of claim 1, wherein the reaction of step (5) 30. The process of claim 1, wherein the compound of is conducted in the presence of a catalyst, wherein the catalyst Formula (II) is 2,2'-(2-(1R)-1-({(2,5-dichlorobenzoyl) is an organic amine base. aminoacetylamino)-3-methylbutyl-5-oxo-1,3,2-diox 15. The process of claim 14, wherein the catalyst is selected aborolane-4.4-diyl diacetic acid. from the group consisting of triethylamine, triethylenedi 31. A process for generating a compound, Form-1, amine, pyridine, collidine, 2.6-lutidine, 4-dimethylaminopy 25 ridine, di-tertbutylpyridine, N-methylmorpholine, N-meth ylpiperidine, tetramethylguanidine, diazabicyclo5.4.0 Form-1 undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, 1.5- diazabicycle 4.3.0non-5-ene, N.N'diisopropylethylamine, C O or any combination thereof. 30 H / 16. The process of claim 1, wherein step (5) further com N B prises cooling the solution. NH ~ 17. The process of claim 16, wherein the cooling comprises O O O H cooling the Solution uncontrolled until the internal tempera 35 ture is about 25° C. C 18. The process of claim 16, wherein the cooling is con ducted in the presence of a co-solvent. 19. The process of claim 18, wherein the co-solvent is the process comprising the steps of selected from the group consisting of heptane, methylcyclo 40 (1) reacting a compound, int-1, with glycine to form a hexane, toluene, tert-butylmethyl ether, ethyl acetate, or any compound, int-2 combination thereof. 20. The process of claim 1, further comprising (6) isolating the compound of Formula (II) as a crystalline C O C O solid. 45 OH: 21. The process of claim 1, wherein PG is a protecting Cl glycine N group selected from the group consisting of an acyl protecting -- H group and a urethane protecting group. O 22. The process of claim 21, wherein PG is a protecting group selected from the group consisting of formyl, acetyl, 50 Succinyl, methoxysuccinyl, tert-butoxycarbonyl (Boc), ben C C Zyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl int-1 int-2 (Fmoc). 23. The process of claim 1, wherein LG is C1. (2) reacting the compound, int-2, with a compound, int-3, 24. The process of claim 1, wherein X is CFCO. 55 to form a compound, int-4 25. The process of claim 1, wherein R is selected from the group consisting of 2,5-dichlorophenyl. 26. The process of claim 1, wherein C O A is 0; R" is Caliphatic; 60 OH + R" is hydrogen, Caliphatic, -(CH2), CH. R. or NH ~ -(CH), CH(R) OR; O each of R'' and R' independently is hydrogen, CO.H. —OH, or a substituted or unsubstituted aliphatic; C each of R and R' independently is hydrogen, Cali 65 phatic, or -(CH2)CO.H; and int-2 p is 0 or 1. US 9, 175,018 B2 89 90 -continued 32. The process of claim 31, wherein the reaction of step (1) is conducted in the presence of NaOH. 33. The process of claim 31, wherein the reaction of step CFCOOH.HN 2. a. -- s (2) is conducted in the presence of TBTU. O 34. The process of claim 31, wherein the reaction of step (2) is conducted in the presence of DIPEA. 35. The process of claim 31, wherein the reaction of step 10 (3) is conducted in the presence of methanol and hexane. int-3 36. The process of claim 31, wherein the reaction of step C O O (4) is conducted in the presence of ethyl acetate. R. 37. The process of claim 31, wherein the reaction of step N N-1 No. 15 (4) comprises cooling the solution uncontrolled until the H internal temperature is about 25°C. O s 38. A process for generating a compound, Form-1, C 2O int-4 Form-1 (3) reacting the compound, int-4, with HCl to form a com O pound, int-5 C O O d O, 25 N N1 Yo H OH | O 1 n-Non HCI N O OH 30 C O S. C the process comprising reacting a compound, int-5, with cit int-4 ric acid to form the compound, Form-1

C O O 35 R. I. N N1 J3 C O O H s N R.N-1 3 citric acid O S. 40 H i C int-5 O s C (4) reacting the compound, int-5, with citric acid to form 45 int-5 the compound, Form-1 C O it. IO O N ~ N1 3 citric acid 50 C O O O.

R. I. r O s 1 N1 O OH C O O OH int-5 55 N O C Form-1 C O O O. 60 N N-"No 39. The process of claim 38, wherein the reaction is con H s OH ducted in the presence of ethyl acetate. O N. O OH 40. The process of claim 38, wherein the reaction further comprises cooling the Solution uncontrolled until the internal C temperature is about 25°C.

k k k k k