USOO958.0471B2 (12) United States Patent (10) Patent No.: US 9,580,471 B2 Bai et al. (45) Date of Patent: Feb. 28, 2017 (54) PROCESS OF PREPARING GUANYLATE 7,041,786 B2 5/2006 Shailubhai et al. CYCLASE CAGONSTS 7.33 E. S. Seaallubha et al. 2006/0160989 A1* 7/2006 Kundu et al. ................. 530/330 (75) Inventors: R EN"S"S (S. 2009/0048175 A1 2/2009 Shailubhai et al. ............ 514f13 steps E" O hai 'S f 2010.0069306 A1 3/2010 Shailubhai et al. (US); Jun Jia, Shanghai (CN), Junfeng 2010/0093635 A1 4/2010 Shailubhai et al. Zhou, Shanghai (CN), Qiao Zhao, 2010, 0120694 A1 5/2010 Shailubhai et al. Shanghai (CN). Guoquing Zhang 2010/0152118 A1 6, 2010 Shailubhai et al. Shanghai (CN); Kunwar Shailubhai, 2010/0221329 A1 9, 2010 Shailubhai et al. Audubon, PA (US); Stephen Comiskey, Doylestown, PA (US); Rong Feng, FOREIGN PATENT DOCUMENTS Langhorne, PA (US) JP H5-508859. A 12/1993 (73) Assignee: SYNERGY PHARMACEUTICALS, sO 3.99, A 1 is: INC., New York, NY (US) WO WOO2/O78683 10, 2002 - WO WO-20081373.18 A1 11, 2008 (*) Notice: Subject to any disclaimer, the term of this WO WO2008,151257 12/2008 patent is extended or adjusted under 35 WO WO2009/149278 12/2009 U.S.C. 154(b) by 0 days. WO WO2009, 1492.79 12/2009 WO WO2010/0093.19 1, 2010 WO WO-2010O27405 A2 3, 2010 (21) Appl. No.: 14/001,638 WO WO2010/065751 6, 2010 WO WO2O11,069038 6, 2011 (22) PCT Filed: Mar. 1, 2012 WO WO2012f1 18972 9, 2012 (86). PCT No.: PCT/US2012/0272.87 OTHER PUBLICATIONS S 371 (c)(1), & 8 (2), (4) Date: Feb. 11, 2014 Currie et al., “Guanylin: An endogenous activator of intestinal guanylate cyclase.” Proc. Natl. Acad. Sci. USA 89:947-951 (1992). (87) PCT Pub. No.: WO2012/118972 Forte, "Guanylin regulatory peptides: structures, biological activi ties mediated by cyclic GMP and pathobiology.” Reg. Pept. 81:25 PCT Pub. Date: Sep. 7, 2012 39 (1999). O O Guba et al., “Guanylin Strongly Stimulates Rat Duodenal HCO (65) Prior Publication Data Secretion: Proposed Mechanism and Comparison With Other US 2014/O155575 A1 Jun. 5, 2014 Secretagogues.” Gastroenterology 1 11:1558-1568 (1996). • al-s Hamra et al., “Uroguanylin: Structure and activity of a second Related U.S. Application Data endogenous peptide that stimulates intestinal guanylate cyclase.” Proc. Natl. Acad. Sci. USA 90:10464-10468 (1993). (60) Provisional application No. 61/447,891, filed on Mar. Schulz et al., “Guanylyl Cyclase Is a Heat-Stable Enterotoxin 1, 2011. Receptor.” Cell 63:941-948 (1990). Joo et al., “Regulation of intestinal C1 and HCO, secretion by (51) Int. Cl. uroguanylin.” Am. J. Physiol. 274:G633-G644 (1998). WR 70s 3.08: (Continued) C07K I4/95 (2006.01) (52) U.S. Cl. Primary Examiner — Christina Bradley CPC .................. C07K 7/64 (2013.01); C07K 7/08 (74) Attorney, Agent, or Firm — Cynthia Kozakiewicz; (2013.01); C07K 14/195 (2013.01) Ivor Elrifi (58) Field of Classification Search CPC ...................................................... CO7K 1/O26 See application file for complete search history. (57) ABSTRACT (56) References Cited The invention provides processes of preparing a peptide including a GCC agonist sequence selected from the group U.S. PATENT DOCUMENTS consisting of SEQ ID NOs: 1-249 described herein. 4,107,158 A * 8/1978 Lefrancier .................... 530,328 5,322.931 A * 6/1994 Hubbs et al. ................. 530.333 5,977.302 A * 11/1999 Palmer et al. ................ 530/339 12 Claims, 3 Drawing Sheets US 9,580.471 B2 Page 2 (56) References Cited OTHER PUBLICATIONS Carpino et al., “Rapid, Continuous Solution-Phase Peptide Synthe sis: Application to Peptides of Pharmaceutical Interest'. Org. Proc. Res. Dev. (2003), 7 (1):28-37. European Application No. 12752488, Extended European Search Report dated Mar. 24, 2015. European Application No. 12752488, Partial European Search Report dated Nov. 19, 2014. PCT/US2012/027287. International Search Report mailed Oct. 23, 2012. PCT/US2012/027287. International Preliminary Report on Patent ability mailed. PCT/US2012/027287. Written Opinion mailed Oct. 23, 2012, 6 pageS. Fujino et al., “Syntheses and biological activities of analogs of luteinizing hormone releasing hormone (LH-RH).” Biochemical and Biophysical Research Communications (1972); 49(3): 698-705. Nyfeler, R., “Peptide synthesis via fragment condensation.” Peptide Synthesis Protocols (1995); 35:303-316. * cited by examiner U.S. Patent Feb. 28, 2017 Sheet 1 of 3 US 9,580,471 B2 V Y NN V N N CO C v g NN N s NN N CN 5. NN SS d O cy) cy) (WM) peulee 96 U.S. Patent Feb. 28, 2017 Sheet 2 of 3 US 9,580,471 B2 U.S. Patent Feb. 28, 2017 Sheet 3 of 3 US 9,580,471 B2 - US 9,580,471 B2 1. 2 PROCESS OF PREPARING GUANYLATE patches of diseased bowel, ulcerative colitis affects only the CYCLASE CAGONSTS innermost lining (mucosa) of the colon in a continuous manner. Depending on which portion of the gastrointestinal RELATED APPLICATIONS tract is involved, Crohn's disease may be referred to as illeitis, regional enteritis, colitis, etc. Crohn's disease and This application is a 35 U.S.C. S371 National Phase ulcerative colitis differ from spastic colon or irritable bowel Application of PCT/US2012/027287, filed on Mar. 1, 2012 syndrome, which are motility disorders of the gastrointes which claims the benefit of and priority to U.S. Provisional tinal tract. Gastrointestinal inflammation can be a chronic Application No. 61/447,891 filed on Mar. 1, 2011, the condition. It is estimated that as many as 1,000,000 Ameri contents of each of which are incorporated by reference in 10 cans are afflicted with inflammatory bowel disease, with their entireties. male and female patients appearing to be equally affected. Most cases are diagnosed before age 30, but the disease can INCORPORATION OF SEQUENCE LISTING occur in the sixth, seventh, and later decades of life. The contents of the text file named “40737 15 IBS and chronic idiopathic constipation are pathological 508001.WO ST25.txt”, which was created on Sep. 11, 2012 conditions that can cause a great deal of intestinal discom and is 113 KB in size, are hereby incorporated by reference fort and distress but unlike the inflammatory bowel diseases, in their entirety. IBS does not cause the serious inflammation or changes in bowel tissue and it is not thought to increase the risk of FIELD OF THE INVENTION colorectal cancer. In the past, inflammatory bowel disease, celiac disease, and IBS were regarded as completely sepa The present invention relates to processes of preparing rate disorders. Now, with the description of inflammation, guanylate cyclase C peptide agonists useful for the treatment albeit low-grade, in IBS, and of symptom overlap between and prevention of various diseases and disorders. IBS and celiac disease, this contention has come under 25 question. Acute bacterial gastroenteritis is the strongest risk BACKGROUND OF THE INVENTION factor identified to date for the subsequent development of postinfective irritable bowel syndrome. Clinical risk factors Guanylate cyclase C is a transmembrane form of gua include prolonged acute illness and the absence of Vomiting. nylate cyclase that is expressed on various cells, including A genetically determined Susceptibility to inflammatory gastrointestinal epithelial cells (reviewed in Vaandrager 30 stimuli may also be a risk factor for irritable bowel syn 2002 Mol. Cell. Biochem. 230:73-83). It was originally drome. The underlying pathophysiology indicates increased discovered as the intestinal receptor for the heat-stable toxin intestinal permeability and low-grade inflammation, as well (ST) peptides secreted by enteric bacteria and which cause as altered motility and visceral sensitivity. Serotonin (5-hy diarrhea. The ST peptides share a similar primary amino acid droxytryptamine 5-HT) is a key modulator of gut function structure with two peptides isolated from intestinal mucosa 35 and is known to play a major role in pathophysiology of IBS. and urine, guanylin and uroguanylin (Currie, et al., Proc. The activity of 5-HT is regulated by c0MP. Nat'l Acad. Sci. USA 89:947-951 (1992); Hamra, et al., While the precise causes of IBS and inflammatory bowel Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993); Forte, diseases (IBD) are not known, a disruption in the process of L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell 63:941 continual renewal of the gastrointestinal mucosa may con 948 (1990); Guba, et al., Gastroenterology 1 11:1558-1568 40 tribute to disease pathology in IBD and aggravate IBS. The (1996); Joo, et al., Am. J. Physiol. 274:G633-G644 (1998)). renewal process of the gastrointestinal lining is an efficient In the intestines, guanylin and uroguanylin act as regula and dynamic process involving the continual proliferation tors of fluid and electrolyte balance. In response to high oral and replenishment of unwanted damaged cells. Proliferation salt intake, these peptides are released into the intestinal rates of cells lining the gastrointestinal mucosa are very lumen where they bind to guanylate cyclase C localized on 45 high, second only to the hematopoietic system. Gastrointes the luminal membrane of enterocytes (simple columnar tinal homeostasis depends on both the proliferation and epithelial cells of the small intestines and colon). The programmed cellular death (apoptosis) of epithelial cells binding of the guanylin peptides to guanylate cyclase C lining the gut mucosa. Cells are continually lost from the induces electrolyte and water excretion into the intestinal Villus into the lumen of the gut and are replenished at a lumen via a complex intracellular signaling cascade that is 50 substantially equal rate by the proliferation of cells in the initiated by an increase in cyclic guanosine monophosphate crypts, followed by their upward movement to the villus.