US 2014O155575A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0155575 A1 Bai et al. (43) Pub. Date: Jun. 5, 2014 (54) PROCESS OF PREPARING GUANYLATE (86). PCT No.: PCT/US 12/27287 CYCLASE CAGONSTS S371 (c)(1), (2), (4) Date: Feb. 11, 2014 (75) Inventors: Juncai Bai, North Augusta, SC (US); Related U.S. Application Data Ruoping Zhang, North Augusta, SC (US); Jun Jian, Shanghai (CN); Junfeng (60) Provisional application No. 61/447,891, filed on Mar. Zhou, Shanghai (CN); Qiao Zhao, 1, 2011. Shanghai (CN); Guoquing Zhang, Publication Classification Shanghai (CN); Kunwar Shailubhai, Audubon, PA (US); Stephen Comiskey, (51) Int. Cl. Doylestown, PA (US); Rong Feng, C07K 7/64 (2006.01) Langhorne, PA (US) C07K 7/08 (2006.01) (52) U.S. Cl. CPC. C07K 7/64 (2013.01); C07K 7/08 (2013.01) (73) Assignee: SYNERGY PHARMACEUTICALS USPC ........................................... 530/321:530/326 INC., New York, NY (US) (57) ABSTRACT (21) Appl. No.: 14/001,638 The invention provides processes of preparing a peptide including a GCC agonist sequence selected from the group (22) PCT Fled: Mar. 1, 2012 consisting of SEQID NOs: 1-249 described herein. Patent Application Publication Jun. 5, 2014 Sheet 1 of 3 US 2014/O155575A1 16d -aw C.CD N.5 CN OCD SRCD SNCN ( . CN C S O d. C-S V 3c : SS-$C O. htCD as hCD S.> a. N N N. NN C N. NN v N s i s Š : CN CN w- w (WM) peulee 9. Patent Application Publication Jun. 5, 2014 Sheet 2 of 3 US 2014/O155575A1 FIG. 2 Patent Application Publication Jun. 5, 2014 Sheet 3 of 3 US 2014/O155575A1 CY) O US 2014/0155575 A1 Jun. 5, 2014 PROCESS OF PREPARING GUANYLATE inflammatory bowel disease, with male and female patients CYCLASE CAGONSTS appearing to be equally affected. Most cases are diagnosed before age 30, but the disease can occur in the sixth, seventh, RELATED APPLICATIONS and later decades of life. 0001. This application claims the benefit of and priority to 0006 IBS and chronic idiopathic constipation are patho U.S. Provisional Application No. 61/447,891 filed on Mar. 1, logical conditions that can cause a great deal of intestinal 2011, the content of which is incorporated by reference in its discomfort and distress but unlike the inflammatory bowel entirety. diseases, IBS does not cause the serious inflammation or changes in bowel tissue and it is not thought to increase the FIELD OF THE INVENTION risk of colorectal cancer. In the past, inflammatory bowel disease, celiac disease, and IBS were regarded as completely 0002 The present invention relates to processes of prepar separate disorders. Now, with the description of inflamma ing guanylate cyclase C peptide agonists useful for the treat tion, albeit low-grade, in IBS, and of symptom overlap ment and prevention of various diseases and disorders. between IBS and celiac disease, this contention has come under question. Acute bacterial gastroenteritis is the strongest BACKGROUND OF THE INVENTION risk factor identified to date for the subsequent development 0003 Guanylate cyclase C is a transmembrane form of of postinfective irritable bowel syndrome. Clinical risk fac guanylate cyclase that is expressed on various cells, including tors include prolonged acute illness and the absence of Vom gastrointestinal epithelial cells (reviewed in Vaandrager 2002 iting. A genetically determined Susceptibility to inflamma Mol. Cell. Biochem. 230:73-83). It was originally discovered tory stimuli may also be a risk factor for irritable bowel as the intestinal receptor for the heat-stable toxin (ST) pep syndrome. The underlying pathophysiology indicates tides secreted by enteric bacteria and which cause diarrhea. increased intestinal permeability and low-grade inflamma The ST peptides share a similar primary amino acid structure tion, as well as altered motility and visceral sensitivity. Sero with two peptides isolated from intestinal mucosa and urine, tonin (5-hydroxytryptamine 5-HT) is a key modulator of gut guanylin and uroguanylin (Currie, et al., Proc. Nat'l Acad. function and is known to play a major role in pathophysiology Sci. USA 89:947-951 (1992); Hamra, et al., Proc. Nat 'l Acad. of IBS. The activity of 5-HT is regulated by c0MP. Sci. USA 90:10464-10468 (1993); Forte, L., Reg. Pept. 81:25 0007 While the precise causes of IBS and inflammatory 39 (1999); Schulz, et al., Cell 63:941-948 (1990); Guba, et al., bowel diseases (IBD) are not known, a disruption in the Gastroenterology 1 11:1558-1568 (1996); Joo, et al., Am. J. process of continual renewal of the gastrointestinal mucosa Physiol. 274:G633-G644 (1998)). may contribute to disease pathology in IBD and aggravate 0004. In the intestines, guanylin and uroguanylin act as IBS. The renewal process of the gastrointestinal lining is an regulators of fluid and electrolyte balance. In response to high efficient and dynamic process involving the continual prolif oral salt intake, these peptides are released into the intestinal eration and replenishment of unwanted damaged cells. Pro lumen where they bind to guanylate cyclase C localized on the liferation rates of cells lining the gastrointestinal mucosa are luminal membrane of enterocytes (simple columnar epithe very high, second only to the hematopoietic system. Gas lial cells of the small intestines and colon). The binding of the trointestinal homeostasis depends on both the proliferation guanylin peptides to guanylate cyclase C induces electrolyte and programmed cellular death (apoptosis) of epithelial cells and water excretion into the intestinal lumen via a complex lining the gut mucosa. Cells are continually lost from the intracellular signaling cascade that is initiated by an increase Villus into the lumen of the gut and are replenished at a in cyclic guanosine monophosphate (cGMP). substantially equal rate by the proliferation of cells in the 0005. The coMP-mediated signaling that is initiated by crypts, followed by their upward movement to the villus. The the guanylin peptides is critical for the normal functioning of rates of cell proliferation and apoptosis in the gut epithelium the gut. Any abnormality in this process could lead to gas can be increased or decreased in a variety of circumstances, trointestinal disorders such as irritable bowel syndrome (IBS) e.g., in response to physiological stimuli such as aging, and inflammatory bowel diseases. Inflammatory bowel dis inflammatory signals, hormones, peptides, growth factors, ease is a general name given to a group of disorders that cause chemicals and dietary habits. In addition, an enhanced pro the intestines to become inflamed, characterized by red and liferation rate is frequently associated with a reduction in Swollen tissue. Examples include ulcerative colitis and turnover time and an expansion of the proliferative Zone. The Crohn's disease. Crohn's disease is a serious inflammatory proliferation index is much higher in pathological states Such disease that predominantly affects the ileum and colon, but as ulcerative colitis and other gastrointestinal disorders. can also occur in other sections of the gastrointestinal tract. Intestinal hyperplasia is a major promoter of gastrointestinal Ulcerative colitis is exclusively an inflammatory disease of inflammation. Apoptosis and cell proliferation together regu the colon, the large intestine. Unlike Crohn's disease, in late cell number and determine the proliferation index. which all layers of the intestine are involved, and in which Reduced rates of apoptosis are often associated with abnor there can be normal healthy bowel in between patches of mal growth, inflammation, and neoplastic transformation. diseased bowel, ulcerative colitis affects only the innermost Thus, both increased proliferation and/or reduced cell death lining (mucosa) of the colonina continuous manner. Depend may increase the proliferation index of intestinal tissue, ing on which portion of the gastrointestinal tract is involved, which may in turn lead to gastrointestinal inflammatory dis Crohn's disease may be referred to as ileitis, regional enteri CaSCS. tis, colitis, etc. Crohn's disease and ulcerative colitis differ 0008. In addition to a role for uroguanylin and guanylinas from spastic colon or irritable bowel syndrome, which are modulators of intestinal fluid and ion secretion, these peptides motility disorders of the gastrointestinal tract. Gastrointesti may also be involved in the continual renewal of gastrointes nal inflammation can be a chronic condition. It is estimated tinal mucosa by maintaining the balance between prolifera that as many as 1,000,000 Americans are afflicted with tion and apoptosis. For example, uroguanylin and guanylin US 2014/0155575 A1 Jun. 5, 2014 peptides appear to promote apoptosis by controlling cellular amino acid units from position 1 through position 6 of SEQ ion flux. Given the prevalence of inflammatory conditions in ID NO: 1 or 9. In another embodiment, h is 7.j is 15, and k is Western societies a need exists to improve the treatment 16. More specifically, the first fragment has a sequence of options for inflammatory conditions, particularly of the gas amino acid units from position 15 through position 16 of SEQ trointestinal tract. ID NO: 1 or 9, the second fragment has a sequence of amino 0009 Peptide agonists of guanylate cyclase C agonists acid units from position 7 through position 14 of SEQID NO: (“GCC agonists”) are described in U.S. Pat. Nos. 7,041,786, 1 or 9, and the protected peptide produced via coupling the 7,799,897, and U.S. Patent Application Publication Nos. first and second fragments has a sequence of amino acid units US2009/0048175, US 2010/0069306, US 2010/0120694, US from position 7 through position 16 of SEQID NO: 1 or 9. In 2010/0093635, and US 2010/0221329. However, the solid one embodiment, the process of the invention further includes phase synthesis of peptides for pharmaceutical application deprotecting an amino group of the amino acid unitat position presents a number of special problems such as an overall low 7 of the protected peptide having the sequence of amino acid yield (e.g., less than 10%).