Lorputinib for use in the treatment of JAK-mediated disorders

Abstract

Disclosed herein are JAK inhibitors with low systemic toxicity. In some aspects, the disclosure includes methods for treating disease states, disorders, and conditions mediated by JAK, such as gastrointestinal cancers, including colorectal cancer and familial adenomatous polyposis.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 159,726, filed March 11, 2021, U.S. Provisional Patent Application No. 63 / 167,287, filed March 29, 2021, and U.S. Provisional Patent Application No. 63 / 232,356, filed August 12, 2021, each of which is incorporated by reference in its entirety herein.

[0002] FIELD OF THEINVENTION The present invention relates to methods of using JAK inhibitors and pharmaceutical compositions comprising such inhibitors, which compounds and pharmaceutical compositions are contemplated as being useful for preventing or treating disease states, disorders, and conditions mediated by JAK. [Background technology]

[0003] Reportedly, approximately one in three women and one in two men will be diagnosed with cancer, and approximately one in four will die from it. See A.Albini,et al.,Clin.Cancer Res.22(17),4322-27(2016) "Cancer prevention and interception: A new era for chemopreventive approaches". A significant number of cancers occur in the following organs of the gastrointestinal tract: stomach, small intestine, colon, rectum, and anus. Stomach cancer, or gastric cancer, is reported to be the fourth most common cancer and the second most common cause of cancer death worldwide. G.Bjelakovic,et al.,The Cochrane Database of Systematic Reviews(3):CD004183(2008) "Antioxidant supplements for preventing gastrointestinal cancers". Reportedly, small intestine cancer affects 9,000 people annually in the United States. More than 22,000 cases of stomach cancer are reportedly diagnosed annually in the United States. Approximately 145,000 cases of colon cancer are reportedly diagnosed annually in the United States. More than 40,000 cases of rectal cancer are reportedly diagnosed annually in the United States. Of these cancers, cancer affecting the colon or rectum, known as colorectal cancer (CRC), accounts for more than 180,000 cases annually in the United States. According to the American Cancer Society, 140,250 new cases of colorectal cancer are expected to be diagnosed in the United States in 2018, and 50,600 deaths from colorectal cancer are expected to occur in the United States in 2018. The stomach, small intestine, and large intestine (the latter including the colon, rectum, and anus) are collectively referred to herein as the gastrointestinal system ("SIS"), and cancer in any of these organs is referred to as gastrointestinal system cancer ("SISC").Preventing the development of SISC (particularly the development of CRC), whether at its onset or at a recurrent stage, is an unmet medical need. This preventative measure is referred to herein as SISC prevention or CRC prevention when referring to the prevention of colorectal cancer.

[0004] FAP is the most common adenomatous polyposis syndrome. It is an autosomal dominant disorder characterized by the early development of hundreds to thousands of adenomatous polyps throughout the colon. If left untreated, nearly all patients with the syndrome will develop colon cancer by age 35-40. Although prophylactic colectomy is the standard of care, patients remain at risk for malignant transformation of duodenal and rectal polyps. Multiple studies with both nonselective and selective cyclooxygenase inhibitors (e.g., sulindac or celecoxib) have shown that anti-inflammatory agents can inhibit the formation of colorectal adenomatous polyps. (RKS Hillips, et al. Gut 50, 857-860 (2002) "A randomized, double blind, placebo controlled study of celecoxib, a selective cyclooxygenase 2 inhibitor, on duodenal polyposis in familial adenomatous polyposis"; P. Rice, et al. Cancer Res. 63, 616-620 (2003) "Sulindac sulfide inhibits epidermal growth factor-induced phosphorylation of extracellular-regulated kinase 1 / 2 and Bad in human colon cancer cells"). The toxicity associated with these agents has hindered their further development. There is a high unmet need for new treatment options to reduce polyp burden, delay or eliminate the need for colectomy, and prevent the development of adenocarcinoma in individuals with FAP. Polyps from patients with FAP, as well as those resulting in sporadic CRC, display inflammation associated with signal transduction and activator of the interleukin (IL)-23 / IL-17 / Janus kinase (JAK) / STAT3 transcription pathway. Adenomatous epithelium is associated with a markedly increased immune infiltration compared with adjacent normal-appearing nonadenomatous epithelium and surrounding immune.This infiltration is characterized by activation of the STAT3 pathway, as indicated by IL-17 and p-STAT3 expression. In addition, the adenomatous epithelium itself may be locally activated. Adenomas with high grade dysplasia may show more activated IL-17 and p-STAT3 immune infiltration, as well as more widely distributed epithelial p-STAT3 expression. These findings suggest that this inflammatory pathway leads to further mutagenesis and tumorigenesis. Both immune and epithelial STAT3 activation have also been reported to contribute to tumorigenesis in mouse models. EC Wick, et al., Inflamm. Bowel Dis. 20(5), 821-34 (2014) "Stat3 activation in murine colitis induced by enterotoxigenic Bacteroides fragilis". Specifically, IL-23 has been associated with tumor growth and progression in CRC, and adenomas with high grade dysplasia showed increased levels of IL-17A and phosphorylated STAT3. (JLLangowski, et al. Nature 442(7101), 461-465(2006) "IL-23 promotes tumor incidence and growth").Excessive inflammatory responses in the gastrointestinal tract are mediated by inflammatory cytokines such as tumor necrosis factor (TNF), interferon-gamma (IFNγ), IL-1, IL-6, IL-12, IL-21, and IL-23, which exert their effects on cells of the innate and adaptive immune systems, including T and B lymphocytes, epithelial cells, macrophages, and dendritic cells (M. Coskun, et al., Pharmacological Research 76, 1-8 (2013), S. Danese, et al. Am J Physiol Gastrointest Liver Physiol. 310, G155-162 (2016) "JAK inhibition using tofacitinib for inflammatory bowel disease treatment: a hub for multiple inflammatory cytokines", Neurath M.Nat.Rev.Immunol.14,329-342(2014) "Cytokines in inflammatory bowel disease", S.Vermeire,et al..Gastroenterology150,S1267(2016) "Filgotinib(GLPG0634),an Oral JAK1 Selective Inhibitor,Induces Clinical Remission in Patients With Moderate-to-Severe Crohn's Disease:Results From the Phase2 FITZROY Study Interim Analysis [DDW abstract812c]"). The JAK family, JAK1, JAK2, JAK3, and Tyk2, are non-receptor tyrosine kinases that play a central role in the response to many such cytokines (J. O'Shea, N. Engl. J. Med. 368, 161-170 (2013) "JAKs and STATs in immunity, immunodeficiency, and cancer").Following receptor ligation, the associated JAK homo- or heterodimers are phosphorylated and activated, allowing the subsequent recruitment, phosphorylation, and activation of STAT family transcription factors. Phosphorylated STATs translocate to the nucleus and induce gene transcription of several chemokines, cytokines, and proteases involved in the pathogenesis of FAP (M.Coskun, et al., Pharmacological Research 76, 1-8 (2013) cited above). This JAK-STAT pathway is the signaling mechanism for many cytokines that play a role in the pathogenesis of GI cancer. (M.Li, et al. PLoS One 10 (5), e0127356 (2015) "Prognostic role of phospho-STAT3 in patients with cancers of the digestive system: a systematic review and meta-analysis"). The JAK-STAT pathway is common to multiple inflammatory cytokine responses, and therefore it offers an attractive therapeutic target for preventing the excessive inflammatory responses associated with FAP and the adenomas associated with sporadic CRC, as well as for restoring mucosal homeostasis to attempt to block the development of adenocarcinoma in the GI tract. Investigational and commercially available orally administered, systemically bioavailable pan-JAK inhibitors have a variety of off-target effects that apparently limit high (higher) doses, such as elevated lipids (low- and high-density lipoproteins) and cytopenias (including neutropenia, lymphopenia, and anemia), as well as elevated liver enzymes. Thus, therapeutic inhibition of JAK with limited exposure to the intestinal tract offers an opportunity to achieve efficacy in JAK-driven diseases of the intestinal tract, including FAP, SISC, and CRC.

[0005] With reference to Figure 1, an orally administered drug can in principle follow the gastrointestinal tract from the mouth to the esophagus (1), to the stomach (2), through the duodenum (3) to the jejunum (4), then to the ileum (5), and then to the colon (6). The relative absorption areas of these various parts are about 60% in the jejunum (4), about 26% in the ileum (5), and about 13% in the colon (6). Absorption through these various gastrointestinal regions can result in the onset of systemic distribution, which can lead to undesirable side effects. The gastrointestinal tract has a very large surface area. For example, HF Helander, et al., Surface area of ​​the digestive tract-revisited, Scandinavian Journal of Gastroenterology 49(6), 681-89 (2014), and KJ Filipski, et al., Intestinal Targeting of Drugs: Rational Design Approaches and Challenges Current Topics in Medicinal Chemistry 13, 776-802 (2013). Such a wide absorption surface area favors systemic distribution of substances that can pass through the walls of various parts of the intestinal tract into the bloodstream, which may then result in undesirable side effects of the systemically distributed substance. Although systemic distribution is shown by the dashed arrow in FIG. 1 as passing through the colon wall for simplified illustration purposes, such distribution is not limited to the colon wall, and can also occur through the walls of other parts of the gastrointestinal tract shown in FIG. 1, such as the wall of the small intestine. It is also understood that the dashed arrows in Figure 1 represent systemic distribution beyond the gastrointestinal tract, as such systemic distribution is known to occur with reference to gastrointestinal physiology, and such dashed arrows are merely referenced to diagrammatically illustrate such systemic distribution. See, for example, Current Topics in Medicinal Chemistry 13, 777-80 (2013), cited above, for a description of intestinal tissue, transport in intestinal tissue, and metabolism. Summary of the Invention

[0006] Since some of the known JAK inhibitors have side effects related to their systemic effects, it is desirable to discover new JAK inhibitors as active agents for the treatment and prevention of intestinal diseases, including FAP, SISC, and CRC.

[0007] Disclosed herein are JAK inhibitors with low systemic toxicity. In some embodiments, the present invention provides JAK inhibitors represented by compounds of formula I: 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide,

[0008] [ka] or its pharma- ceutically acceptable salts, solvates, and polymorphs, and methods of using the compound of formula I in the treatment of disease states, disorders, and conditions mediated by JAK. The term "compound of formula I" is intended to encompass the compound of formula I or its pharma- ceutically acceptable salts, or a solvate of the compound of formula I or its pharma- ceutically acceptable salts, or any polymorphs or co-crystals of the foregoing in a solvent-free form or in any one of the hydrated and / or solvated forms described herein (hereinafter "compound of formula I or its pharma- ceutically acceptable salts, solvates, and polymorphs"). The compound of formula I is also referred to as the "active agent(s)."

[0009] Aspects of the present invention relate to compounds of formula I, pharmaceutical compositions containing same, methods of their use as JAK inhibitors, and methods of their use in the treatment of disease states, disorders and conditions mediated by JAK.

[0010] In some embodiments, a method for treating or preventing the development of gastrointestinal cancer (SISC), particularly the development of colorectal cancer (CRC), comprises administering to a subject a therapeutically effective amount of a composition comprising a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof.

[0011] In some embodiments, a method for treating or preventing a subject suffering from familial adenomatous polyposis ("FAP") comprises administering to the subject a composition comprising a therapeutically effective amount of a compound of formula I, or pharma- ceutically acceptable salts, solvates, and polymorphs thereof.

[0012] Embodiments of the invention exhibit pan-JAK inhibitory effects with local GI effects and low or negligible systemic effects.Furthermore, embodiments of the invention having such characteristics can be administered orally.

[0013] Additional aspects, features, and advantages of the invention will become apparent from the following detailed description as well as through the practice of the invention. [Brief description of the drawings]

[0014] [Figure 1]1 shows a schematic diagram of a portion of the human gastrointestinal tract (shown as a stretched rendering, not to scale). The duodenum (3), jejunum (4), and ileum (5) (all shown diagrammatically) form the small intestine after the stomach (2) and esophagus (1). The large intestine includes the colon (6), which in turn includes the cecum (7) and appendix (not shown), the ascending colon, transverse colon, descending colon, sigmoid colon (annular, also not shown), and rectum (11). The transverse colon is the portion contained between the right colonic flexure (8) and the left colonic flexure (9), with the ascending colon extending from the cecum (7) to the right colonic flexure (8) and the descending colon extending from the left colonic flexure (9) to the rectum (11). For convenience, the various distribution patterns are illustrated with reference to the colon, but they may also refer to other portions of the gastrointestinal tract. Systemic distribution is shown by dashed arrows in Figure 1 as penetrating the colon wall for simplified illustrative purposes, but such distribution is not limited to the colon wall and can also occur through walls of other portions of the gastrointestinal tract shown in Figure 1, such as the wall of the small intestine. Some other permeation distribution is shown by solid arrows in Figure 1 as penetrating colon tissue for simplified illustrative purposes, but such penetration is not limited to colon tissue, as such can also occur in tissues of other portions of the gastrointestinal tract shown in Figure 1, such as the small intestine tissue. The effect of embodiments of JAK inhibitors according to the present invention is illustratively shown as disrupting the JAK / STAT signaling pathway that would otherwise result in JAK-mediated injury, such as inflammation associated with inflammatory bowel disease ("IBD") or colon cancer. [Diagram 2] FIG. 1 is a schematic showing the preparation / interconversion of embodiments of compounds of formula I. [Diagram 3]Overlay of high throughput X-ray powder diffraction (HT-XRPD) patterns for the following embodiments of the compound of formula I: from bottom to top, 1s, 2 (obtained by equilibration at room temperature in 1,4-dioxane), 3b (obtained by thermal cycling in cyclohexanone), 1b+4 (obtained by cooling crystallization at μL scale in methanol / water (50 / 50, v / v)), 5 (obtained by thermal cycling in chloroform), 6 (obtained by cooling crystallization at mL scale in acetonitrile), 7 (obtained by 1s+7, thereby obtained by solvent equilibration in heptane), 7 (obtained by desolvation of 1s+7, thereby obtained by solvent equilibration in heptane), 8 (obtained by desolvation of embodiment 5 by cycling differential scanning calorimetry), and 9 (obtained by desolvation of embodiment 2 by cycling differential scanning calorimetry). [Figure 4] FIG. 1 is an overlay of high throughput X-ray powder diffraction (HT-XRPD) patterns for the following embodiments of the compound of formula I: 1s (starting material), 1a (several forms of the sample of embodiment 1s were subjected to accelerated aging conditions). condition, AAC) (obtained after exposure to 40 °C and 70% relative humidity), 1b (obtained by solvent equilibration in toluene at room temperature), 1c (obtained by cooling crystallization in ethyl acetate / 1,4-dioxane (50 / 50, v / v) at μL scale), 1d (obtained by cooling crystallization in acetonitrile / chloroform (50 / 50, v / v) at μL scale), 1e (obtained by cooling crystallization in ethyl acetate / 1,4-dioxane (50 / 50, v / v) at μL scale), 1f (obtained by solvent equilibration in p-xylene at room temperature), 1g (obtained by solvent equilibration in anisole at 50 °C), 1h (obtained by cooling crystallization in p-xylene at μL scale). [Diagram 5]Overlay of high throughput X-ray powder diffraction (HT-XRPD) patterns for the following embodiments of compounds of formula I: from bottom to top, 1s, 3b (obtained by thermal cycling in cyclohexanone), 3c (obtained by cooling crystallization in 1,4-dioxane on a μL scale), 3d (obtained by cooling crystallization in tetrahydrofuran on a μL screen), and 3e (obtained by thermal cycling in isobutanol). [Figure 6] HR-XRPD diffractograms of embodiment 1s in its initial form ("1s"), after exposure to 40°C and 70% relative humidity for 4 days ("1s 70RH"), and after exposure to 25°C and 100% relative humidity for 4 days ("10"). [Figure 7A] X-ray powder diffraction (XRPD) patterns of embodiment 11(A), embodiment 12(B), embodiment 13(C), embodiment 14(D), and embodiment 11b(E). [Figure 7B] X-ray powder diffraction (XRPD) patterns of embodiment 11(A), embodiment 12(B), embodiment 13(C), embodiment 14(D), and embodiment 11b(E). [Figure 7C] X-ray powder diffraction (XRPD) patterns of embodiment 11(A), embodiment 12(B), embodiment 13(C), embodiment 14(D), and embodiment 11b(E). [Figure 7D] X-ray powder diffraction (XRPD) patterns of embodiment 11(A), embodiment 12(B), embodiment 13(C), embodiment 14(D), and embodiment 11b(E). [Figure 7E] X-ray powder diffraction (XRPD) patterns of embodiment 11(A), embodiment 12(B), embodiment 13(C), embodiment 14(D), and embodiment 11b(E). [Figure 8] 1 is an overlay of X-ray powder diffraction (XRPD) patterns for the following embodiments of a compound of formula I: from bottom to top, embodiment 17, embodiment 18, embodiment 15, and embodiment 16. [Figure 9]Modulated DSC ("mDSC") profile for embodiment 19 showing a glass transition temperature (Tg) at 115.3°C ("Rev" in the vertical axis label stands for "reversible"). [Figure 10A] (A) TGA (thermogravimeteric analysis) of embodiment 18 showing a 6.5% w / w loss between 30° C. and 170° C. (B) DSC (differential scanning calorimetry) of embodiment 18 showing an endotherm of 52.8 J / g between 45° C. and 90° C., an endotherm of 31.0 J / g at 140.6° C., an exotherm of 24.3 J / g at 168.8° C., and an endotherm of 31.3 J / g at 200.0° C. [Figure 10B] (A) TGA (thermogravimeteric analysis) of embodiment 18 showing a 6.5% w / w loss between 30° C. and 170° C. (B) DSC (differential scanning calorimetry) of embodiment 18 showing an endotherm of 52.8 J / g between 45° C. and 90° C., an endotherm of 31.0 J / g at 140.6° C., an exotherm of 24.3 J / g at 168.8° C., and an endotherm of 31.3 J / g at 200.0° C. [Figure 11] 1 is an overlay of X-ray powder diffraction (XRPD) patterns for the following embodiments of a compound of formula I: from bottom to top, embodiment 20 and embodiment 21. [Figure 12A] (A) TGA of embodiment 17 showing a 4.2% w / w loss between 30° C. and 100° C. (B) DSC of embodiment 17 showing an endotherm of 90.3 J / g between 45° C. and 100° C., an endotherm of 35.5 J / g at 143.8° C., an endotherm of 1.6 J / g at 168.3° C., an exotherm of 3.8 J / g at 178° C., and an endotherm of 9.2 J / g at 200.0° C. [Figure 12B] (A) TGA of embodiment 17 showing a 4.2% w / w loss between 30° C. and 100° C. (B) DSC of embodiment 17 showing an endotherm of 90.3 J / g between 45° C. and 100° C., an endotherm of 35.5 J / g at 143.8° C., an endotherm of 1.6 J / g at 168.3° C., an exotherm of 3.8 J / g at 178° C., and an endotherm of 9.2 J / g at 200.0° C. [Figure 13] 1. Overlay of X-ray powder diffraction (XRPD) patterns for the following embodiments of the compound of formula I: (from bottom to top) embodiment 31, embodiment 30, embodiment 17, embodiment 29, embodiment 16, embodiment 26, embodiment 25, embodiment 18, embodiment 24, embodiment 23, embodiment 27, and embodiment 22. [Figure 14] Overlay of X-ray powder diffraction (XRPD) patterns for the following embodiments of compounds of formula I: from bottom to top, embodiment 32, embodiment 33, embodiment 23, embodiment 34, embodiment 35, embodiment 36, embodiment 25, embodiment 38, embodiment 17, embodiment 39, and embodiment 28. [Figure 15] 3 is an overlay of X-ray powder diffraction (XRPD) patterns for the following embodiments of compounds of formula I: from bottom to top, embodiment 46, embodiment 45, embodiment 44, embodiment 43, embodiment 42, embodiment 41, embodiment 40, and embodiment 37. [Figure 16] 47, and 48. An overlay of X-ray powder diffraction (XRPD) patterns for embodiments of compounds of formula I: from bottom to top, embodiment 53, embodiment 52, embodiment 51, embodiment 50, embodiment 49, embodiment 48, and embodiment 47. [Figure 17A] (A) TGA of embodiment 11 showing a loss of 4.7% w / w between 155° C. and 185° C. (B) DSC of embodiment 11 showing a first endotherm of 57.8 J / g at 167.8° C. due to solvent loss and a second endotherm of 90.8 J / g at 194.5° C. due to sample melting. [Figure 17B] (A) TGA of embodiment 11 showing a loss of 4.7% w / w between 155° C. and 185° C. (B) DSC of embodiment 11 showing a first endotherm of 57.8 J / g at 167.8° C. due to solvent loss and a second endotherm of 90.8 J / g at 194.5° C. due to sample melting. [Figure 18] 12 is a Gravimetric Vapor Sorption (GVS) isotherm plot of embodiment 11 showing a mass change of 0.66% from 0 to 90% RH. The mass change on the vertical axis is based on the mass of the starting sample. [Figure 19A] (A) TGA of embodiment 6 showing weight loss at temperatures above 260° C., which is interpreted as associated with sample decomposition. (B) DSC of embodiment 6 showing an endotherm of 95.8 J / g at 194.4° C. due to sample melting. [Figure 19B] (A) TGA of embodiment 6 showing weight loss at temperatures above 260° C., which is interpreted as associated with sample decomposition. (B) DSC of embodiment 6 showing an endotherm of 95.8 J / g at 194.4° C. due to sample melting. [Figure 20A] (A) TGA of embodiment 8 showing a loss of 1.4% w / w between 40° C. and 240° C., corresponding to a loss of 0.07 moles of 1,4-dioxane. (B) DSC of embodiment 8 showing an endotherm of 58.6 J / g at 199.7° C. due to the sample melting. [Figure 20B] (A) TGA of embodiment 8 showing a loss of 1.4% w / w between 40° C. and 240° C., corresponding to a loss of 0.07 moles of 1,4-dioxane. (B) DSC of embodiment 8 showing an endotherm of 58.6 J / g at 199.7° C. due to the sample melting. [Figure 21A] (A) The TGA of embodiment 2 showing a 7.4% w / w loss from 75° C. to 110° C., a 11.9% w / w loss from 110° C. to 130° C., a 2.0% w / w loss from 130° C. to 165° C., and a 2.5% w / w loss from 165° C. to 210° C. (B) The DSC of embodiment 2 showing an endotherm of 86.2 J / g at 92.8° C., an endotherm of 11.1 J / g at 111.5° C., an endotherm of 45.5 J / g at 149.0° C., an exotherm of 20.6 J / g at 165.2° C., an endotherm of 3.7 J / g at 177.1° C., an endotherm of 43.0 J / g at 200.2° C., and an endotherm of 29.3 J / g at 220.6° C. [Figure 21B](A) The TGA of embodiment 2 showing a 7.4% w / w loss from 75° C. to 110° C., a 11.9% w / w loss from 110° C. to 130° C., a 2.0% w / w loss from 130° C. to 165° C., and a 2.5% w / w loss from 165° C. to 210° C. (B) The DSC of embodiment 2 showing an endotherm of 86.2 J / g at 92.8° C., an endotherm of 11.1 J / g at 111.5° C., an endotherm of 45.5 J / g at 149.0° C., an exotherm of 20.6 J / g at 165.2° C., an endotherm of 3.7 J / g at 177.1° C., an endotherm of 43.0 J / g at 200.2° C., and an endotherm of 29.3 J / g at 220.6° C. [Figure 22A] (A) TGA of embodiment 9. (B) DSC of embodiment 9 showing an endotherm at 221.8° C. with 104.4 J / g. [Figure 22B] (A) TGA of embodiment 9. (B) DSC of embodiment 9 showing an endotherm at 221.8° C. with 104.4 J / g. [Figure 23A] (A) TGA of embodiment 16 showing a 5.2% w / w loss between 30° C. and 105° C. (B) DSC of embodiment 16 showing an endotherm of 48.4 J / g between 35° C. and 90° C., an endotherm of 41.8 J / g at 147.0° C., an endotherm of 1.0 J / g at 166.6° C., an exotherm of 4.4 J / g at 180.7° C., and an endotherm of 7.7 J / g at 201.1° C. [Figure 23B] (A) TGA of embodiment 16 showing a 5.2% w / w loss between 30° C. and 105° C. (B) DSC of embodiment 16 showing an endotherm of 48.4 J / g between 35° C. and 90° C., an endotherm of 41.8 J / g at 147.0° C., an endotherm of 1.0 J / g at 166.6° C., an exotherm of 4.4 J / g at 180.7° C., and an endotherm of 7.7 J / g at 201.1° C. [Figure 24] X-ray powder diffraction (XRPD) of embodiment 11 (labeled "11") and X-ray powder diffraction (XRPD) of embodiment 11 after a variable temperature (VT)-XRPD experiment (labeled "11 after VT") and X-ray powder diffraction (XRPD) of embodiment 6. [Diagram 25] X-ray powder diffraction (XRPD) pattern of the compound of formula I in Form 1s. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As used herein, the terms "comprise," "contain," and "include" are used in their open, non-limiting sense.

[0016] Any formula given herein is intended to represent the compound having the structure depicted by the structural formula, as well as specific variants or forms. A specific structure may exist as a tautomer. In addition, the amorphous form, hydrate, solvate, polymorphs, and pseudopolymorphs of such compounds of the present invention, and mixtures thereof, are also envisioned as part of the present invention. An embodiment of the present invention is any one of the solvent-free forms, or hydrated and / or solvated forms as exemplified herein.

[0017] As used herein, the term "about," when preceding a numerical value, means a range of plus or minus 10% of that value, unless the context of this disclosure dictates otherwise or is inconsistent with such an interpretation (e.g., "about 50" means 45-55, "about 25,000" means 22,500-27,500, etc.).

[0018] The terms "administer" or "administered" or "administering" refer to administering a compound of Formula I or a pharmaceutical composition thereof to a subject by any method known to one of skill in the art in light of the present disclosure, such as intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., enteral), intranasal, or intraperitoneal routes of administration. In certain embodiments, a pharmaceutical composition of the invention is administered orally to a subject.

[0019] The term "subject" refers to any animal, particularly a mammal, most particularly a human, that will be or has been treated by a method according to an embodiment of the present invention. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs), such as monkeys or apes, and more particularly humans.

[0020] The term "inhibitor" or "inhibitors" refers to a compound that reduces, prevents, inactivates, desensitizes, or downregulates the expression or activity of JAK.

[0021] The term "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent, including a crystalline form of the present invention, that elicits in a tissue system, animal or human the biological or pharmaceutical response that a researcher, veterinarian, physician, or other clinical professional desires to obtain, including reduction or inhibition of an enzyme or protein, or amelioration of symptoms, mitigation of a disease, retardation or slowing of disease progression, or prevention of a disease.

[0022] As used herein, the terms "treat", "treating" or "treatment" of any disease, condition, syndrome, or disorder, in one embodiment, refer to ameliorating the disease, condition, syndrome, or disorder (i.e., delaying or arresting or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat", "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be discernible by the patient. In a further embodiment, "treat", "treating" or "treatment" refers to modulating the disease, condition, syndrome, or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treat", "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease, condition, syndrome, or disorder.

[0023] A "pharmaceutically acceptable salt" is a salt of a compound that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject.See generally SM Berge, et al., "Pharmaceutical Salts", J.Pharm.Sci.66,1-19(1977), and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002.The compounds of formula (I) may have sufficiently acidic groups, sufficiently basic groups, or both types of functional groups, and therefore may react with many inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts. Examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-diol, hexaphosphate ... Examples of suitable salts include phenyl-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate salts.

[0024] Disclosed herein are compositions of compounds of formula I and methods of using them in the treatment of disease states, disorders, and conditions mediated by one or more of the JAK family of tyrosine kinases. In some embodiments, the methods include administering to a subject a compound having the structure of formula I, comprising an effective amount of 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide,

[0025] [ka] or a pharma- ceutically acceptable salt, or solvate, or polymorph thereof, to a subject.

[0026] Also disclosed herein is a method for treating or preventing a subject suffering from gastrointestinal cancer or colon cancer. The method comprises administering to the subject a composition comprising a therapeutically effective amount of a compound of formula I, or its pharma-ceutically acceptable salts, solvates, and polymorphs. The stomach, small intestine, and large intestine (the latter including the colon, rectum, and anus) are collectively referred to herein as the gastrointestinal system ("SIS"), and cancer in any of these organs is referred to as gastrointestinal cancer ("SISC"). Cancer in any of the large intestines is referred to as colorectal cancer ("CRC").

[0027] Also disclosed herein is a method for treating or preventing a premalignant condition in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of formula I, or its pharma-ceutically acceptable salts, solvates, and polymorphs. The premalignant condition preceding SISC may be present in any one of several regions of the gastrointestinal tract (particularly the colon and / or rectum). Sometimes, such conditions are manifested in the form of polyps, which may be present in small numbers, as in its sporadic manifestations, in large numbers, as in familial adenomatous polyposis ("FAP"), or not at all, as in certain forms of hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome.

[0028] Also disclosed herein is a method of treating, delaying, or preventing any symptoms of disease in a subject suffering from any SISC-associated cancer predisposition syndrome, such as familial adenomatous polyposis ("FAP"). The method includes delaying the need for a surgical procedure, such as a colectomy to prophylactically remove the colon, to reduce the risk of future colon cancer development. The method includes administering to the subject a composition comprising a therapeutically effective amount of a compound of formula I, or a pharmacologic acceptable salt, solvate, and polymorph thereof. The term FAP is used herein to encompass what are sometimes referred to as subtypes of FAP, such as attenuated familial adenomatous polyposis ("AFAP"). Unless otherwise indicated or explicitly referred to as any particular subtype, the term FAP includes subtypes such as AFAP.

[0029] In some embodiments, a method for reducing the number of polyps or polyp burden (the latter defined by considering both the number and individual size of polyps) in a subject comprises administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula I, or a pharma- ceutically acceptable salt, solvate, and polymorph thereof. In some embodiments, reducing the number of polyps comprises reducing the number of polyps in the colon, rectum, postoperative J pouch, and / or duodenum after administration of a compound of Formula I. In some embodiments, the number of polyps with a diameter of ≧2 mm in the colon, rectum, postoperative J pouch, and duodenum is reduced. In some embodiments, the number of polyps with a diameter of ≧5 mm in the colon, rectum, postoperative J pouch, and / or duodenum is reduced. In certain embodiments, the reduction in the number of polyps following administration is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more, or any range therebetween.

[0030] Also disclosed herein is a method for treating or preventing the development of SISC (particularly the development of CRC) at any stage of disease progression, the method comprising administering to a subject a therapeutically effective amount of a composition comprising a compound of formula I, or a pharmacologic acceptable salt, solvate, or polymorph thereof.

[0031] Also disclosed in the present disclosure is a method for treating or preventing any chronic or acute inflammatory disorder in the gastrointestinal system of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof.

[0032] A further aspect of the invention relates to the use of a compound of formula I for administration to a subject who has or has had at least one of sporadic gastrointestinal polyp formation, FAP (or other hereditary polyposis syndromes), and Lynch syndrome (or other hereditary non-polyposis syndromes).

[0033] A further aspect of the invention is a method of treating a subject suffering from or diagnosed with at least one form of localized or metastatic SISC, including at least one of gastric cancer, small intestine cancer, colon cancer, and anal cancer, by administering a therapeutically effective amount of a composition comprising a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof.

[0034] Further aspects of the invention include a method of SISC inhibition in a subject, the method comprising administering a therapeutically effective amount of a composition comprising a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is FAP SISC inhibition. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is Lynch syndrome SISC inhibition. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is sporadic polyp inhibition. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is gastric cancer inhibition. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is small intestine cancer inhibition. Further aspects are provided by any one of the preceding aspects, in which the SISC inhibition is anal cancer inhibition.

[0035] In some SISC inhibition scenarios, drug delivery along the entire or majority of the intestinal tract may be desirable. In other scenarios, it may be desirable to increase the local concentration in any given portion of the gastrointestinal tract. In still other scenarios, a combination of these two delivery modes at different sites in the intestinal tract may be desirable.

[0036] The therapeutically effective dose of a compound of formula I includes a dose range of about 1 mg to about 1000 mg, 10 mg to about 1000 mg, or any specific amount or range therein, particularly about 1 mg to about 100 mg, 10 mg to about 100 mg, or any specific amount or range therein. In some embodiments of the invention, the dose range is about 1 mg to about 5 mg. In some embodiments of the invention, the dose range is about 5 mg to about 10 mg. In some embodiments of the invention, the dose range is about 10 mg to about 15 mg. In some embodiments of the invention, the dose range is about 15 mg to about 20 mg. In some embodiments of the invention, the dose range is about 20 mg to about 25 mg. In some embodiments of the invention, the dose range is about 25 mg to about 30 mg. In some embodiments of the invention, the dose range is about 35 mg to about 40 mg. In some embodiments of the invention, the dose range is about 45 mg to about 50 mg. In some embodiments of the present invention, the dose range is about 55 mg to about 60 mg. In some embodiments of the present invention, the dose range is about 65 mg to about 70 mg. In some embodiments of the present invention, the dose range is about 75 mg to about 80 mg. In some embodiments of the present invention, the dose range is about 85 mg to about 90 mg. In some embodiments of the present invention, the dose range is about 95 mg to about 100 mg. In some embodiments of the present invention, the dose range is about 30 mg to about 1620 mg. In some embodiments of the present invention, the dose range is about 50 mg to about 90 mg. In some embodiments of the present invention, the dose range is about 50 mg to about 80 mg. In some embodiments of the present invention, the dose is about 50 mg. In some embodiments of the present invention, the dose is about 55 mg. In some embodiments of the present invention, the dose is about 60 mg. In some embodiments of the present invention, the dose is about 65 mg. In some embodiments of the present invention, the dose is about 70 mg. In some embodiments of the invention, the dose is about 75 mg. In some embodiments of the invention, the dose is about 80 mg. In some embodiments of the invention, the dose is about 85 mg. In some embodiments of the invention, the dose is about 90 mg. In some embodiments of the invention, the dose is about 95 mg. In some embodiments of the invention, the dose is about 100 mg.In some embodiments of the present invention, each of the dosages is a daily dose. In some embodiments of the present invention, the dosage requires multiple administrations per day, such as twice a day or three times a day. For example, a 100 mg dose is achieved by administering a 50 mg dose twice a day. A 130 mg dose is achieved by administering a 65 mg dose twice a day. A 150 mg dose is achieved by administering a 75 mg dose twice a day. A 200 mg dose is achieved by administering a 100 mg dose twice a day. A 300 mg dose is achieved by administering a 150 mg dose twice a day. A 400 mg dose is achieved by administering a 200 mg dose twice a day. And a 600 mg dose is achieved by administering a 300 mg dose twice a day.

[0037] In some embodiments, the compound of formula I is in a crystalline form selected from forms 1s, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 2, 3b, 3c, 3d, 3e, 5, 6, 7, 8, 9, 10, 11, 11b, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53.

[0038] In some embodiments, a method of treating or preventing a subject from suffering from familial adenomatous polyposis ("FAP") comprises administering to the subject an effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, administration is once daily or twice daily. In some embodiments, treating FAP comprises reducing the polyp burden in the subject. In some embodiments, reducing the polyp burden comprises reducing the number of polyps and / or reducing the size of the polyps.

[0039] In some embodiments, a method for treating or preventing FAP in a subject who has previously undergone therapy for FAP or who is currently undergoing therapy for FAP comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, the therapy can be surgery, radiation therapy, a Cox-2 inhibitor, a non-steroidal anti-inflammatory drug (NSAID), or chemotherapy.

[0040] In some embodiments, the method of treating or preventing a subject from suffering from SISC comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, administration is once daily or twice daily. In some embodiments, treating or preventing SISC comprises reducing polyp burden in the subject. In some embodiments, reducing polyp burden comprises reducing the number of polyps and / or reducing the size of polyps.

[0041] In some embodiments, the method of treating or preventing SISC in a subject previously undergoing therapy or currently undergoing therapy comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, the therapy can be surgery, radiation therapy, or chemotherapy. EGFR inhibitors, VEGF inhibitors, and checkpoint inhibitors.

[0042] In some embodiments, the method of treating or preventing CRC in a subject who has previously undergone therapy or is currently undergoing therapy comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, the therapy can be surgery, radiation therapy, chemotherapy, EGFR inhibitors, VEGF inhibitors, and checkpoint inhibitors.

[0043] In some embodiments, the method of treating or preventing a subject from suffering from CRC comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, administration is once daily or twice daily. In some embodiments, treating or preventing CRC comprises reducing CRC malignancy and / or associated polyp burden in the subject. In some embodiments, reducing polyp burden comprises reducing the number of polyps and reducing the size of polyps. In some embodiments, reducing a polyp parameter comprises reducing the number of polyps or reducing the size of polyps.

[0044] In some embodiments, the method of preventing recurrence of CRC in a subject comprises administering to the subject a therapeutically effective dose of a compound of formula I from about 1 mg to about 100 mg.

[0045]

[67] In some embodiments, a method for preventing recurrence of FAP-associated polyposis in a subject comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I.

[0046] In some embodiments, a method for preventing the progression of symptoms of FAP in a subject comprises administering to the subject a therapeutically effective dose of a compound of formula I from about 1 mg to about 100 mg.

[0047] In some embodiments, a method for slowing the progression of symptoms of FAP in a subject comprises administering to the subject a therapeutically effective dose of a compound of formula I from about 1 mg to about 100 mg.

[0048] In some embodiments, a method for preventing SISC in a subject diagnosed with irritable bowel disease (IBD) comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, the IBD can be ulcerative colitis or Crohn's disease.

[0049] In some embodiments, the method for preventing SISC in a subject previously diagnosed with cancer comprises administering to the subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. In some embodiments, the cancer can be breast cancer, ovarian cancer, uterine cancer, or abdominal cancer.

[0050] In some embodiments, the compounds of formula I are administered as a preventative measure or prevention to treat subjects at high risk of developing CRC, SISC, or FAP. High risk may be due to factors such as irritable bowel syndrome, the presence of certain gut microbiome components, a family history of colon cancer, a personal history of colon cancer, the finding of polyps or pre-malignant lesions during colonoscopy, or other genetic factors such as mutations in the KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and / or APC genes.

[0051] In some embodiments, a method for preventing FAP or CRC in a subject in a high-risk group comprises administering to the subject a therapeutically effective dose of a compound of formula I from about 1 mg to about 100 mg.

[0052] In some embodiments, the method of preventing a pre-malignant condition from becoming a malignant condition comprises administering to a subject a therapeutically effective dose of about 1 mg to about 100 mg of a compound of formula I. The condition can be the presence of adenomatous polyps. In some embodiments, the pre-malignant condition can be Lynch syndrome. In some embodiments, the pre-malignant condition can be early stage CRC and adenoma.

[0053] In some embodiments, the method of treating an excessive inflammatory response in the intestinal tract comprises administering to a subject a therapeutically effective dose of a compound of formula I from about 1 mg to about 100 mg.

[0054] In some embodiments, the method of treating or preventing a subject from suffering from colon cancer comprises administering to the subject a therapeutically effective dose of about 50 mg to about 100 mg of a compound of Formula I (crystalline Form 1s). In some embodiments, administration is once daily or twice daily.

[0055] In some embodiments, the method of treating or preventing a subject from suffering from colon cancer comprises administering to the subject a therapeutically effective dose of about 10 mg to about 50 mg of a compound of Formula I (crystalline Form 1s). In some embodiments, administration is once daily or twice daily.

[0056] In some embodiments, the method of treating or preventing a subject from suffering from colon cancer comprises administering to the subject a therapeutically effective dose of about 30 mg to about 50 mg of a compound of Formula I (crystalline Form 1s). In some embodiments, administration is once daily or twice daily.

[0057] In some embodiments, the method of treating or preventing a subject from suffering from colon cancer comprises administering to the subject a therapeutically effective dose of about 50 mg to about 100 mg of a compound of Formula I (crystalline form 11). In some embodiments, administration is once daily or twice daily.

[0058] In some embodiments, a method of treating a disorder or condition affected by inhibition of JAK tyrosine kinase activity in a subject in need of treatment comprises administering a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, in an amount sufficient to achieve a concentration in colon tissue of about 20 ng / g to about 20,000 ng / g. In some embodiments, the administration achieves a tissue concentration of about 20 ng / g to about 15,000 ng / g, about 20 ng / g to about 10,000 ng / g, about 20 ng / g to about 8,000 ng / g, about 20 ng / g to about 6,000 ng / g, about 20 ng / g to about 4,000 ng / g, or about 20 ng / g to about 1,000 ng / g. In some embodiments, the disorder or condition is colon cancer or FAP.

[0059] In some embodiments, the method of treating a disorder or condition affected by inhibition of JAK in a subject in need of treatment comprises administering a compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, in an amount sufficient to achieve a concentration in rectal tissue of about 20 ng / g to about 20,000 ng / g. In some embodiments, the administration achieves a tissue concentration of about 20 ng / g to about 15,000 ng / g, about 20 ng / g to about 10,000 ng / g, about 20 ng / g to about 8,000 ng / g, about 20 ng / g to about 6,000 ng / g, about 20 ng / g to about 4,000 ng / g, or about 20 ng / g to about 1,000 ng / g. In some embodiments, the disorder or condition is colorectal cancer or FAP.

[0060] Also disclosed herein are methods for determining or predicting response to a JAK inhibitor in a subject in need of treatment. In one aspect, a method for predicting response to a compound of formula I in a subject in need thereof comprises: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) comparing the level of pSTAT-3 in (a) with (b), wherein a decrease in the level of pSTAT-3 in (b) predicts response to the compound of Formula I in the subject.

[0061] In another embodiment, a method of monitoring the effectiveness of ongoing JAK inhibitor therapy in a subject in need thereof includes: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) comparing the level of pSTAT-3 in (a) with (b), wherein a decrease in the level of pSTAT-3 in (b) indicates efficacy of the compound of Formula I in the subject.

[0062] In another aspect, a method for designing a drug regimen for treating familial adenomatous polyposis, cancer, or a JAK-mediated disease in a subject in need thereof includes: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) Comparing the level of pSTAT-3 in (a) with (b); (d) administering a higher dose of the compound of formula I if the level of pSTAT-3 is higher in the test sample as compared to the control sample.

[0063] A method for altering the dose and / or dosing frequency of a JAK inhibitor in a subject suffering from FAP, cancer, or a JAK-mediated disease includes: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) Comparing the level of pSTAT-3 in (a) with (b); (d) increasing the dose of the compound of formula I if the level of pSTAT-3 in the test sample is higher, equal, or slightly decreased as compared to the control sample, and decreasing the dose of the compound of formula I if the level of pSTAT-3 in the test sample is lower as compared to the control sample.

[0064] In some embodiments, the test sample and the control sample are from the same subject. In some embodiments, the control sample can be from the subject prior to administration of the compound of formula I. In some embodiments, the control sample can be from a pool of subject samples prior to administration of the compound of formula I to the subject.

[0065] In some embodiments, the subject's sample (control or test sample) can be any cell or tissue, including blood. In some embodiments, the subject's sample can be normal cells, normal tissue, tumor cells, tumor tissue, or any malignant cells. In some embodiments, the subject's sample can be tissue from different parts of the intestinal tract, such as the colon, rectum, postoperative J-pouch, and duodenum.

[0066] Once a subject control or test sample has been obtained, the level of pSTAT-3 in the subject's sample can be measured using an assay such as flow cytometry, preferably imaging flow cytometry (IFC), luminescence analysis, chemiluminescence analysis, immunohistochemistry, or fluorescence microscopy.

[0067] In some embodiments, comparing the levels of pSTAT-3 in the control sample and the test sample can provide information regarding the effectiveness of the JAK inhibitor in the subject. For example, a decrease in the pSTAT-3 level in the test sample compared to the control sample can indicate that the compound of formula I is effective in the subject. In certain embodiments, the decrease in the pSTAT-3 level in the test sample compared to the control sample is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, or any range therebetween.

[0068] In some aspects, a method of treating CRC or FAP in a subject includes (a) determining a mutation in one or more genes selected from KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC, and (b) administering a therapeutically effective dose of a compound of formula I.

[0069] In some embodiments, a method of diagnosing in a subject whether the subject is at increased risk for developing CRC or FAP includes (a) determining a mutation in one or more genes selected from KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC; and (b) administering a therapeutically effective dose of a compound of formula I.

[0070] In some embodiments, administration of a compound of formula I exhibits pan-JAK kinase inhibitory effects with local GI effects and low or negligible systemic effects, as defined by changes in circulating pSTAT-3 levels. Moreover, embodiments of the invention having such characteristics can be administered orally. In some embodiments, a compound of formula I may be used to prevent and / or control excessive inflammatory responses, the systemic effects of which are eliminated or reduced. In some embodiments, a compound of formula I exhibits local effects on gastrointestinal tissues for the treatment of conditions (such as, but not limited to, IBD) without causing systemic effects or with such systemic effects reduced to an acceptable level.

[0071] Once the improvement of the patient's disease, disorder or condition occurs, the dosage may be adjusted for preventive or maintenance treatment.For example, the dosage or frequency of administration, or both, may be reduced as a function of symptoms to a level at which the desired therapeutic or preventive effect is maintained.Of course, when symptoms are alleviated to an appropriate level, treatment may be stopped.However, if symptoms recur, the patient may require long-term intermittent treatment.

[0072] In addition, it is envisioned that the compound of formula I can be used alone, in combination with one or more other active agents, or in combination with additional active ingredients in the treatment of the conditions discussed herein.Non-limiting examples of active agents that can be combined with the compound of formula I include NSAIDs, Cox-2 inhibitors, anti-EGFR agents (e.g., cetuximab and panitumumab), anti-VEGF / VEGFR agents (e.g., bevacizumab), Ziv-aflibercept, regorafenib, ramucirumab, and immune checkpoint inhibitors (e.g., ipilimumab, nivolumab, and pembrolizumab).The additional active ingredients can be co-administered separately with one compound of formula I, or can be included in a single pharmaceutical composition according to the present invention. In an exemplary embodiment, the additional active ingredient is one known or discovered to be effective in treating a condition, disorder, or disease mediated by JAK activity, e.g., another JAK inhibitor or compound active against another target associated with the particular condition, disorder, or disease. Combinations may serve to increase efficacy (e.g., by including in the combination a compound that enhances the potency or effectiveness of the active agent), reduce one or more side effects, or reduce the required dosage of the active agent.

[0073] In some embodiments, the composition of the compound of formula I comprises a pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient generally used in pharmaceutical compositions is a substance, e.g., an inert substance, that is added to a pharmacological composition or otherwise used as a vehicle, carrier or diluent to facilitate the administration of a drug and is compatible with the drug, non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject.Examples of such excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.

[0074] A pharmaceutical composition delivery form containing one or more dosage units of a compound of formula I can be prepared using pharma- ceutically acceptable excipients and compounding techniques known or available to those skilled in the art. The composition can be administered in the methods of the invention by a suitable delivery route, for example, oral, parenteral, rectal, topical, or ocular, or by inhalation.

[0075] The preparations may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories. The compositions may be formulated for any one of several routes of administration, such as intravenous injection, subcutaneous injection, topical administration, or oral administration. Preferably, the compositions may be formulated for oral administration.

[0076] For oral administration, the active agent can be provided in the form of a tablet, capsule, or bead, or as a solution, emulsion, or suspension. To prepare an oral composition, the active agent can be formulated to provide a dosage of, for example, about 1 to 1000 mg / day for a 70 kg human, in single or multiple dosage units as exemplary ranges.

[0077] Oral tablets may contain the active ingredient mixed with compatible pharma- ceutically acceptable excipients, such as diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorings, and preservatives. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary oral liquid excipients include ethanol, glycerol, water, and the like. Exemplary disintegrants are starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid. Binders may include starch and gelatin. Lubricants, if present, may be magnesium stearate, stearic acid, or talc. If necessary, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the digestive tract, or may be coated with an enteric coating. Additional coatings that can be used may include coatings designed to release a compound or active agent as a function of time, pH, or bacterial content.

[0078] Capsules for oral administration include hard and soft gelatin or (hydroxypropyl) methylcellulose capsules. To prepare hard gelatin capsules, the active ingredient may be mixed with a solid, semi-solid or liquid diluent. Soft gelatin capsules can be prepared by mixing the active ingredient with oils such as peanut oil or olive oil, liquid paraffin, a mixture of mono- and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol. Liquids for oral administration may be in the form of suspensions, solutions, emulsions, or syrups, or may be lyophilized or presented as a dry product to be reconstituted with water or other suitable vehicle before use. Such liquid compositions may optionally contain pharma- ceutically acceptable excipients, such as suspending agents (e.g., sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and the like); non-aqueous vehicles, such as oils (e.g., almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol or water; preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid); wetting agents, such as lecithin; and, optionally, flavorings or coloring agents.

[0079] The composition of the compound of formula I may also be administered by parenteral routes. For example, the composition may be formulated as a suppository for rectal administration. For parenteral applications, including intravenous, intramuscular, intraperitoneal, or subcutaneous routes, the compounds of the present disclosure may be provided in a sterile aqueous solution or suspension buffered to an appropriate pH and isotonicity, or in a parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms will be presented in unit dose forms, such as ampoules or disposable injection devices, multi-dose forms, such as vials from which the appropriate dose can be withdrawn, or in solid forms or pre-concentrates that can be used to prepare injectable formulations. Specific infusion doses may range from about 1-1000 μg / kg / min of the compound mixed with a pharmaceutical carrier over a period ranging from a few minutes to several days.

[0080] All aspects described herein relating to methods for treating are also applicable for use in therapy.

[0081] All aspects described herein relating to methods for treating a disorder or condition are also applicable for use in treating that disorder or condition.

[0082] All aspects described herein relating to use in the treatment of a disorder or condition are also applicable to the method for treating that disorder or condition.

[0083] All aspects described herein relating to methods for treating a disorder or condition are also applicable for use in the method for treating that disorder or condition.

[0084] All aspects described herein relating to use in a method for treating a disorder or condition are also applicable to the method for treating that disorder or condition.

[0085] To further illustrate the present invention and various aspects, the following specific examples are provided.

[0086] In obtaining the compounds described in the Examples below and the corresponding analytical data, the following experimental and analytical protocols were followed, unless otherwise indicated.

[0087] Unless otherwise noted, reaction mixtures were magnetically stirred at room temperature (rt). When solutions are "dried", they are generally dried over a drying agent such as Na2SO4 or MgSO4. When mixtures, solutions, and extracts are "concentrated", they are typically concentrated under reduced pressure on a rotary evaporator.

[0088] Thin-layer chromatography (TLC) was performed using Merck silica gel 60F. 254The experiment was carried out using plates (2.5 cm x 7.5 cm, 250 μm or 5.0 cm x 10.0 cm, 250 μm silica gel pre-coated plates).

[0089] Normal phase flash column chromatography (FCC) was performed on silica gel (SiO2) eluted with 2M NH3 in MeOH / DCM unless otherwise noted.

[0090] Mass spectra (MS) were obtained on an Agilent series 1100 MSD using electrospray ionization (ESI) in positive mode unless otherwise indicated. Calculated masses correspond to exact masses.

[0091] Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker DRX spectrometer. 1 The format of the H NMR data is chemical shift (ppm) downfield relative to tetramethylsilane (multiplicity, coupling constant J (Hz), integral).

[0092] Chemical names were generated by either ChemDraw (CambridgeSoft, Cambridge, MA) or ACD / Name Version 9 (Advanced Chemistry Development, Toronto, Ontario, Canada). As an example, the notation (1r,4r) denotes a trans configuration around the cyclohexyl ring, generated using the naming feature of Chemdraw Ultra Pro 14.0.

[0093] Whenever a yield is given as a percentage, such yield refers to the mass of the entity for which the yield is given relative to the maximum amount of the same entity obtainable under the specified stoichiometric conditions. Reagent concentrations given as percentages refer to mass ratios unless otherwise specified.

[0094] Abbreviations and acronyms used herein include those set forth below: Defined Abbreviations and Acronyms

[0095] [Table 1]

[0096] [Table 2]

[0097] [Table 3]

[0098] Example 1 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide

[0099] [ka]

[0100] Step A: 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide. To ensure dry starting material, ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (Intermediate 3) was heated under vacuum at 50° C. for 18 hours prior to reaction. In a 1 L flask, ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate (Intermediate 3, 52.585 g, 104.01 mmol) was suspended in DMA (50 mL). 1-Amino-2-methylpropan-2-ol (50 mL) was added and the reaction was heated to 110° C. for 45 min, then to 125° C. for 5 h. The reaction was cooled to room temperature and diluted with EtOAc (800 mL). The organic layer was extracted three times with a water / brine solution (the solution consisted of 1 L water and 50 mL brine). The aqueous layer was back extracted with EtOAc (2×600 mL). The combined organic layers were dried over anhydrous MgSO4, concentrated to dryness, and then dried under vacuum for 3 days to give the title compound as a yellow foam (65.9 g, 98% yield). The product was used in the next step without further purification. MS (ESI): Mass calculated C 28 H 32 N6O4S, 548.22; M / z measured, 549.2 [M+H] + . 1H NMR(400MHz, CDCl3): δ8.76(s,1H),8.26-8.19(m,2H),7.84(d,J=4.1Hz,1H),7.60 -7.53(m,1H),7.50-7.44(m,2H),6.84(d,J=4.2Hz,1H),4.76-4.61(m,1H),3.97(s ,2H),3.45(s,1H),3.27(d,J=5.9Hz,2H),2.41(d,J=6.5Hz,2H),2.38-2.25(m,2H) ,2.23-2.12(m,2H),2.09-1.94(m,4H),1.48(qd,J=13.6,4.0Hz,2H),1.21(s,6H).

[0101] Step B: 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide. 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide (65.90 g, 102.1 mmol) was added to a 1 L flask containing a stir bar. 1,4-Dioxane (300 mL) was added followed by aqueous KOH (3M, 150 mL). The reaction was heated at 80° C. for 2 hours. The reaction was cooled to room temperature and the solvent volume was reduced to approximately 200 mL on a rotary evaporator. The residue was treated with a solution of water / brine (100 mL / 100 mL) and then extracted with 10% MeOH in CHCl (2×1 L). The organic layers were combined, dried over anhydrous MgSO and concentrated to dryness to give a yellow solid. The solid was suspended in CHCl (200 mL), stirred vigorously for 30 min and collected by filtration. The solid was rinsed with CHCl (100 mL), dried by pulling air through the filter and then further dried under vacuum at room temperature for 16 h to give the title compound as a white solid (41.59 g, 89% yield). MS (ESI): Mass calculated C 22 H 28NO2, 408.23; m / z measured, 409.2 [M+H] + . 1 H NMR (600 MHz, DMSO-d6): δ 11.85 (s, 1H), 8.50 (s, 1H), 8.21-8.10 (m, 1H), 7.49-7.43 (m, 1H), 6.74-6.65 (m, 1H), 4.53-4.42 (m, 2H), 4.07 (s, 2H), 3.08 (d, J = 6.0 Hz, 2H), 2.58 (d, J = 6.1 Hz, 2H), 2.41-2.28 (m, 2H), 2.09-1.92 (m, 5H), 1.42-1.31 (m, 2H), 1.09 (s, 6H). The synthesis and characterization of the active compounds of each of the embodiments of the present invention are provided herein in the form of examples. The crystal structures of some embodiments of the present invention allow polymorph screening to be performed to further characterize specific forms of any such compounds. This is illustrated in a non-limiting manner for compounds of formula I by the examples under the heading Polymorph Screening.

[0102] The following compounds were prepared with reference to the above syntheses.

[0103] Intermediate 1 2-((1r,4r)-4-((5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

[0104] [ka]

[0105] Step A: tert-Butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate. A 20 L 4-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen was charged with (1r,4r)-4-[[(tert-butoxy)carbonyl]amino]cyclohexane-1-carboxylic acid (1066 g, 4.38 mol, 1.00 equiv.) and THF (10 L). This was followed by the dropwise addition of BH3-Me2S (10 M, 660 mL) over 1 h at -10°C. The resulting solution was stirred at 15°C for 3 h. This reaction was carried out three times in parallel and the reaction mixtures were combined. The reaction was then quenched by the addition of methanol (2 L). The resulting mixture was concentrated under vacuum. This gave tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (3000 g, 99.6%) as a white solid. MS (ESI): Mass calculated C 12 H 23 NO3, 229.32; m / z measured, 215.2 [M-tBu+MeCN+H] + ; 1 H NMR: (300MHz, CDCl3): δ4.40(s,1H),3.45(d,J=6.3Hz,2H),3.38(s,1H),2.05-2.02(m,2H),1.84-1.81(m,2H),1.44(s,11H),1.17-1.01(m,4H).

[0106] Step B: tert-Butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate. A 20 L 4-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen was charged with tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate (1000 g, 4.36 mol, 1.00 equiv), dichloromethane (10 L), pyridine (1380 g, 17.5 mol, 4.00 equiv). This was followed by dropwise addition of MsCl (1000 g, 8.73 mol, 2.00 equiv) at -15°C. The resulting solution was stirred overnight at 25°C. This reaction was carried out three times in parallel and the reaction mixtures were combined. The reaction was then quenched by adding 2 L of water. The aqueous phase was extracted with ethyl acetate (1 x 9 L). The organic layer was separated and washed with 1M HCl (3×10 L), NaHCO3 (sat. aq.) (2×10 L), water (1×10 L), and brine (1×10 L). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. This gave tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (3300 g, 82%) as a white solid. LC-MS: MS(ESI): Mass calcd. C 13 H 25 NO5S, 307.15; m / z measured value 292.1, [M-tBu+MeCN+H] + ; 1 H NMR:(300MHz,CDCl3):δ4.03(d,J=6.6Hz,2H),3.38(s,1H),3.00(s,3H),2.07-2. 05(m,2H),1.87-1.84(m,2H),1.72-1.69(m,1H),1.44(s,9H),1.19-1.04(m,4H).

[0107] Step C: tert-Butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate. A 10 L 4-neck round bottom flask was charged with tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (1100 g, 3.58 mol, 1.00 equiv), DMSO (5500 mL), and NaCN (406 g, 8.29 mol, 2.30 equiv). The resulting mixture was stirred at 90° C. for 5 h. This reaction was run three times in parallel and the reaction mixtures were combined. The reaction was then quenched by adding 15 L of water / ice. The solid was collected by filtration. The solid was washed with water (3×10 L). This gave tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (2480 g, 97%) as a white solid. MS (ESI): Mass calculated C 13 H 22 N2O2, 238.17; m / z measured value 224 [M-tBu+MeCN+H] + ; 1 H NMR:(300MHz,CDCl3):δ4.39(s,1H),3.38(s,1H),2.26(d,J=6.9Hz,2H),2.08-2. 04(m,2H),1.92-1.88(m,2H),1.67-1.61(m,1H),1.44(s,9H),1.26-1.06(m,4H).

[0108] Step D: 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride. A 10 L round bottom flask was charged with tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate (620 g, 2.60 moles, 1.00 equiv.) and 1,4-dioxane (2 L). Then, a solution of HCl in 1,4-dioxane (5 L, 4 M) was added dropwise with stirring at 10° C. The resulting solution was stirred overnight at 25° C. This reaction was carried out four times and the reaction mixtures were combined. The solid was collected by filtration. The solid was washed with 1,4-dioxane (3×3 L), ethyl acetate (3×3 L), and hexane (3×3 L). This gave 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride (1753 g, 96%) as a white solid. MS(ESI): Mass calculation value C8H 14 N2, 138.12; m / z measured value 139.25, [M+H] + ; 1 H NMR:(300MHz,DMSO-d6:δ8.14(s,3H),2.96-2.84(m,1H),2.46(d,J=6.3Hz,2H),1.98(d,J=1 1.1Hz, 2H), 1.79 (d, J = 12.0Hz, 2H), 1.64-1.49 (m, 1H), 1.42-1.29 (m, 2H), 1.18-1.04 (m, 2H).

[0109] Step E: 2-((1r,4r)-4-((5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile. To a 1000 mL round bottom flask containing 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride (29.10 g, 166.6 mmol) was added DMA (400 mL). The resulting suspension was treated with 4-chloro-5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (51.53 g, 152.6 mmol) followed by DIPEA (63.0 mL, 366 mmol). The reaction mixture was placed under N2 and heated at 80° C. for 4 h. The crude reaction mixture was cooled to room temperature and slowly poured into a 2 L flask containing 1.6 L of water and stirred vigorously. The resulting suspension was stirred for 15 minutes at room temperature, then filtered and dried in a vacuum oven with heating at 70° C. for 16 hours to give the title compound (63.37 g, 95%) as a yellow solid. MS (ESI): Mass calculated C 21 H 21 N5O4S, 439.1; m / z measured, 440.1 [M+H] + . 1 H NMR (500MHz, CDCl3): δ9.10(s,1H),8.99(d,J=7.8Hz,1H),8.23-8.15(m,2H),7.66-7.59(m,2H),7.56-7.49(m,2H),6.67(d,J=4 .2Hz,1H),3.95-3.79(m,1H),2.38(d,J=6.2Hz,2H),2.32-2.21(m,2H),2.08-1.98(m,2H),1.88-1.76(m,1H),1.60-1.32(m,4H).

[0110] Intermediate 2 2-((1r,4r)-4-((5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile

[0111] [ka]

[0112] 2-((1r,4r)-4-((5-nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (Intermediate 1, 58.60 g, 133.3 mmol) was dissolved in THF / MeOH (1:1, 4800 mL). The mixture was passed through a continuous flow hydrogenation reactor (10% Pd / C) such as a Thales Nano H-Cube® at 10 mL / min with 100% hydrogen (atmospheric pressure, 80° C.), after which the solution was concentrated to give the product as a purple solid. The solid was triturated with EtOAc (400 mL) and then triturated again with MeOH (200 mL), before filtering and drying in vacuo to give the title compound (50.2 g, 91.9% yield).

[0113] MS(ESI): Mass calculation value C 21 H 23 N5O2S, 409.2; m / z measured, 410.2 [M+H] + . 1 H NMR(400MHz,CDCl3)δ8.10-8.03(m,2H),7.76(s,1H),7.51-7.43(m,1H),7.43-7.34(m,3H),6.44(d,J=4.2Hz,1H),4.61(d,J=8.5Hz,1 H),3.65-3.51(m,1H),2.74(s,2H),2.26(d,J=6.4Hz,2H),2.19-2.05(m,2H),1.97-1.86(m,2H),1.76-1.59(m,1H),1.33-1.12(m,4H).

[0114] Intermediate 3 Ethyl 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)acetate

[0115] [ka]

[0116] To a 1 L round bottom flask containing a stir bar and 2-((1r,4r)-4-((5-amino-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile (Intermediate 2, 58.31 g, 142.4 mmol), ethyl 3-ethoxy-3-iminopropanoate (60.51 g, 309.3 mmol) was added followed by EtOH (600 mL, dried over 3 Å molecular sieves for 48 h). A reflux condenser was attached to the reaction flask, the reaction was purged with N2 and heated at 90 °C for 9 h. The reaction mixture was cooled to room temperature and allowed to stand for 30 h at which time the product crystallized as brown needles. The solids were crushed with a spatula and the reaction mixture was transferred to a 2 L flask. With vigorous stirring, water (1.4 L) was added slowly via a separatory funnel. After the addition of water was complete, the suspension was stirred for 30 min. The brown needles were isolated by filtration and dried by drawing air through the filter for 1 h. The product was transferred to a 500 mL flask and treated with EtOAc (200 mL). A small amount of seed crystals was added to induce the formation of a white solid precipitate. The suspension was stirred for 30 min at room temperature, filtered, rinsed with EtOAc (25 mL), and dried under vacuum to give the product as a white solid (48.65 g, 68% yield). MS (ESI): Mass calculated C 26 H 27 N5O4S, 505.2; m / z measured, 506.2 [M+H] + . 1 H NMR(400MHz,CDCl3)δ8.85(s,1H),8.28-8.19(m,2H),7.84(d,J=4.0Hz,1H),7 .61-7.53(m,1H),7.52-7.43(m,2H),6.84(d,J=4.1Hz,1H),4.32(s,1H),4.20( q,J=7.1Hz,2H),4.09(s,2H),2.44(d,J=6.2Hz,2H),2.40-2.27(m,2H),2.16( d,J=13.3Hz,2H),2.12-1.96(m,3H),1.54-1.38(m,2H),1.27(t,J=7.1Hz,3H).

[0117] Polymorphism Screening Example Some embodiments of the compound of formula I as a free base exhibit multiple crystal structures with complex solid state behavior, some of which may then exhibit significant characteristics between them due to different amounts of incorporated solvent. Some embodiments of the compound of formula I are pseudopolymorphic forms, which are embodiments of the same compound. These exhibit differences in the composition of the crystal lattice due to different amounts of solvent within the crystal lattice itself. In addition, channel solvates may exist in some crystalline embodiments of the compound of formula I. In these, the solvent is incorporated within channels or voids present in the crystal lattice. For example, various crystal configurations have been found for the compound of formula I, as given in Table 2. Due to these characteristics, non-stoichiometric solvates have often been observed, as shown in Table 2. Furthermore, the presence of such channels or voids in the crystal structure of some embodiments according to the invention may allow the presence of water and / or solvent molecules that are held within the crystal structure with various degrees of binding strength. Thus, a change in specific ambient conditions may quickly result in some embodiments according to the invention losing or gaining some amount of water and / or solvent molecules. In each of the embodiments described in Table 2, "solvation" (third column of Table 2) is a formulatable solvation, the actual measurement of which, as a stoichiometry number (fourth column of Table 2), as measured experimentally, is understood to differ slightly from the formulatable solvation depending on the actual ambient conditions. For example, in one embodiment, if about half of the water molecules may be present as hydrogen bound to the active compound in the crystal lattice, while about the other half may be present in the channels or voids of the crystal lattice, the amount of water molecules so loosely contained in the voids or channels may change due to changes in the ambient conditions, resulting in slight differences between the formulatable solvation, as assigned, for example, according to single crystal diffraction, and the stoichiometry as determined, for example, by thermogravimetry coupled with mass spectrometry.

[0118] [Table 4]

[0119] The compound obtained as described in Example 1 was further crystallized by preparing a slurry in DCM (1:3, e.g. 10 g compound in 30 mL DCM) which was stirred at 40° C. for 4 hours and further stirred at 25° C. for 14 hours, then heptane was slowly added at 25° C. (1:2, e.g. 20 mL heptane in compound / DCM slurry / solution), stirred at 40° C. for 4 hours, cooled to 25° C. and further stirred at 25° C. for 14 hours. Subsequent filtration gave the compound of formula I in the form of an off-white solid, which was identified as a monohydrate (1s embodiment).

[0120] The embodiments 1-10 in Table 2 and FIG. 2 are crystalline. The embodiments 1s and 1a-1h are isostructural. The embodiment 1s crystallizes in the point-symmetric triclinic space group P-1. The term "embodiment 1" refers collectively to the isostructural embodiments 1s and 1a through 1h. Any one of such embodiments 1s and 1a through 1h is optionally referred to as being an isostructural element of embodiment 1, or simply an element of embodiment 1. The embodiments 3b, 3c, 3d, and 3e are isostructural and crystallize in the monoclinic space group C2 / c. The term "embodiment 3" refers collectively to the isostructural embodiments 3b, 3c, 3d, and 3e. Any one of such embodiments 3b, 3c, 3d, and 3e is optionally referred to as being an isostructural element of embodiment 3, or simply an element of embodiment 3. Isomorphous embodiments are those that have different chemical compositions (i.e., different solvent and / or water molecules incorporated into the crystal lattice) but similar crystal structure properties (same symmetry, and similar unit cell parameters and crystal packing). Due to the different compositions (solvent or water incorporated into the crystal structure), the unit cell parameters in isomorphous embodiments may differ slightly. The embodiments referenced in Table 2 were prepared and / or interconverted as shown generally in FIG. 2 and described in more detail in the screening techniques below.

[0121] Screening included crystallization procedures such as solvent equilibration with pure solvent, evaporative crystallization, cooling crystallization with hot filtration, crushing crystallization with antisolvent, and crystallization by thermal cycling. Solids were analyzed by HT-XRPD. Where applicable, the mother liquor was completely evaporated and the remaining solids were also analyzed by HT-XRPD. The predominant solid form identified was starting material embodiment 1s as the monohydrate.

[0122] Solvent equilibration at 25°C and 50°C Long-term slurry experiments were performed by suspending compound embodiment 1s in 20 pure solvents and stirring at room temperature for 2 weeks and at 50° C. for 1 week. At the end of the equilibration period, the residual solid was separated from the mother liquor. The solid was dried under ambient conditions and dried under vacuum (5 mBar) before being analyzed by HT-XRPD. The solid was then exposed to accelerated aging conditions (40° C. / 70% relative humidity) for 2 days and analyzed again by HT-XRPD.

[0123] Most of the crystallization solvents gave starting material as embodiment 1s. Some of the crystallization solvents showed HT-XRPD patterns similar to the pattern of the first embodiment 1s. In most of these diffraction patterns, peak shifts and / or additional peaks were identified. Each of these patterns corresponds to one of the embodiments labeled as 1a through 1h, and based on the similarity of the HT-XRPD diffraction patterns of such embodiments, they are presented as embodiments that are isomorphous structural elements of embodiment 1. After exposure to 40°C and 75% RH for 2 days, all isomorphous structural elements of embodiment 1 were converted to embodiment 1a.

[0124] Upon exposure to 100% RH at 25° C., embodiment 1s transformed into hydrated embodiment 10. Nevertheless, embodiment 10 was not physically stable at ambient conditions. It was found that embodiment 1s crystallized in triclinic space group P-1, whereas embodiment 10 crystallized in monoclinic space group C 2 / c. Embodiment 10 had limited physical stability under ambient conditions and transformed into another embodiment, such as 1s or 1a. This behavior is due to the inhomogeneously strong binding of all hydrated / solvated molecules. In this case, embodiment 10 has a second, less strongly bound water molecule that is lost under ambient conditions. More precisely, the physical stability of embodiment 1s was investigated in a climate chamber by exposing a 20 mg sample of such embodiment to 40° C. and 70% relative humidity for 4 days, and another 20 mg sample of the same embodiment was also exposed to 25° C. and 100% relative humidity for 4 days. After 4 days, various solid samples were analyzed by HR-XRPD to measure the crystal lattice parameters and index the diffractograms. The diffractograms are shown in FIG. 6. From bottom to top, the first diffractogram in FIG. 6 corresponds to embodiment 1s as starting material, the second corresponds to the same form after 4 days of exposure to 40° C. and 70% relative humidity, and is labeled "1s 70RH" in the same figure. This analysis revealed that the initial embodiment 1s had been recovered, albeit with a small amount of a second crystalline form, which was probably another hydrated embodiment with a higher water content. It was not possible to index such a form due to the small amount present. The third diffractogram corresponds to embodiment 1s after 4 days of exposure to 25° C. and 100% relative humidity, and is labeled "10" in the same figure. These conditions resulted in the conversion of embodiment 1s to embodiment 10, with a small amount of contamination of the initial embodiment 1s and with the solvation characterized in Table 2. Upon dehydration, both embodiments 1s and 10 recrystallized to anhydrous forms with melting points of 148°C.

[0125] Solvent equilibration at room temperature gave embodiment 1b, which does not include toluene as a crystallization solvent, and embodiment 1f, which does not include p-xylene as a crystallization solvent.

[0126] Three additional solid embodiments were identified and designated as embodiments 2, 3, and 7. The embodiment 2, whose TGA and DSC are shown in Figures 21A and 21B, respectively, was identified from a solvent equilibration experiment carried out in 1,4-dioxane at room temperature, while the embodiment 7 was found as a mixture with embodiment 1s in a single solvent equilibration experiment at 50°C from heptane. Multiple non-identical but similar diffractograms were identified, grouped as embodiments 3b, 3c, 3d, and 3e, which are isomorphous structural elements of embodiment 3. The isomorphous structural elements of embodiment 3 were found to be mixed with elements of embodiment 1. Mixtures containing elements of embodiment 3 were sometimes converted to embodiment 1a or to mixtures of embodiments 1a and 3e. The embodiment 7 appeared to be physically stable, while the embodiment 2 was converted to embodiment 3e after exposure to AAC for 2 days.

[0127] Evaporative crystallization The mother liquor stored from the solvent equilibration experiment, carried out at room temperature, was used for the slow evaporative crystallization experiment. The mother liquor was filtered to remove any particulate matter and allowed to slowly evaporate under ambient conditions. The resulting solid was analyzed by HT-XRPD and again after exposure to AAC for 2 days.

[0128] Due to insufficient solubility of the compound of formula I in some of the solvents, no solids were recovered when using such solvents. In experiments where solids precipitated, amorphous residues or isomorphous structural elements of embodiment 1 or 3 were recovered. During stability testing, different elements of embodiment 1 transformed into embodiment 1a, while samples of embodiment 3 appeared to be physically stable. In some cases, the amorphous solids remained amorphous after stability testing, became soluble, or showed some signs of crystallinity.

[0129] Cooling crystallization The mother liquor from the solvent equilibration experiment carried out at 50° C. was filtered at 50° C. to remove any particulate matter. The suspension was filtered at 50° C. using a 0.2 μm PTFE filter and the solution was placed at 5° C. and aged for 72 hours. When solids precipitated during aging, these solids were separated from the liquid, dried under ambient conditions and under vacuum, and analyzed by HT-XRPD. The remaining mother liquor was allowed to slowly evaporate and the remaining solids were analyzed by HT-XRPD. Samples where no precipitation occurred were placed under vacuum and the dried solids were analyzed by HT-XRPD. All solids were then exposed to AAC (40° C. / 70% RH for 2 days) and analyzed again by HT-XRPD.

[0130] No solids precipitated upon cooling of some of the solutions, and in these cases the solutions were allowed to evaporate under ambient conditions. Due to the low solubility of the compound of formula I in some solvents, no solids were obtained from some of the solutions.

[0131] Precipitation occurred from four solvents (2-propanol, 2-butanone, acetonitrile, and methanol). On a mL scale, after evaporation of a single cooling crystallization experiment at 800 μL of acetonitrile, 25 mg / mL concentration, embodiment 6 was identified. After 2 days of AAC, embodiment 6 appeared to be in a stable solid state and was a non-solvated embodiment.

[0132] Cooling / evaporation crystallization on the μL scale Cooling / evaporation crystallization experiments were carried out in 96-well plates at the μL scale using 12 pure solvents and 12 solvent mixtures and applying four temperature profiles. In each well, 4 mg of embodiment 1s was dosed as solids. Then, crystallization solvent (80 μL) and solvent mixture were added to reach a concentration of 50 mg / mL, and the plate with each well individually sealed was then passed through one of four temperature profiles. Upon completion of the temperature profile, the solvent was evaporated at low ambient pressure (24 hours), and the remaining solids were analyzed by HT-XRPD before and after exposure to AAC (40° C. / 70% RH) for 2 days.

[0133] Elements of embodiment 1 and 3 were found from most of the solvent systems and temperature profiles. However, certain trends in solid state and temperature profile were observed. Embodiment 1b was mainly identified from a short temperature profile (3 hours aging). Nevertheless, the same solvent system with a long aging time led to the identification of element of embodiment 1f, element of embodiment 3, or a mixture of elements of embodiment 1 and 3. Embodiment 3c was obtained from a temperature profile of 1,4-dioxane as crystallization solvent and 50°C as initial temperature, held for 60 minutes, cooled at a rate of 1°C / h (final temperature 20°C) and held for 48 hours. Embodiment 3d was obtained with tetrahydrofuran as crystallization solvent and the same temperature profile as embodiment 3c.

[0134] Embodiment 4 was identified in experiments carried out with methanol / water (50 / 50, v / v), THF, and DCM / IPA (50 / 50, v / v) when applying short aging conditions. Embodiment 4 was obtained by treating embodiment 1s with a mixture of water and methanol (50 / 50) and a temperature profile of 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 20° C. / h (final temperature 5° C.) and held for 3 hours (together with embodiment 1b). Embodiment 4 was also obtained by treating embodiment 1s with a mixture of water and methanol (50 / 50) and a temperature profile of 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 20° C. / h (final temperature 20° C.) and held for 3 hours (together with embodiment 1b). Embodiment 4 did not appear to be physically stable under ambient conditions. A cooling crystallization experiment yielded embodiment 1c using a temperature profile of ethyl acetate / 1,4-dioxane (50 / 50, v / v) as the crystallization solvent and 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 1° C. / h (final temperature 5° C.), and held for 48 hours. A cooling experiment yielded embodiment 1d using a temperature profile of acetonitrile / chloroform (50 / 50, v / v) as the crystallization solvent and 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 1° C. / h (final temperature 5° C.), and held for 48 hours. A cooling experiment yielded embodiment 1e using a temperature profile of ethyl acetate / 1,4-dioxane (50 / 50, v / v) as the crystallization solvent and 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 1° C. / h (final temperature 20° C.), and held for 48 hours.

[0135] Embodiment 5 was identified in an experiment conducted with chloroform as the crystallization solvent and a temperature profile of 50° C. as the initial temperature, held for 60 minutes, cooled at a rate of 1° C. / h (final temperature 20° C.), and held for 48 hours.

[0136] Similar conversions were seen during stability testing previously observed with other crystallization methods: in most cases, all solid forms converted to embodiment 1a or mixtures containing embodiment 1a.

[0137] Evaporative crystallization from solid mixtures. In evaporative crystallization using a solvent / anti-solvent mixture, a clear solution of a compound is prepared from which the solvent (high vapor pressure) first evaporates, causing the compound to precipitate in some crystalline form. These crystals then act as seeds as the anti-solvent (low vapor pressure) evaporates.

[0138] The compound of Formula I was not completely soluble in each of the solvent systems, and for this reason all experiments included filtration prior to evaporation.

[0139] The results of HT-XRPD analysis showed that upon evaporation of the solvent mixture, the compound of formula I crystallized mainly as embodiment 1s. This was observed in the following solvent / anti-solvent systems: tetrahydrofuran / water, acetonitrile / water, chloroform / ethanol, methanol / ethyl acetate, 2-butanone / isopropanol, and heptane / acetone. From two systems (acetone / cumene and 1,4-dioxane / ethyl formate), isomorphous embodiments 3b and 3e were identified, which transformed after AAC into different mixtures of embodiments 1a and 3d, and 1s and 3e, respectively.

[0140] Antisolvent crystallization A saturated solution of the compound of formula I was prepared in neat solvent. Anti-solvent addition was performed by forward and reverse addition. In forward addition, the anti-solvent was added to the compound solution in three aliquots. Reverse addition was performed by adding a volume of the compound solution to a large excess of anti-solvent (20 mL).

[0141] After precipitation, the solids were separated from the liquid, dried under ambient conditions and dried under vacuum (5 mbar) before being analyzed by HT-XRPD. Experiments in which no precipitation occurred upon anti-solvent addition were stored at 5° C. for 48 hours to induce precipitation. The precipitated solids were then separated and analyzed by HT-XRPD. If no solids were obtained, the solids were evaporated under mild conditions and the residual solids were analyzed by HT-XRPD. All solids were exposed to AAC (2 days at 40° C. / 70% RH) and analyzed again by HT-XRPD.

[0142] Sequential antisolvent crystallization showed precipitation in all cases. All solids could be classified as isomorphous structural elements of embodiment 1 (1s, 1b, 1j, 1f) or isomorphous structural elements of embodiment 3 (3b, 3d, 3f). After exposure to AAC, all solid samples converted to embodiment 1a, except for one that converted to a mixture of embodiments 1a and 3e.

[0143] Inverse anti-solvent crystallization experiments carried out with DMSO as solvent yielded different solid forms depending on the anti-solvent used. Using dichloromethane or p-xylene, the isomorphous structural elements (1s and 1b) of embodiment 1 were identified, whereas using MTBE yielded an amorphous residue. Evaporation of two solutions with heptane and water as anti-solvents that did not precipitate upon addition of the anti-solvent yielded an oil. After AAC, the transformation of embodiment 1a was observed, with the amorphous residue becoming crystalline.

[0144] High-temperature filtration experiment Cooling crystallization experiments with hot filtration were performed from supersaturated solutions of the compound of formula I prepared in different solvent mixtures at 50° C. The hot filtered solutions were passed through a 48 hour cooling profile. Vials with precipitated solids after the temperature profile were centrifuged to separate the solids from the liquid and analyzed by HT-XRPD (after drying under vacuum). If no solids precipitated, the solution was evaporated under vacuum and the solids were analyzed by HT-XRPD. All solids were exposed to AAC (2 days at 40° C. / 70% RH) and analyzed again by HT-XRPD. In half of the hot filtration experiments, no precipitation occurred and not enough solids were recovered upon evaporation of the solvent due to insufficient solubility of the compound of formula I in these solvent systems. In three experiments, the amorphous residue was converted and crystallized after AAC to a mixture of elements of embodiment 1 (1s or 1a) and 3 (3e) or became soluble. Embodiment 5 was identified from experiments with acetone / chloroform (50 / 50, v / v). This embodiment appeared to be physically unstable, as conversion to embodiment 1a was observed after AAC.

[0145] Thermal cycle experiment A suspension of about 6 mg of embodiment 1s was prepared in 10 solvents at room temperature. The suspension was cycled from 5° C. to 50° C. Upon completion of the thermal cycle, the solids were separated by centrifugation and dried under ambient conditions and under vacuum (5 mbar) before being analyzed by HT-XRPD. All solids were subsequently exposed to AAC for 2 days and analyzed again by HT-XRPD. Thermal cycling experiments usually facilitate the formation of more stable polymorphic forms. All vials contained solids after the thermal profile, except for the experiment performed with cyclohexanone. The cyclohexanone solution was slowly evaporated under medium vacuum. Elements of embodiment 1, 3, or mixtures thereof were identified primarily in the wet solids. Upon drying of these solids, conversion to embodiment 1s was observed. Embodiments 3b and 3e were obtained by thermocycling 300 μL of cyclohexanone (3b) at a concentration of 51 mg / mL, and 400 μL of isobutanol (3e) at a concentration of 37.3 mg / mL.Embodiment 5 was obtained by thermocycling 800 μL of chloroform at a concentration of 18.6 mg / mL.

[0146] Figures 3, 4, and 5 show overlays of HT-XRPD patterns for the embodiments described in Table 2 and referenced in the screens above.

[0147] The embodiment 1s was converted from the majority of the crystallization experiments. It is a channel hydrate with many water molecules and / or other solvents incorporated, depending on the ambient conditions. Conversion to embodiment 1a was observed. This form contained slightly more water (1.3 molecules of water). After exposure to 40° C. and 75% RH for 2 days, all isomorphous structural elements of embodiment 1 were converted to embodiment 1a. The shift of some diffraction peaks in the XRPD pattern for the elements of embodiment 1 may be due to different solvents or water molecules incorporated in the crystal lattice. FIG. 4 shows an overlay of HT-XRPD patterns of the elements of embodiment 1. The diffractogram 1s corresponds to the compound of formula I as the starting material for the form of embodiment 1s. Diffractogram 1a corresponds to embodiment 1a obtained after exposing some samples of embodiment 1s to AAC. Diffractogram 1b corresponds to embodiment 1b obtained from a solvent equilibration experiment at room temperature in toluene. Diffractogram 1c corresponds to embodiment 1c obtained from cooling crystallization experiments in ethyl acetate / 1,4-dioxane (50 / 50, v / v) on the μL scale. Diffractogram 1c corresponds to embodiment 1d obtained from cooling crystallization experiments in acetonitrile / chloroform (50 / 50, v / v) on the μL scale. Diffractogram 1e corresponds to embodiment 1e obtained from cooling crystallization experiments in ethyl acetate / 1,4-dioxane (50 / 50, v / v) on the μL scale. Diffractogram 1f corresponds to embodiment 1f obtained from solvent equilibration experiments at room temperature with p-xylene. Diffractogram 1g corresponds to embodiment 1g obtained from solvent equilibration experiments at 50° C. with anisole. Diffractogram 1h corresponds to embodiment 1h obtained from cooling crystallization experiments in p-xylene on the μL scale.

[0148] Diffractograms for elements of embodiment 3 are shown in FIG. 5. The shifts observed in the different HT-XRPD patterns can be mostly attributed to different solvent molecules incorporated into the crystal lattice. Embodiment 3 was obtained by heating embodiment 2 to 40° C. and 70% RH for 4 days. Embodiments 3b-3e are solvated forms containing non-stoichiometric amounts of solvent (0.3-0.7 molecules) depending on the solvent incorporated into the crystal structure. Mixtures containing elements of embodiment 3 were unstable upon exposure to AAC, and they transformed in some cases into embodiment 1a, or mixtures of embodiment 1a and 3e. The transformation to embodiment 1a is due to the exchange of solvent molecules with water molecules and recrystallization into hydrated embodiment 1a upon exposure to high relative humidity.

[0149] By heating embodiment 2 to a temperature of about 200° C., followed by cooling to 25° C., and also by DSC 25-200-25-300° C. cycles, embodiment 9 was obtained.

[0150] Embodiment 9 was also obtained by further procedures. One such procedure was a two-step procedure: embodiment 1s (1.5 g) was treated with 1,4-dioxane (10 volumes) at RT. Seeds of embodiment 2 (5 mg) were added and the sample was stirred at RT for 24 hours. The resulting suspension was filtered and the sample was air-dried for 1.5 hours. By XRPD, the sample was determined to be embodiment 2. In the second step of this two-step procedure, embodiment 2 was heated to 210° C. at 10° C. / min and held at 210° C. for 30 minutes. The sample was then allowed to cool to RT. By XRPD analysis, the resulting solid was determined to be embodiment 9. Another such procedure was also a two-step procedure to obtain embodiment 9. In this procedure, embodiment 1s (1.5 g) was treated with 1,4-dioxane (10 volumes). Seeds of embodiment 2 (5 mg) were added and the sample was stirred at RT for 24 hours. The resulting suspension was filtered and the sample was air-dried for 1.5 hours. By XRPD, the sample was determined to be embodiment 2. In the second step of the procedure, embodiment 2 was heated to 150° C. at 10° C. / min, followed by further heating to 170° C. at 2° C. / min. The sample was then allowed to cool to RT. By XRPD analysis, the resulting solid was determined to be embodiment 9. The TGA and DSC of embodiment 9 are shown in Figures 22A and 22B, respectively.

[0151] Embodiment 1s was obtained by slurrying embodiment 9 in the following solvents at 50° C. for 6 days: 2-butanone, acetone / water (90 / 10, v / v) and acetonitrile / water (90 / 10, v / v). Embodiment 1s was also obtained when the same experiment was carried out at room temperature.

[0152] By heating embodiment 5 to a temperature of about 175° C., embodiment 8 was obtained. Embodiment 8 was also obtained by further procedures. One such procedure was a two-step procedure: embodiment 1s (1.5 g) was treated with 1,4-dioxane (10 volumes) and stirred at RT for 72 hours. The resulting suspension was filtered and the resulting solid was dried in a vacuum oven at RT for 16 hours. The solid obtained from this first step was determined to be embodiment 3c by XRPD. In the second step, embodiment 3c (100 mg) was heated to 150° C. at 10° C. / min, followed by heating to 180° C. at a slower rate of 2° C. / min. The sample was then allowed to cool back to RT. The resulting solid was determined to be embodiment 8 by XRPD. Another such procedure was also a two-step procedure to obtain embodiment 8. In this procedure, embodiment 19 (300 mg) was treated with 1,4-dioxane (3 volumes) and shaken at 60° C. for 24 hours. The resulting suspension was filtered and the solid obtained from this first step was determined by XRPD to be embodiment 3c. In the second step, embodiment 3c (300 mg) was heated to 180° C. at 10° C. / min. The sample was then allowed to cool back to RT. The solid obtained was determined by XRPD to be embodiment 8. The TGA and DSC of embodiment 8 are shown in Figures 20A and 20B, respectively.

[0153] In addition to the preparation of embodiment 6 above, this embodiment was obtained by heating embodiment 11 (80-100 mg) (the preparation of which is described below) by thermogravimetric analysis from ambient temperature to 185°C at 10°C / min, which was held isothermally for 3 minutes. The sample was then allowed to cool to RT. Embodiment 6 was also obtained from embodiment 11 by subjecting it to a slurry experiment. The slurry experiment was carried out as follows: solvent was added to embodiment 11 (50 mg) and the mixture was stirred at the specified temperature for 0.5 hours. Form 9 seed crystals (5 mg) were added and the mixture was stirred at the specified temperature overnight. The solid was isolated by centrifugation and analyzed by XRPD using isopropyl acetate (0.5 mL) at both 30°C and 50°C. The formation of embodiment 6 was confirmed by XRPD. The TGA and DSC of embodiment 6 are shown in Figure 19A and Figure 19B, respectively.

[0154] Further embodiments of the compounds of formula I were obtained as follows.

[0155] Example 5 was converted to Example 9 by subjecting it to a slurry experiment. The slurry experiment was carried out as follows, using various solvents at the specified temperatures: the solvent was added to Example 5 (50 mg), and the mixture was stirred at the specified temperature for 0.5 hours. Seed crystals of Form 9 (5 mg) were added, and the mixture was stirred at the specified temperature overnight. The solid was isolated by centrifugation and analyzed by XRPD. The following solvents were used to carry out the slurry experiment at 50° C.: TBME (0.75 mL) and a 33:67 mixture of isopropyl acetate:heptane (0.5 mL). The following solvents were used to carry out the slurry experiment at 75° C.: isopropyl acetate (0.5 mL) and methyl ethyl ketone (0.5 mL).

[0156] Embodiment 11 was obtained as follows: A suspension of embodiment 1s (45 g) in ethanol (absolute, water content <0.1%, 300 mL) at 50° C. was stirred for 16.5 hours. The suspension was then cooled to 5° C. at 0.25° C. / min. The solution was then stirred at 5° C. for 3 hours. The solid was then filtered off, washed with cold (5° C.) ethanol (absolute, water content <0.1%, 90 mL) and dried under vacuum at 40° C. for 17 hours to obtain about 39 g of embodiment 11. The TGA and DSC of embodiment 11 are shown in FIG. 17A and FIG. 17B, respectively.

[0157] Embodiment 11 was also obtained as follows: Absolute ethanol (170 mL) was added to embodiment 1s (19 g) and heated to near the boiling point of the solvent. A small amount of solid (5%) did not dissolve and was removed by hot filtration. The filtered solid was determined to be embodiment 1s. Therefore, the solid was added back to the filtrate and the mixture was heated until all the solid was dissolved. Heptane (535 mL) was added dropwise to the hot solution via a separatory funnel. The hot solution was vigorously stirred during the dropwise addition of heptane. After the addition of heptane was complete, the flask containing the hot solution / heptane mixture was immersed in an ice-water bath and vigorously stirred for 1 hour. The solid was then collected by filtration and the white solid filter cake was dried by pulling air for 15 minutes. It was further dried by heating at 70° C. under high vacuum for 16 hours, and then heated at 80° C. for 18 hours to obtain 16.3 g of embodiment 11. The diffractogram of embodiment 11 is shown in FIG. 7.

[0158] In the hygroscopicity test of embodiment 11, it was found to be only slightly hygroscopic, with a mass change of 0.66% between 0-90% RH in the GVS analysis shown in FIG. 18. XRPD analysis after GVS analysis showed that the material was physically stable. Variable temperature (VT)-XRPD was performed to evaluate the stability of embodiment 11 upon heating. When exposed to a temperature of about 175° C., the material remained unchanged as shown by XRPD analysis, but above 180° C., the sample was transformed to embodiment 6. The diffractograms of embodiment 11 before and after the VT-XRPD experiment, along with the diffractogram of embodiment 6, are shown in FIG. 24. Embodiment 11 was also subjected to static storage analysis at 40° C. / 75% RH for up to 48 days. Samples were analyzed by XRPD and Karl Fisher (Karl Fisher, KF) after 2 days, 5 days, and 48 days. The embodiment 11 remained unchanged as shown by XRPD analysis, with a total water uptake rate of 1.2% after 48 days. After 48 days of static storage, the material 1 H-NMR showed that the material retained 0.36 molar equivalents of ethanol. 1 H-NMR showed it to contain 0.46 molar equivalents of ethanol.

[0159] Embodiment 11b was obtained from embodiment 1s as follows: 10 mL of dry methanol was added to 3.3 g of embodiment 1s. The mixture was subjected to the following temperature cycling: the mixture was heated at 40° C. for 1 hour, followed by increasing the temperature to 60° C. over 2 hours. The mixture was then stirred at 60° C. for 1 hour. The temperature was then reduced to 40° C. over 2 hours. This temperature cycling regime was repeated for a total of about 20 hours. At this point, the mixture was cooled to 5° C. over 2 hours. The solid was isolated at 5° C. by vacuum filtration and then dried under vacuum at ambient temperature for about 66 hours. Alternatively, embodiment 11b was obtained from embodiment 1s using the following procedure: 1 mL of dry methanol was added to 330 mg of embodiment 1s. The mixture was subjected to the following temperature cycling: the mixture was heated at 40° C. for 1 hour, followed by increasing the temperature to 60° C. over 2 hours. The mixture was then stirred at 60° C. for 1 hour. The temperature was then reduced to 40° C. over 2 hours. This temperature cycling regime was repeated for a total of about 18 hours. At this point, the solids were isolated by centrifugation and then dried under vacuum at ambient temperature for about 33 hours. Molecular sieves (3 Å, 100° C., activated under vacuum for at least 24 hours) were used to dry the methanol for the above experiments. The diffractogram of embodiment 11b is shown in FIG. 7E.

[0160] Embodiment 12 was obtained from embodiment 1s, which was exposed to humidity conditions of less than 10% RH at 25° C. to provide embodiment 12. The diffractogram of embodiment 12 is shown in FIG. 7B.

[0161] Example 13 was obtained as follows: A sample of Example 1s was added to a 250 mL 4-neck flask at 25±5° C. The flask was then charged with MeOH (4.0V, 40 mL) and purified water (10 mL, 1.0V) and stirred until all solids were dissolved. N2 was bubbled through the mixture for 1 h, and then the mixture was cooled to 0-5° C. A cooled solution (0-5° C.) of 0.225 mL volume of NaBH4 / water (0.006 equiv., 2.5% w / w) was prepared (0-5° C.) using purified water (40 mL) charged to a 100 mL 4-neck flask at 0° C., followed by the addition of NaBH4 (1.0 g). The mixture was stirred at 0° C. until all NaBH4 was dissolved. The NaBH4 solution was added to a 250 mL flask, cooled (0-5° C.), and stirred at 0-5° C. The color of the reaction mixture changed to yellow. Purified water (40 mL, 4.0 V, degassed with N2 before use) was added dropwise over 1 h at 0-5° C. The reaction was stirred under N2 at 0-5° C. for 4 h. Additional purified water (30 mL, 3.0 V, degassed) was added dropwise over 1 h at 0-5° C. and the reaction mixture was stirred under N2 at 0-5° C. for an additional 16 h. The reaction was then filtered and the resulting solid was washed with purified water (20 mL, 2 V, degassed with N2 before use) under N2 in a glove box environment (O2 content is 200 ppm). The solid was dried under vacuum with humidified nitrogen at 35±5° C. to give embodiment 13 as an off-white solid. The diffractogram of embodiment 13 is shown in FIG. 7C.

[0162] Example 14 was prepared as follows: 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide (48.15 kg, prepared as in Example 2, step B), EtOH (technical grade, 481 L), and KOH (6.613 kg) were stirred at 10-20° C. for 9 hours. The reaction was then quenched with acetic acid (6.74 L) while maintaining the temperature at 10-20° C. Acetonitrile (240 L) was added and the solvent was evaporated under reduced pressure to a volume of approximately 240 L. This addition and evaporation of acetonitrile was repeated two more times. The resulting mixture was heated to 60-70°C for 5 hours, then cooled to 10-15°C and stirred for 2 hours. The solids in the mixture were filtered off and washed twice with acetonitrile (48 L). The solids were then added to water (240 L) and the reaction mixture was heated to 45-50°C for 3-5 hours, then cooled to 15-20°C for 4 hours. The remaining solids were filtered off and the filter cake was washed with water (96 L, 2 times). The filter cake was dried at 45°C to obtain embodiment 14 (26.28 kg). The diffractogram of embodiment 14 is shown in Figure 7D.

[0163] Further embodiments of the compounds of formula I were obtained as follows.

[0164] Solubility evaluation Embodiment 1s (15 mg) was treated with increasing volumes of solvent until the material was completely dissolved or until a maximum of 100 mL of solvent had been added. Solvent was added in increments of 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 70 mL, and 100 mL. After each addition of solvent, the system was held at 50° C. for 5 minutes with gentle stirring and visually evaluated for the presence of solids. This process was continued until a total of 100 mL of solvent had been added. If no solids remained, no additional solvent was added. After the evaluation was completed, the solution was held at 50° C. for 1 hour and then cooled from 50° C. to 5° C. at 0.1° C. / min with stirring. If solids were present, the mixture was filtered using a 96-well plate under vacuum and analyzed by XRPD. If a clear solution was obtained, the solution was left to evaporate at RT. The following solvents (total amount added is given in parentheses immediately after the solvent) at temperatures of 5° C. and 50° C. were used according to this procedure (degree of dissolution is given in parentheses after each temperature) to obtain the described embodiments: with water (100 mL) at 5° C. (suspension) and 50° C. (suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with methanol (10 mL) at 5° C. (suspension) and 50° C. (solution), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with ethanol (30 mL) at 5° C. (suspension) and 50° C. (solution), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with 2-propanol (30 mL) at 5° C. (suspension) and 50° C. (solution), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5 1-propanol (30 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 1s, whose diffractogram is shown in FIG. 5; acetone (100 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 1s, whose diffractogram is shown in FIG. 5; ethyl acetate (100 mL) at 5° C. (suspension) and 50° C. (turbid) gives embodiment 1s, whose diffractogram is shown in FIG. 5; acetonitrile (100 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 6, whose diffractogram is shown in FIG. 3; toluene (100 mL) at 5° C. (partially dissolved) and 50° C. (turbid) gives embodiment 1s, whose diffractogram is shown in FIG. 5;Isopropyl acetate (100 mL) at 5° C. (suspension) and 50° C. (turbid) gave embodiment 1s, the diffractogram of which is shown in FIG. 5; methyl t-butyl ether (100 mL) at 5° C. (suspension) and 50° C. (suspension) gave embodiment 1s, the diffractogram of which is shown in FIG. 5; 2-butanone (100 mL) at 5° C. (suspension) and 50° C. (solution) gave embodiment 1s, the diffractogram of which is shown in FIG. 5; THF (70 mL) at 5° C. (partially dissolved) and 50° C. (solution) gave embodiment 1s, the diffractogram of which is shown in FIG. s is obtained; DMSO (5 mL) at 5° C. (solution, sample was frozen and left to evaporate at RT) and at 50° C. (solution) gives embodiment 1s whose diffractogram is shown in FIG. 5; N-methylpyrrolidinone (5 mL) at 5° C. (solution, left to evaporate at RT) and at 50° C. (solution) gives embodiment 1s whose diffractogram is shown in FIG. 5; diethyl ether (100 mL) at 5° C. (suspension) and at 50° C. (suspension) gives embodiment 1s whose diffractogram is shown in FIG. 5; methyl ether at 5° C. (suspension) and at 50° C. (suspension) gives embodiment 1s whose diffractogram is shown in FIG. Isobutyl ketone (100 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; DCM (100 mL) at 5° C. (suspension) and 50° C. (suspension) gave embodiment 1s, whose diffractogram is shown in FIG. 5; heptane (100 mL) at 5° C. (suspension) and 50° C. (suspension) gave embodiment 18, whose diffractogram is shown in FIG. 8; 1,4-dioxane (100 mL) at 5° C. (partially dissolved, sample frozen and left to evaporate at RT) and 50° C. (suspension) gave the dry form, whose diffractogram is shown in FIG. 5. in nitromethane (100 mL) at 5° C. (suspension) and 50° C. (suspension), a less crystalline embodiment (diffractogram not shown) is obtained; in 1-methoxy-2-propanol (20 mL) at 5° C. (solution) and 50° C. (solution), a dry form of embodiment 20 is obtained, whose diffractogram is shown in FIG. 11; in 2-methyl-THF (100 mL) at 5° C. (suspension) and 50° C. (suspension), embodiment 18 is obtained, whose diffractogram is shown in FIG. 8 and whose TGA and DSC are shown in FIGS. 10A and 10B, respectively;Tetralin (100 mL) at 5° C. (suspension) and 50° C. (turbidity) gives the mixture of embodiment 4 and embodiment 1b, whose diffractogram is shown in FIG. 3; 3-methyl-1-butanol (100 mL) at 5° C. (suspension) and 50° C. (solution) gives the mixture of embodiment 17, whose diffractogram is shown in FIG. 8 and whose TGA and DSC are shown in FIG. 12A and FIG. 12B, respectively; anisole (100 mL) at 5° C. (suspension) and 50° C. (turbidity) gives the mixture of embodiment 4 and embodiment 1b, whose diffractogram is shown in FIG. 3; t-butanol / water (1:1, 10 mL) at 5° C. (solution) and 50° C. (solution) gives the mixture of embodiment 19, in the dry form, whose modulated DSC is shown in FIG. 9; 1,2-dimethoxyethane (100 mL) at 5° C. (suspension) and 50° C. (turbidity) gives the mixture of embodiment 4 and embodiment 1b, whose diffractogram is shown in FIG. 3; Cumene (100 mL) at 5° C. (turbid) gives the mixture of embodiment 4 and embodiment 1b, whose diffractogram is shown in FIG. 3; diisopropyl ether (100 mL) at 5° C. (suspension) and 50° C. (suspension) gives embodiment 18, whose diffractogram is shown in FIG. 8; morpholine (5 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 21, whose dry form, whose diffractogram is shown in FIG. 11; ethanol:water (95:5, 10 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 21, whose dry form, whose diffractogram is shown in FIG. 11; ethanol:water (9:1, 5 mL) at 5° C. (solution) and 50° C. (solution) gives embodiment 1s, whose dry form, whose diffractogram is shown in FIG. 5; acetonitrile:water (95:5, 30 mL) at 5° C. (suspension) and 50° C. (solution) gives embodiment 21, whose dry form, whose diffractogram is shown in FIG.

[0165] Incubation at 5°C Several experiments of incubation at 5° C. were carried out by treating 30 mg of embodiment 1s with each solvent and the mixture was slurried at 5° C. for 48 hours. Aliquots were taken and immediately analyzed by XRPD. Each aliquot was dried for 16 hours and reanalyzed by XRPD. The air-dried samples were then placed in a vacuum oven (RT) for 24 hours before being further analyzed by XRPD. The following solvents (total amount of solvent added in parentheses immediately following the solvent followed by the degree of dissolution) were used according to this procedure to obtain the described embodiments: water (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; methanol (5 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; ethanol (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; 2-propanol (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; 1-propanol (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; acetone (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; ethyl acetate (30 mL, suspension) gave embodiment 1s whose diffractogram is shown in FIG. 5; acetonitrile (30 mL, suspension) gave crystalline with toluene (30 mL, suspension) embodiment 15, the diffractogram of which is shown in FIG. 8; with isopropyl acetate (30 mL, suspension) embodiment 17, the diffractogram of which is shown in FIG. 8; with methyl t-butyl ether (30 mL, suspension) embodiment 18, the diffractogram of which is shown in FIG. 8; with 2-butanone (30 mL, suspension) embodiment 1s, the diffractogram of which is shown in FIG. 5; with THF (30 mL, suspension) embodiment 17, the diffractogram of which is shown in FIG. 8; with diethyl ether (30 mL, suspension) embodiment 1s, the diffractogram of which is shown in FIG. 5; with methyl isobutyl ketone (30 mL, suspension) embodiment 17, the diffractogram of which is shown in FIG. 8; with DCM (30 mL, suspension) embodiment 1s, the diffractogram of which is shown in FIG. 5;Heptane (30 mL, suspension) gives embodiment 1s, whose diffractogram is shown in FIG. 5; 1,4-dioxane (30 mL, suspension) gives embodiment 3c, whose diffractogram from this experiment is shown in FIG. 5; nitromethane (30 mL, suspension) gives embodiment 1s in a less crystalline form (diffractogram not shown); propylene glycol (30 mL, suspension) gives embodiment 1s in a less crystalline form (diffractogram not shown); 2-methyl-tetrahydrofuran (30 mL, suspension) gives embodiment 18, whose diffractogram is shown in FIG. 8; tetralin (30 mL, suspension) gives embodiment 1s in a less crystalline form (diffractogram not shown); 3-methyl-1-butanol (30 mL, suspension) gives embodiment 18, whose diffractogram is shown in FIG. 8; anisole (30 mL, suspension) gives embodiment 1s, whose diffractogram shows some additional peaks. With 1,2-dimethoxyethane (30 mL, suspension), embodiment 1s was obtained whose diffractogram is similar to embodiment 1s (shown in FIG. 5) except that it shows some additional peaks; with cumene (30 mL, suspension), embodiment 1s was obtained whose diffractogram is similar to embodiment 1s (shown in FIG. 5) except that it shows some additional peaks; with diisopropyl ether (30 mL, suspension), embodiment 17 was obtained whose diffractogram is shown in FIG. 8; with ethanol:water (95:5, 30 mL, suspension), embodiment 1s was obtained whose diffractogram is shown in FIG. 5; with acetonitrile:water (95:5, 30 mL, suspension), embodiment 1s was obtained whose diffractogram is shown in FIG. 5; with acetonitrile:water (95:5, 30 mL, suspension), embodiment 1s was obtained whose diffractogram is shown in FIG.

[0166] heat / cool maturation A suspension of embodiment 1s (30 mg) in each solvent was placed in a platform shaker incubator and subjected to a series of heat-cool cycles from ambient temperature to about 50° C. over a period of 24 hours. This was achieved by switching the heat on and off every 4 hours. Shaking was maintained throughout. An aliquot was taken from each sample and allowed to air dry for 2 hours. The air-dried solids were analyzed by XRPD, then vacuum dried using a vacuum oven (RT, 24 hours) and reanalyzed by XRPD. Each sample from this experiment was vacuum dried, and after vacuum drying, each sample was analyzed by XRPD incubation at elevated temperature. The following solvents (the total amount of solvent added is given in parentheses immediately following the solvent) were used according to this procedure to obtain the described embodiments: water (20 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; methanol (5 mL) gave embodiment 22, whose diffractogram is shown in FIG. 13; ethanol (5 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; 2-propanol (10 mL) gave embodiment 27, whose diffractogram is shown in FIG. 13 for this experiment; 1-propanol (10 mL) gave embodiment 23, whose diffractogram is shown in FIG. 13; acetone (20 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; ethyl acetate (20 mL) gave the less crystalline form of embodiment 1s (diffractogram not shown); acetonitrile ( 13) gives the less crystalline embodiment 24, whose diffractogram is shown in FIG. 13; toluene (20 mL) gives the less crystalline embodiment 1s (diffractogram not shown); isopropyl acetate (20 mL) gives the less crystalline embodiment 18, whose diffractogram is shown in FIG. 13; methyl t-butyl ether (20 mL) gives the less crystalline embodiment 1s (diffractogram not shown); 2-butanone (20 mL) gives the less crystalline embodiment 26, whose diffractogram is shown in FIG. 13; THF (20 mL) gives the less crystalline embodiment 18, whose diffractogram is shown in FIG. 13; diethyl ether (20 mL) gives the less crystalline embodiment 1s (diffractogram not shown); methyl isobutyl ketone (20 mL) gives the less crystalline embodiment 25, whose diffractogram is shown in FIG. 13;DCM (20 mL) gave embodiment 1s with a less crystalline form (diffractogram not shown); heptane (20 mL) gave embodiment 1s with a diffractogram shown in FIG. 5; 1,4-dioxane (20 mL) gave embodiment 27 with a diffractogram shown in FIG. 13 for this experiment; xylene (20 mL) gave embodiment 1s with a less crystalline form (diffractogram not shown); propylene glycol (5 mL) gave embodiment 1s with a less crystalline form (diffractogram not shown); 2-methyl-tetrahydrofuran (20 mL) gave embodiment 18 with a diffractogram shown in FIG. 13; tetralin (20 mL) gave embodiment 1s with a diffractogram shown in FIG. 5; 3-methyl-butanol (20 mL) gave embodiment 1s with a diffractogram shown in FIG. with anisole (20 mL) embodiment 16, the diffractogram of which is shown in FIG. 13, and the TGA and DSC of which are shown in FIG. 23A and FIG. 23B, respectively; with 1,2-dimethoxyethane (20 mL) embodiment 29, the diffractogram of which is shown in FIG. 13; with cumene (20 mL) embodiment 1s, the diffractogram of which is shown in FIG. 5; with diisopropyl ether (20 mL) embodiment 17, the diffractogram of which is shown in FIG. 13; with ethanol:water (95:5, 20 mL) embodiment 30, the diffractogram of which is shown in FIG. 13; with acetonitrile:water (95:5, 20 mL) embodiment 1s, the less crystalline form (diffractogram not shown); with polyethylene glycol (5 mL) embodiment 31, the diffractogram of which is shown in FIG. 13.

[0167] Incubation of embodiment 1s at 60° C. Embodiment 1s (30 mg) was treated with solvent and shaken at 60° C. for 24 hours. An aliquot was removed and air dried for 16 hours. The dried sample was then analyzed by XRPD. The following solvents (the total amount of solvent added is given in parentheses immediately following the solvent) were used according to this procedure to obtain the described embodiments: water (10 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; ethanol (10 mL) gave embodiment 32, whose diffractogram is shown in FIG. 14; 2-propanol (10 mL) gave embodiment 33, whose diffractogram is shown in FIG. 14; 1-propanol (10 mL) gave embodiment 23, whose diffractogram is shown in FIG. 14; acetone (10 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; ethyl acetate (10 mL) gave embodiment 34, whose diffractogram is shown in FIG. 14; acetonitrile (10 mL) gave embodiment 35, whose diffractogram is shown in FIG. 14; toluene (10 mL) gave embodiment 36, whose diffractogram is shown in FIG. 14; isopropyl acetate (10 mL) gave embodiment 25, whose diffractogram for this experiment is shown in FIG. 14; methyl t-butyl acetate (10 mL) gave embodiment 26, whose diffractogram is shown in FIG. ether (10 mL) gives embodiment 35, whose diffractogram is shown in FIG. 14; 2-butanone (10 mL) gives embodiment 38, whose diffractogram is shown in FIG. 14; THF (10 mL) gives embodiment 33, whose diffractogram is shown in FIG. 14 for this experiment; diethyl ether (10 mL) gives embodiment 1s, whose diffractogram is shown in FIG. 5; methyl isobutyl ketone (10 mL) gives embodiment 25, whose diffractogram is shown in FIG. 14 for this experiment; DCM (10 mL) gives embodiment 1s, whose diffractogram is shown in FIG. 5; heptane (10 mL) gives embodiment 1s, whose diffractogram is shown in FIG. 5; 1-4-dioxane (10 mL) gives embodiment 33, whose diffractogram is shown in FIG. 14; nitromethane (10 mL) gives embodiment 1s, whose diffractogram is shown in FIG. 5; propylene glycol (10 mL) gives embodiment 28, whose diffractogram is shown in FIG. 14 for this experiment;2-Methyl-tetrahydrofuran (10 mL) gave embodiment 33, whose diffractogram is shown in FIG. 14; tetralin (10 mL) gave the mixture of embodiment 1s, whose diffractogram is shown in FIG. 5, and embodiment 19, whose modulated DSC profile is shown in FIG. 9 (the diffractogram of the mixture is not shown); 3-methyl-1-butanol (10 mL) gave embodiment 33, whose diffractogram is shown in FIG. 14; anisole (10 mL) gave embodiment 36, whose diffractogram is shown in FIG. 14; 1,2-dimethoxyethane (10 mL) gave embodiment 34, whose diffractogram is shown in FIG. 14; cumene (10 mL) gave embodiment 1s, whose diffractogram is shown in FIG. 5; diisopropyl ether (10 mL) gave embodiment 17, whose diffractogram is shown in FIG. 8; water (95:5, 10 mL) gave embodiment 28, whose diffractogram is shown in FIG. 14, and polyethylene glycol (5 mL) gave embodiment 39, whose diffractogram for this experiment is shown in FIG. 14. ;

[0168] high temperature maturation Samples of each of the embodiments 19 (25 mg) were treated with the amount of solvent specified below, in turn, to give samples, each of which was stirred for 24 hours at 60° C. The solids from each sample were isolated, air-dried for 16 hours, and analyzed by XRPD. The following solvents (the total amount of solvent added in parentheses immediately after the solvent, followed by the degree of dissolution) were used according to this procedure to obtain the described embodiments: with water (125 μL, suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with methanol (125 μL, suspension), embodiment 1s was obtained, whose diffractogram is similar to that of embodiment 1s shown in FIG. 5, except that it shows some additional peaks; with ethanol (125 μL, suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with 2-propanol (75 μL, suspension), embodiment 37 was obtained, whose diffractogram is shown in FIG. 15; with 1-propanol (75 μL, suspension), embodiment 40 was obtained, whose diffractogram is shown in FIG. 15; with acetone (75 μL, suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with ethyl acetate (75 μL, suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with acetonitrile (75 μL, suspension), toluene (75 μL, suspension) gives embodiment 1s, the diffractogram of which is shown in FIG. 5; isopropyl acetate (75 μL, suspension) gives embodiment 37, the diffractogram of which is shown in FIG. 15; methyl t-butyl ether (75 μL, suspension) gives embodiment 33, the diffractogram of which is shown in FIG. 14; 2-butanone (75 μL, suspension) gives embodiment 1s, the diffractogram of which is shown in FIG. 5; THF (75 μL, diethyl ether (150 μL, suspension) gives embodiment 37, whose diffractogram is shown in FIG. 15; diethyl ether (150 μL, suspension) gives embodiment 1s, whose diffractogram is shown in FIG. 5; methyl isobutyl ketone (150 μL, suspension) gives embodiment 33, whose diffractogram is shown in FIG. 14; DCM (75 μL, suspension) gives embodiment 1s, whose diffractogram is shown in FIG. 5; heptane (150 μL, suspension) gives embodiment 41, whose diffractogram is shown in FIG. 15; 1.With 4-butanone (75 μL, suspension), embodiment 3c is obtained, the diffractogram of which is shown in FIG. 5; with nitromethane (75 μL, suspension), embodiment 42 is obtained, the diffractogram of which is shown in FIG. 15; with propylene glycol (75 μL, suspension), embodiment 43 is obtained, the diffractogram of which is shown in FIG. 15; with 2-methyl-tetrahydrofuran (150 μL, suspension), embodiment 33 is obtained, the diffractogram of which is shown in FIG. 14; with tetralin (150 μL, suspension), embodiment 33 is obtained, the diffractogram of which is shown in FIG. 14; with 3-methyl-1-butanol (75 μL, suspension), embodiment 33 is obtained, the diffractogram of which is shown in FIG. 14; with anisole (150 μL, suspension), embodiment 33 is obtained, the diffractogram of which is shown in FIG. With 1,2-dimethoxyethane (75 μL, suspension), embodiment 1s was obtained, whose diffractogram is shown in FIG. 5; with cumene (150 μL, suspension), embodiment 44 was obtained, whose diffractogram is shown in FIG. 15; with diisopropyl ether (150 μL, suspension), embodiment 33 was obtained, whose diffractogram is shown in FIG. 14; with ethanol:water (95:5, 75 μL, suspension), embodiment 45 was obtained, whose diffractogram is shown in FIG. 15; with acetonitrile:water (95:5, 75 μL, suspension), embodiment 1s was obtained, whose diffractogram shows cell growth compared to the diffractogram shown in FIG. 5; with polyethylene glycol (75 μL, suspension), embodiment 46 was obtained, whose diffractogram is shown in FIG. 15.

[0169] Thermal cycling Samples of embodiment 19 (25 mg) were each treated with the amount of solvent specified below to obtain multiple samples in turn, each of which was matured by thermal cycling (40-60° C., 4 hour cycles) for 24 hours. The solids were isolated, air-dried for 16 hours, and analyzed by XRPD. The following solvents (total amount of solvent added in parentheses immediately following the solvent, followed by the appearance observed at 24 hours) were used according to this procedure to obtain the described embodiments: water (125 μL, greenish solid) gave embodiment 1s, whose diffractogram is shown in FIG. 5; methanol (75 μL, clear solid) gave embodiment 11, whose diffractogram showed peaks shifted at higher angles compared to the diffractogram in FIG. 7; ethanol (100 μL, greenish solid) gave embodiment 1s, whose diffractogram is shown in FIG. 5; 2-propanol (75 μL, yellowish solid) gave embodiment 33, whose diffractogram is shown in FIG. 14; 1-propanol (75 μL, white suspension) gave embodiment 33, whose diffractogram is shown in FIG. 14; acetone (75 μL, greenish solid) gave embodiment 47, whose diffractogram is shown in FIG. 16; ethyl acetate (75 μL , white suspension) gives embodiment 33, the diffractogram of which is shown in FIG. 14; acetonitrile (75 μL, white suspension) gives embodiment 6, the less crystalline embodiment, the diffractogram of which is shown in FIG. 3; toluene (75 μL, clear solid) gives embodiment 36, the diffractogram of which is shown in FIG. 14; isopropyl acetate (75 μL, white solid) gives embodiment 33, the diffractogram of which is shown in FIG. 14; methyl t-butyl ether (75 μL, white suspension) gives embodiment 6, the diffractogram of which is shown in FIG. 3; 2-butanone (75 μL, off-white solid) gives embodiment 33, the diffractogram of which is shown in FIG. 14; THF (75 μL, off-white solid) gives embodiment 48, the diffractogram of which is shown in FIG. 16; diethyl ether (150 μL, off-white solid) gives embodiment 49, the diffractogram of which is shown in FIG. 16;Methyl isobutyl ketone (150 μL, off-white solid) gave embodiment 25, whose diffractogram is very similar to that of embodiment 25 shown in FIG. 13; DCM (125 μL, suspension) gave embodiment 1s, whose diffractogram is shown in FIG. 5; heptane (150 μL, white solid) gave embodiment 19, whose modulated DSC profile is shown in FIG. 9; 1,4-dioxane (75 μL, white solid) gave embodiment 3c, whose diffractogram is shown in FIG. 5; nitromethane (75 μL, propylene glycol (75 μL, cream suspension) yields embodiment 10, the diffractogram of which is very similar to that of embodiment 10 (shown in FIG. 16), except that the diffractogram shows an amorphous halo; 2-methyl-tetrahydrofuran (150 μL, white solid) yields embodiment 48, the diffractogram of which is shown in FIG. 16; and tetralin (150 μL, white solid) yields a less crystalline embodiment (diffractogram not shown); 3-methyl-1-butanol (75 μL, white suspension) gives embodiment 25, whose diffractogram is shown in FIG. 13; anisole (150 μL, white suspension) gives embodiment 51, whose diffractogram is shown in FIG. 16; 1,2-dimethoxyethane (75 μL, white suspension) gives embodiment 52, whose diffractogram is shown in FIG. 16; cumene (150 μL, white solid) gives a less crystalline embodiment (diffractogram not shown); diisopropyl ether (150 μL, white solid). gives embodiment 6, whose diffractogram is shown in FIG. 3; ethanol:water (95:5, 75 μL, clear solids) gives embodiment 11, whose diffractogram is shown in FIG. 7; acetonitrile:water (95:5, 75 μL, clear solids) gives embodiment 53, whose diffractogram is shown in FIG. 16; and polyethylene glycol (75 μL, light pink suspension) gives embodiment 31, whose diffractogram is very similar to that of embodiment 31 (shown in FIG. 13), except that the diffractogram shows an amorphous halo.

[0170] Any one of the embodiments 11, 11b, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53 of the compound of formula I, and any combination thereof, are embodiments of the compound according to the invention. Further other embodiments of the compound according to the invention include the compound of formula I as a non-hygroscopic solvate (e.g., embodiment 11 of the compound of formula I). ​​Further other embodiments of the compound according to the invention include the compound of formula I in amorphous form (e.g., embodiment 19 of the compound of formula I). ​​Any one of the embodiments 11, 16, 17, and 18 of the compound of formula I, and any combination thereof, are embodiments of the compound according to the invention. A further embodiment of the invention comprises a compound according to the invention in the form of a pharma- ceutically acceptable co-crystal.An additional embodiment of the invention comprises a compound according to the invention in the form of a pharma- ceutically acceptable salt.

[0171] An embodiment of the present invention includes a compound of formula I in at least one of Forms 1s, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 2, 3b, 3c, 3d, 3e, 5, 6, 7, 8, 9, and 10. An embodiment of the present invention includes a compound of formula I in the form of a pharma- ceutically acceptable co-crystal.

[0172]

[0023] Embodiments of the present invention include a compound of formula I in at least one of Forms 1s, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 2, 3b, 3c, 3d, 3e, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53.

[0024] Embodiments of the present invention include a compound of formula I in the form of a pharma- ceutically acceptable co-crystal.

[0173] The XRPD of Form 1s of the compound of Formula I is shown in Figure 25. The list of major peaks includes peaks at 2θ values ​​of 6.13±0.2, 9.88±0.2, 10.26±0.2, 13.39±0.2, 14.52±0.2, 16.64±0.2, 18.24±0.2, 19.98±0.2, 20.58±0.2, and 22.01±0.2.

[0174] The compound of formula I was tested in enzyme and cellular assays. The results of the enzyme assays and descriptions thereof are shown in Table 4, entitled "Enzyme Inhibition Assay Results," and in columns 51-54 of U.S. Patent No. 10,294,226, issued May 21, 2019, which is incorporated by reference in its entirety. The compound was also tested in three cellular assays: IL-2 pSTAT5 (JAK1 / JAK3), IFNα pSTAT-4 (JAK1 / TYK2), and GM-CSF pSTAT5 (JAK2 / JAK2), and the results and descriptions of the assays are shown in Table 5, entitled "Cell-Based Assay Data," and in columns 53-55 of U.S. Patent No. 10,294,226, which is incorporated by reference in its entirety.

[0175] The compound of formula I was tested in solubility and permeability assays. The results of the solubility assay are shown in Table 6, entitled "Solubility Assay Data," and the results of the permeability assay are shown in Table 7, entitled "MDCK-MDR1 Permeability Data," and in columns 55-58 of U.S. Patent No. 10,294,226, which is incorporated herein by reference in its entirety.

[0176] The compounds of formula I were tested according to the protocols set forth in columns 21-22 and 58-59 and Table 1a of US Pat. No. 10,294,226, which is incorporated herein by reference in its entirety.

[0177] The compounds of Formula I are described and provided in Table 8, Column 59 of US Pat. No. 10,294,226, which is incorporated herein by reference in its entirety, and have been further characterized by their physicochemical properties.

[0178] The compound of formula I is further characterized by the description of embodiments 11, 11b, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53 as described and provided in Figures 7A, 7B, 7C, 7D, 7E, and 8-23, and paragraphs

[0328] to

[0348] of U.S. Patent Application No. 2020 / 0165250, published May 28, 2020, which is incorporated by reference in its entirety.

[0179] The amounts in mg in the following examples refer to the amount of active agent in the form of the compound of formula I. If what is administered is the active agent in another form, such as a pharma- ceutically acceptable salt, appropriate corrections should be made.

[0180] Example 2: In vivo testing The compound of formula I showed high colon exposure and tissue target binding in mice, and low systemic exposure after oral administration in an IL-12 / IL-18-induced STAT3 phosphorylation assay in colon tissue and in an ex vivo cytokine-stimulated colon explant study. Oral administration of the compound of formula I in mice resulted in minimal systemic exposure and a lack of consistent dose-dependent pharmacodynamic responses in blood, as determined by ex vivo whole blood stimulation with either interferon-alpha (IFNα) or IL-21.

[0181] Compounds of formula I show low levels of systemic exposure after oral administration. To understand the relationship between systemic exposure and target binding in human studies, the inhibition of cytokine-induced pSTAT-3 in human whole blood was measured using such compounds. In human whole blood, compounds of formula I inhibited JAK1, JAK2, JAK3, and Tyk2 homo- and heterodimer downstream of IFNα (JAK1 / Tyk2), thrombopoietin (TPO) (JAK2 / JAK2), and IL-21 (JAK1 / JAK3) signaling in a concentration-dependent manner. The average IC50 values ​​of IFNα, TPO, and IL-21-mediated STAT3 phosphorylation by compounds of formula I were 105.3 nM, 490.4 nM, and 579.2 nM, respectively, as shown in Table 3. In Table 3, the results for compounds of formula I are reported.

[0182] [Table 5] (a) CI, confidence interval; (b) IFNα, interferon alpha; (c) n, number of trials; (d) ND, undecided, not convergent; (e) TPO, thrombopoietin.

[0183] Mouse whole blood IC 50 Values ​​were also generated to understand the relationship between systemic exposure and target binding in mouse models. In vitro activity of the compound of formula I in inhibiting IFNα-(JAK1 / Tyk2), IL-21-(JAK1 / 3), and IL-12-(JAK2 / Tyk2)-induced pSTAT responses in mouse whole blood was measured. The results shown in Table 3 demonstrated that the compound of formula I inhibited IFNα and IL-21 responses with IC50 values ​​of 298.6 nM and 415.1 nM, respectively. A reliable IC50 was obtained for inhibition of IL-12-induced pSTAT-4 in mouse whole blood. 50 could not be reported.

[0184] To understand the potential of local gut-selective JAK inhibitors to inhibit STAT signaling in colonic tissue, two pharmacokinetic / pharmacodynamic (PK / PD) studies were developed. The first involves the combined intraperitoneal administration of IL-12 and IL-18 in mice to induce a systemic inflammatory response that leads to activation of the JAK / STAT pathway in colonic tissue. This can be monitored by robust phosphorylation of STAT3 3 hours after challenge. The second PK / PD model involves ex vivo stimulation of mouse colonic tissue with a cocktail of cytokines (IL-22, IL-6, and IFNα) and measures inhibition of STAT3 phosphorylation after oral administration of a compound of formula I.

[0185] Combined intraperitoneal administration of IL-12 and IL-18 induces colonic inflammation in mice (Chikano S, et al. Gut 47(6), 779-786 (2000) "IL-18 and IL-12 induce intestinal inflammation and fatty liver in mice in an IFN-gamma dependent manner", Nold-Petry C, et al. Front Immunol. 8, 1531 (2017) "Gp96 Antagonist Protects in Murine-Intestinal-Inflammation"). Inflammatory responses in colonic tissue can be monitored by the strong phosphorylation of STAT3 3 hours after challenge with IL-12 / IL-18. In this non-GLP study, the effect of orally administered compound of formula I was evaluated on the STAT3 response in mouse colonic tissue induced by systemic administration of both IL-12 and IL-18.

[0186] C57BL / 6 mice (6-8 weeks old) were randomized by weight and assigned to various treatment groups for each of the two studies. Mice were challenged intraperitoneally with a combination of IL-12 (250 ng) and IL-18 (1 μg). To examine the effect of the compound of formula I on IL-12 / IL-18-induced pSTAT-3 responses, groups of mice were orally administered the compound of formula I 1 hour prior to IL-12 / IL-18 challenge. Three hours after IL-12 / IL-18 challenge, mice were euthanized by CO2 asphyxiation, blood was collected by cardiac puncture for PK analysis, and colon tissue was collected to measure pSTAT-3 responses and drug levels. Mice orally administered 20% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were used as vehicle controls. Two independent studies (S-1 and S-2) were conducted to examine the effect of the compound of formula I on IL-12 / IL-18-induced colonic pSTAT-3 responses in mice. In both studies, a significant increase in colonic pSTAT-3 was observed after IL-12 / IL-18 challenge. In both experiments, the compound of formula I showed a clear trend for dose-related inhibition of pSTAT-3 responses: 3.9% and 33.1% inhibition at 10 mg / kg; 25.8% and 61.2% inhibition at 25 mg / kg; and 49.7% and 66.1% inhibition at 50 mg / kg (Table 4). Statistical analysis showed a lack of significance in inhibition in S-1, while significant inhibition was achieved at 25 mg / kg and 50 mg / kg doses in S-2. Serum levels were measured 30 minutes and 4 hours (terminal) after dosing. As shown in Tables 5 and 6 below, the compound of formula I showed low systemic exposure at 30 minutes at all doses tested (nM, mean ± SEM; 7.4 ± 1.4 and 7.0 ± 0.7 at 10 mg / kg; 21.8 ± 5.5 mg / kg and 70.5 ± 60.4 at 25 mg / kg; 121.7 ± 87.2 mg / kg and 38.8 ± 7.4 at 50 mg / kg dose). The observed serum exposure of the compound of formula I was significantly lower than the mouse whole blood IC50 for IL-21-induced pSTAT-3, IFNα-induced pSTAT-3, and pSTAT-4, as shown in Table 3. Colonic drug levels were measured 4 hours after administration.Higher drug levels were observed in colon samples of mice treated with compound of formula I compared to serum (Tables 5 and 6). Colon exposure of compound of formula 1 increased in a dose-dependent manner. At a dose of 25 mg / kg, colon exposure of compound of formula I was 30814.5±5552.5 ng / g tissue and 18256.5±4118.8 ng / g tissue. Taken together, serum and colon exposure data suggest that the pharmacological response of compound of formula I appears to be driven primarily by tissue exposure of the drug. Compound of formula I demonstrates in vivo target binding via inhibition of IL-12 / IL-18-induced pSTAT-3 in colon with high colon exposure and low systemic exposure. Oral administration of compound of formula I in mice resulted in minimal systemic exposure and lack of consistent dose-dependent pharmacodynamic response in blood, as shown in other studies.

[0187] [Table 6] The percentage of inhibition of colonic pSTAT-3 was determined using log-transformed data after removing outliers.

[0188] [Table 7] Data are presented as mean ± SEM; N = 3–7 / group. BLOQ, below limit of quantification; min, minutes; h, hours.

[0189] [Table 8] Data are presented as mean ± SEM; N = 3–7 / group. BLOQ, below limit of quantification; min, minutes; h, hours.

[0190] In addition to the above IL-12 / IL-18 model, the local efficacy of the compound of formula I was further established in a mouse cytokine mixture (IL-22 / IL-6 / IFNα) colon explant model. The combination of IL-22, IL-6, and IFNα induced a rapid and robust pSTAT-3 response in ex vivo mouse colon explants 1 hour after challenge. This study aimed to investigate the dose response and duration of action of the compound of formula I on IL22 / IL6 / IFNα-induced pSTAT-3 response in an ex vivo mouse colon explant model.

[0191] C57BL / 6 mice (6-8 weeks old) were randomized by body weight and assigned to various treatment groups for each study. Mice were orally administered the indicated doses of a compound of formula I. At the end of the study, blood was collected via cardiac puncture and serum and washed proximal colon were prepared for PK analysis.

[0192] Distal colon explant (approximately 3 mm 2) were either untreated or treated with cytokines (IL-6, IL-22, IFNα at 100 ng / mL each). After 1 hour of stimulation, tissues were snap frozen for homogenization and assayed for pSTAT-3. In two independent experiments, SS-1 and SS-2, the compound of formula I dose-dependently inhibited colonic pSTAT-3. In SS-1, the compound of formula I inhibited basal pSTAT-3 responses by 54.3, 74.4, 78.6, and 89.5% at 0.5, 2.5, 5, and 25 mg / kg, respectively. IL-22 / IL-6 / IFNα-stimulated pSTAT-3 responses were similarly inhibited by 42.7, 74.9, 83.8, and 93.8% at 0.5, 2.5, 5, and 25 mg / kg, respectively, of the compound of formula I. In SS-2, basal pSTAT-3 was inhibited by 36.7, 66.2, 71.3, and 83.8% at 0.5, 2.5, 5, and 25 mg / kg doses of the compound of formula I, respectively. IL-22 / IL-6 / IFNα-stimulated pSTAT-3 was inhibited by 5.5, 62.0, 68.1, and 89.8% at 0.5, 2.5, 5, and 25 mg / kg, respectively, by the compound of formula I. Thus, the compound of formula I demonstrated tight target binding in ex vivo colon tissues via inhibition of IL-22 / IL-6 / IFNα-induced pSTAT-3 responses.

[0193] As shown in Tables 7 and 8, the effect of the compound of formula I was accompanied by low systemic (serum) exposure (≦10.3±6.9nM) and high colonic exposure of the compound of formula I (27488.1±6128.6ng / g and 22929.6±4146.6ng / g in SS-1 and SS-2, respectively, at 25mg / kg). The high colonic exposure and low systemic exposure observed for the compound of formula I imply the role of colonic drug levels as an important driver of the observed pharmacology. The compound of formula I showed target binding in colonic tissue 4 hours after oral administration.

[0194] [Table 9] Data are presented as mean ± SEM; N = 5 / group. BLOQ, below limit of quantification.

[0195] [Table 10] Data are presented as mean ± SEM; N = 5 / group. BLOQ, below limit of quantification.

[0196] A limited tissue (liver, intestine) distribution study was performed in female C57Bl / 6 mice (N=3 / time point) 0.5, 1, 2, 3, 5, 7, and 24 hours after oral administration of the compound of formula I at a dose of 10 mg / kg formulated as a solution in 20% HP-β-CD. Plasma and the following tissues were collected for compound concentration analysis: liver, whole ileum, and whole colon. Prior to homogenization, the ileum and colon were flushed with saline. Feces were collected over a 24-hour period. All tissues (except plasma) were homogenized in sterile water and the concentration of the compound of formula I was determined using LC-MS / MS. The results of this study demonstrated that the highest tissue concentration was found in the ileum (>colon>liver>plasma). (See Table 9). The concentration of the compound of formula I in feces was 125.6 μg 24 hours after administration.

[0197] [Table 11] a Values ​​are means ± SD (N=3 animals). b BLOQ(1ng / mL) c N=2 (SD not calculated) BLOQ = below limit of quantification; N = number of animals; PO = oral administration; SD = standard deviation.

[0198] Preliminary tissue distribution studies were conducted in male Sprague-Dawley rats (N = 3 / time point) at 0.5, 1, 2, 3, 5, 7, and 24 hours after oral administration of the compound of Formula I at a dose of 25 mg / kg. The following tissues were collected for compound concentration analysis: liver, kidney, brain, muscle, epididymal fat, duodenum, jejunum, ileum, colon (both luminal contents and tissue), and plasma. Compound concentrations in various tissues are shown in Tables 10 and 11. The results of this study showed low systemic exposure and high local concentrations (intestinal tract).

[0199]

Table 12

[0200]

Table 13

[0201] Example 3: A Phase 1b trial to evaluate the efficacy and safety of the compound of Formula I, a Janus kinase (JAK) inhibitor, in participants with familial adenomatous polyposis. The compounds of formula I are potent, oral, small molecule pan-Janus kinase (JAK) inhibitors with favorable gut-selective properties based on permeability and solubility. Inhibition of this group of cytoplasmic tyrosine kinases prevents phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. Phosphorylated STATs (pSTATs) translocate to the nucleus and induce gene transcription of several chemokines, cytokines, and proteases involved in the pathogenesis of rheumatoid arthritis, inflammatory bowel disease, familial adenomatous polyposis (FAP), and other inflammatory diseases.

[0202] Objectives and Evaluation Items The primary objective of this study is to determine the effect of the compound of Formula I on colonic polyp burden (total polyp burden) in participants with FAP. Key secondary objectives are to evaluate safety, other measures of efficacy, local and systemic pharmacokinetics (PK), and pharmacodynamics (PD) in polyps.

[0203] Overall Plan This is a Phase 1b multicenter study to evaluate the efficacy and safety of the compound of formula I in adult participants with FAP. The study is designed to determine whether the compound of formula I has clinical activity in the colon and duodenum, as assessed by reduction in the number of polyps and reduction in JAK signaling in polyps over 24 weeks. Participants can be either pre- or post-colectomy, but all participants must have colon or rectal polyps. All participants are required to have a genetic diagnosis of classic FAP (i.e., adenomatous polyposis coli (APC)) germline mutation or obligate carrier with colonic disease. Participants with attenuated FAP are not eligible.

[0204] Number of participants The study will enroll approximately 40 participants (approximately 20 each post-colectomy and pre-colectomy).

[0205] Treatment Group and Duration All participants will receive 75 mg twice daily of the compound of Formula I. The total duration of study participation for each participant will be approximately 32 weeks, consisting of screening (30 days), treatment (24 weeks), and a follow-up visit (approximately 30 days after the last dose of study drug). The compound of formula I will be manufactured and provided under the responsibility of the sponsor as 75 mg tablets.

[0206] Efficacy evaluation All participants will undergo efficacy assessments at Week 24. Assessments will include lower GI polyp burden, duodenal polyp burden, and disease response, which will be performed by the investigator.

[0207] Pharmacokinetic evaluation Venous blood samples and tissue samples from the lower GI tract (intestinal mucosa and polypectomy) are taken for determination of plasma and tissue concentrations of the compound of formula I at the times indicated on the activity schedule.

[0208] Pharmacodynamic and biomarker evaluation Biomarkers are used to assess the effect of compounds of Formula I on molecular and cellular effectors of the JAK / STAT pathway in blood, tissue biopsy, and fecal samples taken from FAP patients. The relationship between dose regimen, PK, biomarker changes, and efficacy is then evaluated to guide dose optimization when additional doses are evaluated.

[0209] Safety assessment Assessments will include evaluation of adverse events (AEs), serious adverse events (SAEs), infectious events including tuberculosis (TB), laboratory blood tests (complete blood count and serum chemistry), vital signs, endoscopy, and concomitant medication review. In addition to local examinations, central cholesterol testing will be performed.

[0210] statistical methods No formal statistical hypothesis testing will be performed in this study. The statistical analysis plan provides specific details of the statistical methods. A sample size of about 20 for the cohort of post-colectomy participants was calculated by assuming that polyp burden would decrease by at least 25% on average from baseline to week 24. A dropout rate of 20% was assumed. A standard deviation of 30 percentage points was assumed for the percent change in polyp burden from baseline to week 24. With these assumptions, the 95% confidence interval for the percent change in polyp burden from baseline to week 24 has about a 90% or greater probability that its upper limit is less than 0%, thereby providing evidence that treatment with a compound of formula I reduces polyp burden on average. A sample size of about 20 was similarly determined for the cohort of pre-colectomy patients.

[0211] [Table 14]

[0212] Aspects: Aspect 1: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing familial adenomatous polyposis in a subject, comprising administering to the subject a therapeutically effective amount of the compound of formula I.

[0213] Aspect 2: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing gastrointestinal cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0214] Aspect 3: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing colon cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0215] Aspect 4: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in the treatment or prevention of familial adenomatous polyposis or colorectal cancer in a subject at high risk, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0216] Aspect 5: A compound of Formula I, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing familial adenomatous polyposis or colorectal cancer in a subject, comprising: (a) determining a mutation in one or more genes selected from KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC; and (b) administering a therapeutically effective dose of a compound of Formula I.

[0217] Aspect 6: A compound of Formula I, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use in diagnosing whether a subject is at increased risk for developing colorectal cancer or familial adenomatous polyposis, comprising: (a) determining a mutation in one or more genes selected from KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC; and (b) administering a therapeutically effective dose of a compound of Formula I.

[0218] Aspect 7: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing a disorder or condition affected by inhibition of JAK in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0219] Aspect 8: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing recurrence of CRC in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0220] Aspect 9: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in treating or preventing recurrence of FAP in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0221] Aspect 10: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in the treatment or prevention of SISC in a subject diagnosed with irritable bowel disease (IBD), comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0222] Aspect 11: A compound of formula I, or a pharma- ceutically acceptable salt, solvate, or polymorph thereof, for use in preventing a pre-malignant condition from becoming a malignant condition in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I.

[0223] Aspect 12: The use according to any one of aspects 1 to 11, wherein the therapeutically effective amount of the compound of formula I is from about 10 mg to about 1000 mg.

[0224] Aspect 13: The use according to any one of aspects 1 to 12, wherein the therapeutically effective amount of the compound of formula I is from about 1 mg to about 100 mg.

[0225] Embodiment 14: Use according to any one of embodiments 1 to 13, wherein the compound of formula I is administered once a day.

[0226] Embodiment 15: Use according to any one of embodiments 1 to 14, wherein the compound of formula I is administered twice daily.

[0227] Embodiment 16: The use according to any one of embodiments 1 to 15, wherein the subject has previously undergone therapy or is currently undergoing therapy.

[0228] Embodiment 17: The use according to any one of embodiments 1 to 16, wherein the therapy can be surgery, radiation therapy, chemotherapy, NSAIDs, Cox-2 inhibitors, EGFR inhibitors, VEGF inhibitors, and checkpoint inhibitors.

[0229] Embodiment 18: The use according to any one of embodiments 1 to 17, wherein treating FAP comprises reducing the polyburden in the subject.

[0230] Embodiment 19: The use according to any one of embodiments 1 to 18, wherein reducing the polyp burden comprises a reduction in the number of polyps and a reduction in the size of the polyps.

[0231] Aspect 20: The use according to any one of aspects 1 to 19, wherein the high risk subjects include those with irritable bowel syndrome, the presence of a gut microbiome, a family history of colorectal cancer, a personal history of colorectal cancer, findings of polyps or precancerous lesions during colonoscopy, or other genetic factors such as mutations in the KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC genes.

[0232] Aspect 21: The use of any one of aspects 1 to 20, further comprising administering a second active agent in combination with the compound of formula I, wherein the second active agent is selected from an NSAID, a Cox-2 inhibitor, cetuximab, panitumumab, bevacizumab, Ziv-aflibercept, regorafenib, ramucirumab, ipilimumab, nivolumab, and pembrolizumab.

[0233] Embodiment 22: A method of predicting a response to a compound of formula I in a subject in need thereof, comprising: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) comparing the level of pSTAT-3 in (a) with (b), wherein a decrease in the level of pSTAT-3 in (b) predicts response to a compound of Formula I in the subject.

[0234] Embodiment 23. A method for monitoring the efficacy of ongoing JAK inhibitor therapy in a subject in need thereof, comprising: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula: (c) comparing the level of pSTAT-3 in (a) with (b), wherein a decrease in the level of pSTAT-3 in (b) indicates efficacy of the compound of Formula I in the subject.

Claims

1. A pharmaceutical composition for treating or preventing familial adenomatous polyposis in a subject, comprising a therapeutically effective amount of a compound of formula I, or a pharma- ceutical acceptable salt, solvate or polymorph thereof. 【Chemistry 1】

2. A pharmaceutical composition for treating or preventing a subject suffering from gastrointestinal cancer, comprising a therapeutically effective amount of a compound of formula I, or a pharma- ceutical acceptable salt, solvate or polymorph thereof. 【Chemistry 2】

3. A pharmaceutical composition for treating or preventing colon cancer in a subject, comprising a therapeutically effective amount of a compound of formula I, or a pharma- ceutical acceptable salt, solvate or polymorph thereof. 【Chemistry 3】

4. A pharmaceutical composition for preventing familial adenomatous polyposis or colorectal cancer in a subject at high risk, comprising a therapeutically effective amount of a compound of formula I, or a pharma- ceutical acceptable salt, solvate or polymorph thereof. 【Chemistry 4】

5. 1. A pharmaceutical composition for treating colorectal cancer or familial adenomatous polyposis in a subject, comprising a therapeutically effective amount of a compound of formula I, or a pharma- ceutically acceptable salt, solvate or polymorph thereof, the pharmaceutical composition comprising: administered after determining a mutation in one or more genes selected from KRAS, TP53, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, and APC; Pharmaceutical compositions. 【Chemistry 5】

6. 1. A method of testing a subject for whether the subject is at increased risk for developing colorectal cancer or familial adenomatous polyposis, comprising determining a mutation in one or more genes selected from KRAS, EGFR, STK11 (LKB1), PTEN, BMPR1A, SMAD4 (MADH / DPC4), MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH (MYH), POLD1, POLE, TP53, and APC.

7. A pharmaceutical composition for preventing a disorder or condition affected by inhibition of JAK in a subject in need of treatment, comprising a compound of formula I, or a pharma- ceutical acceptable salt, solvate or polymorph thereof. 【Chemistry 6】

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the therapeutically effective amount of the compound of formula I is from about 10 mg to about 1000 mg.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the therapeutically effective amount of the compound of formula I is from about 1 mg to about 100 mg.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the compound of formula I is administered once daily.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the compound of formula I is administered twice daily.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the subject has previously undergone therapy or is currently undergoing therapy.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the therapy can be surgery, radiation therapy, chemotherapy, NSAIDs, Cox-2 inhibitors, EGFR inhibitors, VEGF inhibitors, and checkpoint inhibitors.

14. The pharmaceutical composition of any one of claims 1 to 13, administered in combination with a second active agent, wherein the second active agent is selected from an NSAID, a Cox-2 inhibitor, cetuximab, panitumumab, bevacizumab, Ziv-aflibercept, regorafenib, ramucirumab, ipilimumab, nivolumab, and pembrolizumab.

15. 1. A method of predicting response to a compound of formula I in a subject in need thereof, comprising: 【Chemistry 7】 (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula; (c) comparing the level of pSTAT-3 in (a) with (b); Including, (b) a decrease in the level of pSTAT-3 predicts response to the compound of formula I in the subject. method.

16. 1. A method of monitoring the efficacy of ongoing JAK inhibitor therapy in a subject in need thereof, comprising: (a) measuring the level of pSTAT-3 in a control sample from a subject not exposed to a compound of formula I; 【Chemistry 8】 (b) measuring the level of pSTAT-3 in a test sample from a subject exposed to a compound of formula; (c) comparing the level of pSTAT-3 in (a) with (b); Including, (b) a decrease in the level of pSTAT-3 indicates efficacy of the compound of formula I in the subject. method.

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