EA037347B1 - Small molecule inhibitors of the jak family of kinases - Google Patents

Abstract

2-((1r,4r)-4-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile compounds, pharmaceutical compositions containing them, methods of making them, and methods of using them including methods for treating disease states, disorders, and conditions mediated by JAK, such as inflammatory bowel disease.

Description

Scope of the invention

The present invention relates to certain imidazopyrrolopyridine compounds, pharmaceutical compositions containing them, methods for their preparation and methods of using them as JAK inhibitors for the treatment of JAK-mediated diseases, disorders and conditions.

Background of the invention

Intrinsic factors, extrinsic factors, or a combination of these, may cause or may be associated with the development of abnormal immune responses in the body. As a result, pathological conditions develop in which constituents such as substances and tissues that are normally present in the body are subject to this immune response. These conditions are essentially called diseases of the immune system. Because the body's immune system is involved and the damage affects body tissue, these diseases are also called autoimmune diseases. Since such a system and tissue are part of the same body, the terms autoimmune disease and immune system disease are used interchangeably herein, regardless of what causes the abnormal immune response. Moreover, the nature or mechanism of the underlying immune problem is not always clear. See, for example, D.J. Marks, et al., Crohn's disease: An immune deficiency state, Clinical Reviews in Allergy and Immunology 38 (1), 20-30 (2010); J.D. Lalande, et al, Mycobacteria in Crohn's disease: How innate immune deficiency may result in chronic inflammation, Expert Reviews of Clinical Immunology 6 (4), 633-41 (2010); J.K. Yamamoto-Furusho, et al., Crohn's disease: Innate immunodeficiency, World Journal of Gastroenterology, 12 (42), 6751-55 (2006). As used herein, the term autoimmune disease does not exclude conditions whose causes include external factors or agents such as environmental or bacterial factors, as well as internal factors such as genetic susceptibility. Accordingly, a condition such as Crohn's disease (CD) is referred to herein as an autoimmune disease, whether initiated by the body itself or by external factors. See, for example, J. L. Casanova, et al., Revisiting Crohn's disease as a primary immunodeficiency of macrophages, J. Exp. Med. 206 (9), 1839-43 (2009).

Among the various undesirable effects caused by autoimmune diseases, at least one of the following is most often observed: damage and sometimes destruction of tissues and changes in organs, which can affect the growth and function of organs. Examples of autoimmune diseases affect most major organs, endocrine and exocrine glands, blood and muscles, and many systems such as the digestive, vascular, connective tissue, and nervous systems. Immunosuppressive drugs are often used to treat autoimmune diseases.

There are several theories that explain how autoimmune diseases arise, some of which focus on endogenous factors, while others also involve exogenous factors. At the molecular level, the Janus kinase signaling pathway / signal transducer and transcription activator (JAK / STAT) is thought to play an important role in the transmission of information from extracellular chemical signals to the cell nucleus, regulating genes involved in cellular functions such as immunity. Cytokines are an example of an extracellular molecule that plays an important role in cellular signaling. Leukocytes such as neutrophils are recruited by cytokines and chemokines to ultimately cause tissue damage in chronic inflammatory diseases.

The Janus kinase (JAK) family of proteins consists of 4 tyrosine kinases, JAK1, JAK2, JAK3 and Tyk2, which are the main elements of the intracellular signaling system mediated by type I and type II cytokine receptors. The term JAK refers to JAK1, JAK2, JAK3, or Tyk2, or any combination thereof. Each JAK selectively binds to receptor subunits, which dimerize (or multimerize) to form functional receptors. According to J.D. Clark, et al., Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases, J. Med. Chem. 57 (12), 5023-38 (2014), the activation stage occurs when the cytokine binds to its receptor, inducing multimerization (dimerization or the formation of higher order complexes) of the receptor subunits. This leads to the fact that JAKs, bound to each subunit proximally to each other, trigger a series of phosphorylation events, ultimately leading to phosphorylation and activation of signal transducers and transcriptional activators (STAT proteins). The phosphorylated STAT dimer then travels to the cell nucleus, where it binds to target genes that modulate their expression. Once in the nucleus, STAT proteins regulate gene transcription of numerous mediators involved in the inflammatory process by binding to specific recognition sites on DNA. See, for example, J. Med. Chem. 57 (12), 5023-38 (2014) above. There is a wealth of data available demonstrating the importance of the JAK / STAT pathway in inflammatory, autoimmune diseases and cancer. See, for example, M. Coskun, et al., Involvement of JAK / STAT signaling in the pathogenesis of inflammatory bowel disease, Pharmacological Research 76, 1-8 (2013); and J.J. O'Shea, et al., JAKs and STATs in immunity, immunodeficiency, and cancer, The New England Journal of Medicine 368, 161-70 (2013).

Inflammatory bowel disease, including Crohn's disease and ulcerative colitis (UC), is characterized by recurrent intestinal inflammation, impaired epithelial barrier, and microbiotic dysbiosis. The excessive inflammatory response in the gastrointestinal tract is mediated by several pro-inflammatory cytokines, including TNFa, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL17, IL-21 and IL-23, which affect cells of the innate and adaptive immune system, including T- and B-lymphocytes, epithelial cells, macrophages and dendritic cells (DC). See, for example, Pharmacological Research 76, 1-8 (2013) cited above, S. Danese, et al., JAK inhibition using tofacitinib for inflammatory bowel treatment disease: A hub for multiple inflammatory cytokines, American Journal of Physiology, Gastrointestinal and Liver Physiology 310, G155-62 (2016); and M.F. Neurath, Cytokines in inflammatory bowel disease, Nature Reviews Immunology 14, 329-42 (2014).

Prevention and / or reduction of such an excessive inflammatory response is desirable. In light of the mechanism of this response described above, inhibition of JAK (see the serrated arrow image of FIG. 1 showing the effects of a universal JAK inhibitor on the JAK / STAT signaling pathway and inflammation) prevents or reduces the excessive inflammatory response. JAK inhibitors that inhibit many of these JAK proteins are referred to herein as universal JAK inhibitors. Examples of therapeutic benefits of such prevention or control are seen with tofacitinib, which is an orally bioavailable universal JAK inhibitor approved in the United States for the treatment of rheumatoid arthritis and is currently in clinical development for the treatment of ulcerative colitis. In a Phase 2 clinical trial, the available data evaluated clinical efficacy in 194 patients with moderate to severe ulcerative colitis. See, for example, W.J. Sandborn, et al., Tofacitinib, an oral Janus kinase inhibitor, in active ulcerative colitis, The New England Journal of Medicine 367, 616-24 (2012). Published information from this study shows that patients receiving doses of 0.5, 3, 10 and 15 mg twice daily (BID) had clinical response rates of 32, 48, 61 and 78%, respectively, compared with 42% observed with placebo use. Additionally, the secondary clinical remission endpoint (Mayo score <2) was reported to be 13%, 33%, 48%, and 41% compared to 10% observed in the placebo group. See, for example, The New England Journal of Medicine 367, 616-24 (2012), cited above.In the UC Phase 3 clinical trial, 88 of 476 patients reportedly experienced clinical remission after 8 weeks of tofacitinib (10 mg BID) compared with 10 of 122 patients treated with placebo. See W.J. Sandborn, et al. Efficacy and safety of oral tofacitinib as induction therapy in patients with moderate-to-severe ulcerative colitis: results from 2 phase 3 randomized controlled trials, J. Crohns Colitis 10, S15-S (2016). Crohn's disease publications indicate that tofacitinib was also under development for the treatment of CD; however, development was reportedly discontinued due to lack of clinical efficacy in a 4-week phase 2 clinical trial of moderate to severe CD. See W.J. Sandborn, et al., A phase 2 study of tofacitinib, an oral Janus kinase inhibitor, in patients with Crohn's disease, Clinical gastroenterology and hepatology: The official clinical practice journal of the American Gastroenterological Association 12, 1485-93 e2 (2014). Based on information obtained from the publicly available literature, it is currently unclear whether the ineffectiveness of tofacitinib in CD is related to clinical trial design, mechanistic differences between UC and CD, or to dose-limiting systemic adverse events. See the above publications Pharmacological Research 76, 1-8 (2013); Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 12, 1485-93 e2 (2014); and C.J. Menet, et al., Triazolopyridines as selective JAK1 inhibitors: from hit identification to GLPG0634, J. Med. Chem. 57, 9323-42 (2014). In light of the features of this JAK inhibitor, it is desirable to find additional JAK inhibitors to prevent and / or reduce the excessive inflammatory response.

In phase 2 and phase 3 clinical trials of inflammatory bowel disease (IBD) with tofacitinib, systemic adverse events have been reported. See, supra, The New England Journal of Medicine 367, 616-24 (2012); Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association 12, 1485-93 e2 (2014); and J. Panes, et al. Efficacy and safety of oral tofacitinib for induction therapy in patients with moderateto-severe Crohn's disease: results of a Phase 2b randomized placebo-controlled trial, J. Crohns Colitis 10, S18S19 (2016). These adverse events include a decrease in the absolute neutrophil count (ANC), an increase in total cholesterol (low and high density lipids), intestinal perforation, and infection. Such adverse events are consistent with the results observed after tofacitinib treatment of patients with rheumatoid arthritis (RA) (see, for example, JM Kremer, et al. The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: Results of a double- blind, placebocontrolled phase IIa trial of three dosage levels of CP-690,550 versus placebo, Arthritis and Rheumatism 60, 1895-905 (2009)), some of which are likely due to JAK2-dependent inhibition of EPO, TPO, and colony-stimulating factors (csf- 2 and GM-CSF (granulocyte macrophage colony stimulating factor)) and / or JAK1-dependent inhibition of IL-6. See Arthritis and Rheumatism 60, 1895-905 (2009), cited above; and he. Nielsen, et al., Will novel oral formulations change the management of inflammatory bowel disease? Expert Opinion on Investigational Drugs 25, 709-18 (2016).

As shown in FIG. 1, an orally administered drug can in principle pass through the gastrointestinal tract from the oral cavity to the esophagus (1), 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 for such different parts are approximately 60% for the jejunum (4), approximately 26% for the ileum (5) and approximately 13% for the colon (6). Absorption in these different areas of the gastrointestinal tract can lead to the onset of systemic distribution, which in turn can lead to unwanted side effects. The gastrointestinal tract has a very large surface area. See, for example, H.F. Helander, et al., Surface area of the digestive tract - revisited, Scandinavian Journal of Gastroenterology 49 (6), 681-89 (2014); and K.J. Filipski, et al., Intestinal Targeting of Drugs: Rational Design Approaches and Challenges Current Topics in Medicinal Chemistry 13, 776-802 (2013). Such a large absorption surface area promotes the systemic distribution of substances that can pass through the walls of various parts of the gastrointestinal tract and into the bloodstream, and in turn can lead to undesirable side effects of the systemically distributed substance. For simplicity, the system distribution is represented by the dashed arrows in FIG. 1 in the form of penetration through the walls of the colon, but this distribution is not limited to the walls of the colon, as it can also occur through the walls of other parts of the gastrointestinal tract shown in FIG. 1, for example the wall of the small intestine. It should also be understood that the dashed arrows in FIG. 1 represent the systemic distribution outside the gastrointestinal tract, since such systemic distribution is known to occur in connection with the physiology of the gastrointestinal tract, and that such dashed arrows simply schematically illustrate such a systemic distribution. For a description of gut tissue, gut transport and metabolism, see, for example, Current Topics in Medicinal Chemistry 13, 777-80 (2013), cited above.

One of the main reasons for the decline in potential drugs is safety and tolerability. See, for example, I. Kola, et al., Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery 3,711-5 (2004); M.J. Waring, et al., An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nature Reviews Drug Discovery 14, 475-86 (2015); M. Hay, et al., Clinical development success rates for investigational drugs, Nature Biotechnology 32, 40-51 (2014); and M.E. Bunnage, Getting pharmaceutical R&D back on target, Nature Chemical Biology 7, 335-9 (2011). Increasing the local tissue concentration of a compound in the desired target tissue while limiting exposure to other tissues can reduce unwanted side effects. See, for example, V.P. Torchilin, Drug targeting. European Journal of Pharmaceutical Sciences: Official Journal of the European Federation for Pharmaceutical Sciences 11 Suppl 2, S81-91 (2000). This concept has gained widespread acceptance for certain diseases and tissues such as the eye (see, for example, R. Gaudana, et al., Ocular drug delivery, The AAPS Journal 12, 348-60 (2010)), skin (see, for example, R. Folster-Holst, et al., Topical hydrocortisone 17-butyrate 21-propionate in the treatment of inflammatory skin diseases: pharmacological data, clinical efficacy, safety and calculation of the therapeutic index, Die Pharmazie 71, 115-21 ( 2016)) and lung (see, for example, JS Patil, et al., Pulmonary drug delivery strategies: A concise, systematic review, Lung India: official organ of Indian Chest Society 29, 44-9 (2012)). Similar to these tissue targeting approaches, increasing the intestinal drug concentration while limiting unwanted drug levels in other tissues can increase the safety range. See, for example, I.R. Wilding, et al., Targeting of drugs and vaccines to the gut, Pharmacology & Therapeutics 62, 97-124 (1994); D. Charmot, Non-systemic drugs: a critical review, Current Pharmaceutical Design 18, 143445 (2012); and Current Topics in Medicinal Chemistry 13, 780 (2013) above. Tissue-selective modulation of targets in the tissues of the gastrointestinal tract by compounds with limited systemic effects can potentially improve the therapeutic index of such compounds in the treatment of diseases of the gastrointestinal tract, including ulcerative colitis and Crohn's disease. See, for example, O. Wolk, et al., New targeting strategies in drug therapy of inflammatory bowel disease: mechanistic approaches and opportunities, Expert Opin. Drug Deliv. 10 (9), 1275-86 (2013). The term systemic effects is used in this document to refer to systemic exposure and the consequences of any such systemic exposure, even if they are not always the same.

Since some of the known JAK inhibitors have undesirable effects associated with their systemic effects, it is desirable to find new JAK inhibitors as active substances for the prevention and / or reduction of excessive inflammatory response, the systemic effects of which are absent or reduced. In addition, it would be desirable to find JAK inhibitors with a local effect on gastrointestinal tissue for the treatment of conditions such as, but not limited to, IBD, with reduced systemic effects. Because of the role played by various JAK proteins, it is also desirable to find universal JAK inhibitors.

Intestinal tissue targeting can, in principle, be carried out according to different

- 3 037347 strategies. See, for example, Current Topics in Medicinal Chemistry 13, 780-95 (2013), cited above, for approaches including physicochemical property-based, transport-mediated, prodrug-based, and composition and technology-based approaches. However, it is believed that there are a number of problems and disadvantages that are inherent in tissue targeting programs and, in particular, for gut targeting compounds, as described in the publication Current Topics in Medicinal Chemistry 13, 795 (2013), cited above.

Conditions for IBD can spread to different parts of the gastrointestinal tract. While for simplicity in the descending colon, FIG. 1 shows only the section (10) of colon disease, inflammatory bowel disease can affect any part of the gastrointestinal tract, as in the case of Crohn's disease, or the rectum and colon, as in the case of ulcerative colitis. See, for example, NIDDK (National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, <http://spotidoc.com/doc/71780/crohns-disease-national- digestive-diseases-information>, accessed Nov. 29, 2016. Sites of IBD can be, for example, ileal (localized in the ileum), ileal (affecting parts of the ileum and colon), and colon (localized in the colon, as shown as an example in Fig. 1 in the descending colon.) Thus, in some cases of the disease, it may be desirable to deliver the drug to all or a large part of the gastrointestinal tract. In other cases of the disease, it may be desirable to increase the local concentration in any particular part of the In still other embodiments, it may be desirable to combine the two forms of delivery to different sites in the intestinal tract.

One such option is to deliver an active substance that has a limited systemic effect due to limited absorption as it passes through the gastrointestinal tract, as shown by the solid arrows in FIG. 1, and can simultaneously act in different parts of the gastrointestinal tract (GIT), i.e. possesses a feature referred to herein as local LCD effects. Due to the reduced systemic effects for such a substance, a wider range of doses can be estimated. It would also be desirable for such an active substance to have a low permeability, so that only a small amount of it passes through the intestinal wall into the vascular bed in order to limit undesirable side effects when the substance reaches non-target areas.

In addition, JAK inhibitors can be considered as potential treatments for other diseases. They can be used to treat eye diseases, including dry eye syndrome (B. Colligris, et al., Recent developments on dry eye disease treatment compounds, Saudi J. Ophthalmol. 28 (1), 19-30 (2014)), myeloproliferative neoplasms , myeloproliferative disorders (EJ Baxter, et al., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders, Lancet 365, 1054-1061 (2005); C. James, et al., A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera, Nature 434, 1144-1148 (2005); R. Kralovics, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders, N. Engl. J. Med. 352, 1779-1790 (2005 ); RL Levine, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis, Cancer Cell 7, 387-397 (2005); G. Wernig, et al., Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced p olycythemia vera, Cancer Cell 13, 311-320 (2008)), myeloproliferative syndrome, acute myeloid leukemia, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, juvenile idiopathic arthritis (H.W. Li, et al., Effect of miR-19a and miR-21 on the JAK / STAT signaling pathway in the peripheral blood mononuclear cells of patients with systemic juvenile idiopathic arthritis, Exp. Ther. Med. 11 (6), 2531-2536 (2016)), type III hypersensitivity reactions, type IV hypersensitivity, aortic inflammation, iridocyclitis / uveitis / optic neuritis, juvenile spinal muscular atrophy, diabetic retinopathy, diabetic kidney disease including diabetic nephropathy (FC Brosius, et al., JAK inhibition in the treatment of diabetic kidney disease, Diabetologia 59 (8), 1624-7, (2016); CC Berthier, et al., Enhanced expression of Janus kinase-signal transducer and activator of transcription pathway members in human diabetic nephropathy, Diabetes 58 (2), 469-77, (2009); EN Gurzov, et al., The JAK / STAT pathway in obesity and diabetes, FEBS J. 283 (16), 3002-15 (2016) )), microangiopathy, inflammation (M. Kopf, et al., Averting inflammation by targeting the cytokine environment, Nature Reviews Drug Discovery 9, 703-718 (2010); JJ O'Shea, et al., A new modality for immunosuppression : targeting the JAK / STAT pathway, Nature Rev. Drug Discov. 3, 555-564 (2004)), chroni inflammatory bowel disease, including ulcerative colitis (UC) and Crohn's disease (R.H. Duerr, et al., A genome-wide association study identifies IL23R as an inflammatory bowel disease gene, Science 314, 1461-1463 (2006); M. Coskun, et al., Involvement of JAK / STAT signaling in the pathogenesis of inflammatory bowel disease, Pharmacol. Res. 76, 1-8 (2013); M.J. Waldner, et al., Master regulator of intestinal disease: IL-6 in chronic inflammation and cancer development, Semin. Immunol. 26 (1), 75-9 (2014); S. Danese, et al., JAK inhibition using tofacitinib for inflammatory bowel disease treatment: a hub for multiple inflammatory cytokines, Am. J. Physiol. Gastrointest. Liver Physiol. 310 (3), G155-62 (2016); W. Strober, et al., Proinflammatory cytokines in the pathogenesis of inflammation

- 4 037347 matory bowel diseases, Gastroenterology 140, 1756-1767 (2011)), allergic diseases, vitiligo, atopic dermatitis (R. Bissonnette, et al., Topical tofacitinib for atopic dermatitis: a phase IIa randomized trial, Br. J. Dermatol. 175 (5), 902-911 (2016)); W. Amano, et al., JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation, J. Dermatol. Sci. 84, 258-265 (2016); T. Fukuyama, et al., Topically Administered Janus-Kinase Inhibitors Tofacitinib and Oclacitinib Display Impressive Antipruritic and Anti-Inflammatory Responses in a Model of Allergic Dermatitis, J. Pharmacol. Exp. Ther. 354 (3), 394-405 (2015)), alopecia areata (AK Alves de Medeiros, et al., JAK3 as an Emerging Target for Topical Treatment of Inflammatory Skin Diseases, PLoS One 11 (10) (2016); L. Xing, et al., Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition, Nat. Med. 20 (9), 1043-9 (2014)), dermatitis, scleroderma, acute or chronic immune disease associated with organ transplantation (PS Changelian, et al. Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor, Science 302, 875-878 (2003); F. Behbod, et al. Concomitant inhibition of Janus kinase 3 and calcineurindependent signaling pathways synergistically prolongs the survival of rat heart allografts, J. Immunol, 166, 3724-3732 (2001); S. Busque, et al., Calcineurin-inhibitor-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients, Am. J. Transplant, 9, 1936-1945 (2009)), psoriatic arthropathy, arthropathy n in ulcerative colitis, autoimmune bullous disease, autoimmune hemolytic anemia, rheumatoid arthritis (J.M. Kremer, et al., A randomized, doubleblind placebo-controlled trial of 3 dose levels of CP-690,550 versus placebo in the treatment of active rheumatoid arthritis, Arthritis Rheum. 54 (annual meeting abstract), L40 (2006); W. Williams, et al., A randomized placebo-controlled study of INCB018424, a selective Janus kinase1 & 2 (JAK1 & 2) inhibitor in rheumatoid arthritis (RA), Arthritis Rheum. 58, S431 (2008); N. Nishimoto, et al., Study of active controlled monotherapy used for rheumatoid arthritis, an IL-6 inhibitor (SAMURAI): evidence of clinical and radiographic benefit from an x ray reader-blinded randomized controlled trial of tocilizumab, Ann. Rheum. Dis. 66 (9), 1162-7 (2007)), interstitial pulmonary disease associated with rheumatoid arthritis, systemic lupus erythematosus (A. Goropevsek, et al., The Role of STAT Signaling Pathways in the Pathogenesis of Systemic Lupus Erythematosus, Clin. Rev. Allergy Immunol. (Preliminary web publication) <http://www.docguide.com/role-stat-signaling-pathways-pathogenesis-systemic-lupus-erythematosus?tsid=5> May 23, 2016; M. Kawasaki , et al., Possible role of the JAK / STAT pathways in the regulation of T cellinterferon related genes in systemic lupus erythematosus, Lupus. 20 (12), 1231-9 (2011); Y. Furumoto, et al., Tofacitinib ameliorates murine lupus and its associated vascular dysfunction, Arthritis Rheumatol., (on-line prepublication) <https://www.ncbi.nlm.nih.gov/pubmed/27429362> Jul. 18, 2016)), pulmonary disease associated with systemic lupus erythematosus, a pulmonary disease associated with dermatomyositis / polymyositis (K. Vale, Targeting the JAK / STAT pathwa y in the treatment of 'Th2-high' severe asthma, Future Med. Chem. 8 (4), 405-19 (2016)), ankylosing spondylitis (AS) (C. Thompson, et al., Anti cytokine therapy in chronic inflammatory arthritis, Cytokine 86, 92-9 (2016)), AS-associated pulmonary diseases, autoimmune hepatitis, autoimmune hepatitis type 1 (classic autoimmune or lupus hepatitis), autoimmune hepatitis type 2 (hepatitis with antibodies to liver and kidney microsomes), autoimmune-mediated hypoglycemia, psoriasis (CL Leonardi, et al. ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with psoriasis: 76-week results from a randomized, double-blind, placebo-controlled trial (PHOENIX 1), Lancet 371, 1665-1674 (2008); G. Chan, et al., Dose-dependent reduction in psoriasis severity as evidence of immunosuppressive activity of an oral Jak3 inhibitor in humans, Am. J. Transplant. 6, S87 (2006); KA Papp, et al., Efficacy and safety of tofacitinib, an oral Janus kinase inhibitor, in the treatment of psoriasis: a phase 2b randomized placebo-controlled dose-ranging study, Br. J. Dermatol. 167,668-677 (2012); M. Cargill, et al. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes, Am. J. Hum. Genet. 80, 273-290 (2007)), type 1 psoriasis, type 2 psoriasis, plaque psoriasis, moderate to severe chronic plaque psoriasis, autoimmune neutropenia, autoimmune reaction against spermatozoa, multiple sclerosis (all subtypes, V.M. Segal, et al ., Repeated subcutaneous injections of IL12 / 23 p40 neutralizing antibody, ustekinumab, in patients with relapsing-remitting multiple sclerosis: a phase II, double-blind, placebo-controlled, randomized, dose-ranging study, Lancet Neurol. 7, 796- 804 (2008); Z. Yan, et al., Role of the JAK / STAT signaling pathway in regulation of innate immunity in neuroinflammatory diseases, Clin. Immunol. (Preliminary publication on the Internet) <https: //www.ncbi.nlm .nih.gov / pubmed / 27713030>, accessed Oct 3, 2016; EN Benveniste, et al., Involvement of the janus kinase / signal transducer and activator of transcription signaling pathway in multiple sclerosis and the animal model of experimental autoimmune encephalomyelitis, J Interferon Cytoki ne Res. 34 (8), 577-88 (2014) .; Y. Liu, et al., Therapeutic efficacy of suppressing the Jak / STAT pathway in multiple models of experimental autoimmune encephalomyelitis, J. Immunol. 192 (1), 59-72 (2014)), acute rheumatic fever, Sjogren's syndrome, pulmonary disease associated with Sjogren's syndrome / disease (T. Fujimura, et al., Significance of Interleukin-6 / STAT Pathway for the Gene Expression of REG Ia, and New Autoantigen in Sjogren's Syndrome Patients, in Salivary Duct Epithelial Cells, Clin. Rev. Allergy Immunol. (preliminary web publication) <https://www.ncbi.nlm.nih.gov/pubmed/27339601> Jun 24, 2016), autoimmune thrombocytopenia, neuro

- 5,037347 inflammations, including Parkinson's disease (published by Z. Yan, et al., Oct. 3, 2016, cited above). According to the available data, JAK inhibitors, in addition to inflammatory diseases, are used for the treatment of cancer (SJ Thomas, et al., The role of JAK / STAT signaling in the pathogenesis, prognosis and treatment of solid tumors, British J. Cancer 113, 365-71 ( 2015); A. Kontzias, et al., Jakinibs: A new class of kinase inhibitors in cancer and autoimmune disease, Current Opinion in Pharmacology, 12 (4), 464-70 (Aug. 2012); M. Pesu, et al ., Therapeutic targeting of JANUS kinases, Immunological Reviews, 223, 132-42 (Jun. 2008); P. Norman, Selective JAK inhibitors in development for rheumatoid arthritis, Expert Opinion on Investigational Drugs, 23 (8), 1067-77 ( Aug. 2014)). In addition, JAK inhibitors can be used to prevent colorectal cancer, as reducing inflammation in the colon can prevent cancer in that organ.

Summary of the invention

The present invention relates to the following compounds:

2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- (2- hydroxy2-methylpropyl) acetamide;

2 - ((1d, 4d) -4- (2- (1H-imidazol-4-yl) imidazo [4,5—

d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile;

- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N (cyclopropylmethyl) acetamide;

V- (2-cyanoethyl) -2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -

1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) acetamide;

2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -V- ( tetrahydro2H-pyran-4-yl) acetamide;

2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -V- ( (tetrahydro2H-pyran-4-yl) methyl) acetamide;

V- (2-cyano-2-methylpropyl) -2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) acetamide;

2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N - ((1hydroxycyclobutyl ) methyl) acetamide;

2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -V- (1- methyl-1Hpyrazol-4-yl) acetamide;

V- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1- ((1d, 4d) -

4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-

B] pyridin-2-yl) acetamide;

- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- (1H-pyrazole3 -yl) acetamide;

2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -V- ( (1hydroxycyclopropyl) methyl) acetamide; and pharmaceutically acceptable salts of such compounds and combinations thereof.

The terms compounds of the invention and the compound of the invention include at least one compound selected from the above group of compounds, either in solvent-free form or in any one of the hydrated and / or solvated forms as shown herein.

Embodiments of the present invention relate to compounds, pharmaceutical compositions containing them, methods for their preparation and purification, methods of using them as JAK inhibitors, and methods of using them in the treatment of JAK-mediated diseases, disorders and conditions.

Embodiments of the present invention demonstrate the effects of universal JAK inhibition as well as local GI effects and little or no systemic effects. Moreover, embodiments of the present invention with such features can be administered orally.

An additional embodiment of the present invention is a method of treating a patient diagnosed with a disease, disorder or medical condition or suffering from a JAK mediated disease, disorder or medical condition with the compounds of the invention or active agents of the invention.

Additional embodiments, features and advantages of the invention will be apparent from the following detailed description and practice of the invention.

Brief description of the figures

FIG. 1: An enlarged schematic representation of a portion of the human gastrointestinal tract, not to scale. The duodenum (3), the jejunum (4) and the ileum (5) (all shown schematically) form the small intestine after the stomach (2) and esophagus (1). The large intestine contains the colon (6), which in turn includes the cecum (7) and appendix (not shown), the ascending colon, the transverse colon, the descending colon, the sigmoid colon (the loop is not shown in it), and the rectum intestine (11). The transverse colon is the area between the right (8) and left (9) bends of the colon, the ascending colon runs from the cecum (7) to the right bend (8) of the colon, and the descending colon runs from the left bend ( 9) colon to rectum (11). Different types of distribution are shown for convenience in the colon, but they may also apply to other parts of the gastrointestinal tract. For simplicity, the system distribution is represented by the dashed arrows in FIG. 1 in the form of penetration through the walls of the colon, but this distribution is not limited to the walls of the colon, as it can also occur through the walls of other parts of the gastrointestinal tract shown in FIG. 1, for example the wall of the small intestine. Distribution with some tissue penetration is shown for simplicity by solid arrows in FIG. 1 that penetrate the colon tissue, but such penetration is not limited to the colon tissue as it can also occur in the tissue of other parts of the gastrointestinal tract shown in FIG. 1, for example, tissue from the small intestine. The effect of an embodiment of a JAK inhibitor according to the present invention is shown in the figure as interrupting the JAK / STAT signaling pathway that would otherwise lead to inflammation associated with inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis. By way of example and not limitation, the disease site is shown as a colon (10) portion in the descending colon for illustrative purposes.

FIG. 2: Schematic diagram showing acquisition / interchanging of Ex 1 connection embodiments.

FIG. 3: Overlay of high performance powder X-ray diffractometry (HT-XRPD) diffraction patterns for the following embodiments of Ex 1 compound, 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 in the microliter range in a methanol / water mixture (50/50, v / v), 5 (obtained by thermal cycling in chloroform), 6 (obtained by cooling crystallization in the microliter range in acetonitrile ), 7 (obtained from 1s + 7, which in turn was obtained by equilibration of a solution in heptane), 7 (obtained by desolvation of 1s + 7, which in turn was obtained by equilibration of a solution in heptane), 8 (obtained by desolvation of an embodiment 5 by cycling differential scanning calorimetry)) and 9 (obtained by desolvation of embodiment 2 by cycling differential scanning calorimetry).

FIG. 4: Overlay of high throughput powder X-ray diffraction (HT-XRPD) diffraction patterns for the following embodiments of Ex 1 compound, from bottom to top: 1s (starting material), 1a (obtained after multiple sample forms of embodiment 1s were subjected to accelerated aging conditions (AAC) (40 ° C and 70% relative humidity)), 1b (obtained by equilibration of a solution at room temperature in toluene), 1c (obtained by cooling crystallization in the microliter range in ethyl acetate / 1,4-dioxane (50/50 , v / v)), 1d (obtained by cooling crystallization in the microliter range in a mixture of acetonitrile / chloroform (50/50, v / v)), 1e (obtained by cooling crystallization in the microliter range in a mixture of ethyl acetate / 1 , 4-dioxane (50/50, v / v)), 1f (obtained by equilibration of a solution at room temperature in p-xylene), 1g (prepared by equilibration of a solution at 50 ° C in anisole), 1n (obtained by cooling cr ustallization in the microliter range in p-xylene).

FIG. 5: Overlay of high-performance powder X-ray diffractometry (HT-XRPD) diffraction patterns for the following embodiments of the compound Ex. 1, from bottom to top: 1s, 3b (obtained by thermal cycling in cyclohexanone), 3c (obtained by cooling crystallization in the microliter range in 1.4- dioxane), 3d (obtained by cooling crystallization in the microliter range in tetrahydrofuran) and 3e (obtained by thermal cycling in isobutanol).

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FIG. 6: Diffraction grams of HR-XRPD of embodiment 1s in its original form (1s) after four days of exposure to 40 ° C and 70% relative humidity (1s 70 OB) and after four days of exposure to 25 ° C and 100% relative humidity (10) ...

Detailed description of the invention

The terms including, containing, consisting of are used in an open, unrestricted meaning.

Any of the formulas provided herein are intended to represent both compounds with the structures shown by this structural formula, and some variations or forms of such structures. Some structures can exist as tautomers. In addition, amorphous form, hydrates, solvates, polymorphs and pseudopolymorphs of such compounds of the present invention, and mixtures thereof are also contemplated as parts of the present invention. Embodiments of the present invention are in non-solvent form or in any of the hydrated and / or solvated forms as shown herein.

Reference to a compound herein means reference to any of the following: (a) the literal form of such a compound and (b) any form of such a compound in the environment where the compound is at the time of mention. For example, reference in this document to a compound such as RCOOH includes reference to any of the following forms, for example, R-COOH ( _tv ,), R-COOH ( _sol .), And R-COO ^- (sol.). In this example, R-COOHiT [; l refers to a solid compound and may, for example, be contained in a tablet or some other solid pharmaceutical composition or preparation; K-COOH, - | ms | b1 refers to the non-dissociated form of the compound in a solvent; R-COO ^- (sol,) refers to the dissociated form of a compound in a solvent, such as the dissociated form of a compound in an aqueous medium, whether the dissociated form is derived from R-COOH, a salt thereof, or any other substance that gives R-COO ^- after dissociation in the considered environment. In another example, an expression such as the effect of a substance on a compound of the formula R-COOH means the effect of such a substance on the form or forms of the compound R-COOH that exists or that exist in the environment where such exposure occurs. In yet another example, an expression such as the reaction of a substance with a compound of the formula R-COOH refers to the reaction: (a) such a substance in the appropriate chemical form or forms that exist or that exist in the environment where the described reaction is carried out; with (b) an appropriate chemical form or forms of the compound R-COOH that exists or that exist in the environment where such a reaction is carried out. Thus, if such a substance is, for example, in an aqueous medium, it is understood that the R-COOH compound is in the same environment and, therefore, such a substance is acted upon by such forms as R-COOH (waterH,) and / or R- COO ^- (water _H ,), where the subscript (water.) Means water in accordance with its generally accepted meaning in chemistry and biochemistry. In these nomenclature examples, a carboxylic acid functional group was chosen; however, this choice is not a deliberate limitation, but is merely an illustration.

It is understood that similar examples can be given for other functional groups, including, without limitation, groups such as a hydroxyl group, nitrogen basic groups, for example, as in amines, as well as any other groups that react or rearrange in a known manner in a containing connection environment. Such interactions and rearrangements include, without limitation, dissociation, association, tautomerism, solvolysis, including hydrolysis, solvation, including hydration, protonation, and deprotonation. This document does not provide further examples in this regard, since these interactions and rearrangements in each particular environment are well known to any person skilled in the art.

Any of the formulas provided herein also represent both unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures shown by the formulas provided herein, with the exception of the replacement of one or more atoms with an atom having a selected atomic mass or mass number in an enriched form. Examples of isotopes that can be incorporated into the compounds of the present invention in a non-naturally occurring form include isotopes of hydrogen, carbon, nitrogen, and oxygen such as ² H, ³ H, ¹¹ C, ^{13 C, 14} C, ¹⁵ N, ¹⁸ O and ¹⁷ O, respectively. Such isotopically labeled compounds can be used in metabolic studies (preferably with ¹⁴ C), kinetic studies of reactions (for example, with deuterium (i.e. D or ² H); or tritium (i.e. T or ³ H)), methods detection or imaging [such as positron emission tomography (PET) or single photon emission computed tomography (SPECT)], including drug or substrate tissue distribution tests, or radiotherapy of patients. In particular, compounds labeled with ¹⁸ F or 11C may be particularly preferred for PET or SPECT studies. In addition, substitution with heavier isotopes such as deuterium (i.e., ² H) may provide some therapeutic benefits due to the greater metabolic stability of the compounds, for example, a longer local in vivo half-life or a reduced required dosage. The isotopically labeled compounds of the present invention can be substantially prepared by carrying out the procedures described below in the schemes or examples and preparation methods described below, by replacing a readily available reagent containing no isotopically labeled atoms with a labeled isotope reagent

- 8 037347 atoms.

The term tautomers refers to compounds that are interchangeable forms of the structure of a particular compound and differ in the displacement of hydrogen atoms and electrons. Thus, two structures with the element H in different positions can be in equilibrium without violating the valence rules. For example, enols and ketones are tautomers because they quickly convert to each other when treated with acid or base.

In describing any formula provided herein, selecting a particular functional group from a list of options for a specified variable does not mean determining that option for a variable in other formulas. In other words, if a variable is present in a formula in more than one place, then the choice of a variant from the specified list in one place does not depend on the choice of a variant for the same variable elsewhere in the formula, unless otherwise indicated.

As a first example of substituent terminology, if the substituent S ^{1 of the} example is one of S1 and S2, and the substituent S ^{2 of the} example is one of S3 and S ₄ , these designations refer to the embodiments of the present invention given in accordance with the variants where S ¹ example is S1 and S ² example is S3; S ¹ example is S1 and S ² example is S ₄ ; S ¹ example is S2 and S ² example is S3; S ¹ example is S2 and S ² example is S ₄ ; as well as the equivalents of each of these options. More concise terminology S ¹ example is one of S1 and S2, and S ² example is one of S3 and S _4, respectively, are used herein for brevity and not limitation. The above first example of the terminology used to denote substituents, given in general terms, is intended to illustrate the various variants of the designation of substituents described herein.

Moreover, if more than one designation is given for any element of the chemical structure or substituent, embodiments of the present invention comprise various, independently adopted combinations of variants of the listed designations, as well as their equivalents. As a second example of substituent terminology, if it is described herein that if the substituent S _example is one of S ₁ , S2 and S3, then this listing refers to embodiments of the present invention for which S _example is S ₁ ; S _example is S2; S _example is S3; S _example is one of S ₁ and S2; S _example is one of S ₁ and S3; S _example is one of S2 and S3; S _example is one of S ₁ , S2 and S3; and S _example is any equivalent of each of these options. For brevity, but not limitation, abbreviated terminology is appropriately used herein: the S _example is one of S ₁ , S2, and S3. The above second example in terms of the terminology used to denote substituents, given in general terms, is intended to illustrate the various options for denoting substituents described in this document.

The following JAK inhibitors are exemplary embodiments of the present invention:

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- (1- ((lr, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- (2-hydroxy2 -methylpropyl) acetamide;

2 - ((1d, 4d) -4- (2- (1H-imidazol-4-yl) imidazo [4,5d] pyrrolo [2, 3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile;

- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N (cyclopropylmethyl) acetamide;

N- (2-cyanoethyl) -2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) 1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridine-2 -yl) acetamide;

2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -11- (tetrahydro2H- pyran-4-yl) acetamide;

- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- ((tetrahydro2H- pyran-4-yl) methyl) acetamide;

N- (2-cyano-2-methylpropyl) -2- (1- ((1d, 4d) -4 (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3 - b ] pyridin-2-yl) acetamide;

- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N - ((1hydroxycyclobutyl) methyl) acetamide;

2- (1- ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -11- (1- methyl-1Hpyrazol-4-yl) acetamide;

11- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1- ((1d, 4d) 4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5- d] pyrrolo [2,3-

B] pyridin-2-yl) acetamide;

2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- (1Н- pyrazol3-yl) acetamide; and

2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) -11- ((1hydroxycyclopropyl ) methyl) acetamide.

Additional embodiments of the invention are pharmaceutically acceptable salts of the above compounds.

Additional embodiments of the invention are pharmaceutical compositions, each of which contains an effective amount of at least one of the above compounds or a pharmaceutically acceptable salt thereof.

A pharmaceutically acceptable salt is a salt of a compound that is non-toxic, biologically tolerable, or otherwise biologically acceptable for administration to a patient. See essentially S.M. 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 the invention may have a sufficiently acidic group, a sufficiently basic group or both types of functional groups and react appropriately with a variety of inorganic or organic bases, as well as inorganic or organic acids, to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, hepates, isobutyrates, isobutytes , propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexine-1,6-dioates, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phthalates, sulfenoxybenzoates, phthalates, sulfenzoates, phthalates , phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolate, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates and mandelates.

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If the compound of the invention contains at least one basic nitrogen atom, then the desired pharmaceutically acceptable salt can be obtained by any appropriate method known in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, or an organic acid such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosidic acid such as glucuronic acid or galacturonic acid, alpha hydroxy acid such as mandelic acid, citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid, sulfonic acid such as lauryl sulfonic acid, p-toluenesulfonic acid , methanesulfonic acid, ethanesulfonic acid, any compatible mixture of acids such as those exemplified herein, and any other acid or mixture of acids that are considered equivalent or acceptable substitutes in the light of this technology.

Not all embodiments of the pharmaceutically acceptable salts of the compounds of the present invention may be equally suitable for their development, since compounds that are sufficiently weak bases (e.g., pK _a about 4) may not form sufficiently stable salts for development purposes. See, for example, GA Stephenson, et al., J. Pharm. Sciences 100 (5), 1607-17 (2011) Physical stability of salts of weak bases in the solid state. Certain embodiments of the invention are contemplated to encompass co-crystallized forms of a compound of the present invention with a suitable co-crystal forming agent. The structure and properties of co-crystals for pharmaceutical use and methods for their preparation and description are described, for example, in N. Shan, et al., Drug Discovery Today, 13 (9/10), 440-46 (2008) The role of cocrystals in pharmaceutical science ; N. Qiao, et al., Intl. J. Pharmaceutics, 419, 1-11 (2011) Pharmaceutical cocrystals: An overview; R. Thakuria, et al., Intl. J. Pharmaceutics, 453, 101-25 (2013) Pharmaceutical cocrystals and poorly soluble drugs. acid-containing forms of the compound of the present invention.

The compounds of the invention, including their pharmaceutically acceptable salts, alone or in combination (collectively referred to as active agent or active agents), can be used as JAK inhibitors in the methods of the invention. Such methods of modulating JAK activity include exposing JAK to an effective amount of at least one chemical compound of the present invention.

In some embodiments, a JAK inhibitor is used in a patient who is diagnosed with a disease, disorder, or medical condition, or who suffers from a disease, disorder, or medical condition mediated by JAK activity, such as, for example, as described herein. Symptoms or conditions of a disease are intended to be included within the scope of a disease, disorder, or medical condition.

Accordingly, the present invention relates to methods of using the active agents described herein to treat patients diagnosed with a disease, disorder, or medical condition, or who are suffering from a JAK-mediated disease, disorder, or medical condition. As used herein, the term “treat or treat” refers to the administration of an active agent or composition in accordance with the present invention to a patient to obtain the desired therapeutic or prophylactic benefit by modulating JAK activity. Treatment includes reversing the course, ameliorating, ameliorating, slowing the progression, reducing the severity, or preventing a disease, disorder or condition, or one or more symptoms of such a disease, disorder, or condition mediated by modulation of JAK activity. The term patient refers to a mammalian patient in need of such treatment, such as a human. The term inhibitors or inhibitor refers to compounds that reduce, prevent, deactivate expression, desensitize, or down-regulate JAK expression or activity.

Embodiments of the present invention provide JAK inhibitors for preventing and / or reducing an excessive inflammatory response. Embodiments of JAK inhibitors in accordance with the present invention are generic JAK inhibitors.

Unless otherwise indicated, the term "physicochemical properties of a JAK inhibitor" means the following corresponding mentioned properties: described in the description for compounds of Ex 1-12 in the case of molar masses; defined according to the appropriate definitions in the case of the number of hydrogen bond donors and acceptors and rotatable bonds; and measured with reference to table. 1a (column 2) for plasma concentrations and table. 7 (columns 3 and 4) in the case of permeability coefficients A-B in the presence of a P-glycoprotein inhibitor and permeability coefficients BA.

In embodiments of the present invention, methods for inhibiting JAK are provided,

- 11 037347 including the effect on the JAK receptor of a JAK inhibitor, which is characterized by the following physicochemical properties: plasma concentration in the range from about 0.1 to about 60 ng / ml, cLog P in the range from about 0.1 to about 2.8 , the permeability coefficient A-B in the presence of the inhibitor P glycoprotein in the range from about 0.1 to about 2.5, the permeability coefficients BA in the range from about 0.5 to about 20, the topological polar surface area (tPSA) in the range from about 85 to about 120.

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, the plasma concentration ranges from about 10 to about 20 ng / ml.

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, cLogP ranges from about 0.8 to about 1.4.

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, the permeability coefficient AB in the presence of the P-glycoprotein inhibitor ranges from about 0.6 to about 1.5.

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, the permeability coefficient BA is in the range from about 0.5 to about 5.

In other embodiments, the implementation of the methods for inhibiting JAK in accordance with the present invention tPSA ranges from about 100 to about 120.

In additional embodiments, implementation of the present invention provides methods for inhibiting JAK, including exposing the JAK receptor to a JAK inhibitor, which is further characterized by the following physicochemical properties: molar mass in the range from about 300 to about 500 ghmol ^-1 , the number of hydrogen bond donors in the range from about 2 to about 3, the number of hydrogen bond acceptors ranging from about 4 to about 5, and the number of rotatable bonds ranging from about 3 to about 6, in addition to plasma concentrations, clogP values, permeability factors, and tPSA values described above for methods of JAK inhibition in accordance with the present invention.

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, the molar weight ranges from about 340 ghmol ^-1 to about 430 ghmol ^-1 .

In other embodiments of the methods for inhibiting JAK in accordance with the present invention, the number of rotatable bonds ranges from about 5 to about 6.

In embodiments of the present invention, methods of treating inflammation in the gastrointestinal tract of a patient are provided, comprising administering to the patient a pharmaceutically effective amount of a JAK inhibitor that has the following physicochemical properties: plasma concentration in the range of about 0.1 to about 60 ng / ml, cLog P in the range from about 0.1 to about 2.8, the permeability coefficient AB in the presence of the P-glycoprotein inhibitor in the range from about 0.1 to about 2.5, the permeability coefficients BA in the range from about 0, 5 to about 20, the topological polar surface area (tPSA) ranges from about 85 to about 120.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, the plasma concentration ranges from about 10 ng / ml to about 20 ng / ml.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, cLogP ranges from about 0.8 to about 1.4.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, the permeability ratio AB in the presence of the P glycoprotein inhibitor ranges from about 0.6 to about 1.5.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, the permeability coefficient BA is in the range from about 0.5 to about 5.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, the tPSA ranges from about 100 to about 120.

In additional embodiments, implementation of the present invention provides methods of treating inflammation in the gastrointestinal tract of a patient, in which the physicochemical properties of the JAK inhibitor are further characterized by the following physicochemical properties of the JAK inhibitor: molar mass ranging from about 300 to about 500 ghmol ^-1 hydrogen bond donors in the range of about 2 to about 3, the number of hydrogen bond acceptors in the range of about 4 to about 5 and the number of rotatable bonds in the range of about 3 to about 6, in addition to plasma concentrations, cLogP values, coefficients permeability and tPSA values described above for the methods of treating inflammation in accordance with the present invention.

In other embodiments of the methods of treating inflammation in the gastrointestinal tract in accordance with the present invention, the molar mass ranges from about 350 ghmol ^-1 to about 430 ghmol ^-1 .

In other embodiments, the implementation of the methods for treating inflammation in the gastrointestinal tract in

12,037347 In accordance with the present invention, the number of rotatable bonds ranges from about 5 to about 6.

Embodiments of JAK inhibitors in accordance with the present invention have the following physicochemical properties of JAK: plasma concentration in the range of about 0.1 to about 60 ng / ml, cLogP in the range of about 0.1 to about 2.8, permeability coefficient AB in the presence of a P-glycoprotein inhibitor in the range of about 0.1 to about 2.5, permeability coefficients BA in the range of about 0.5 to about 20, and tPSA in the range of about 85 to about 120.

In additional embodiments of the JAK inhibitors of the present invention, the plasma concentration ranges from about 10 to about 20 ng / ml.

In additional embodiments of the JAK inhibitors of the present invention, cLogP values range from about 0.8 to about 1.4.

In additional embodiments of the JAK inhibitors of the present invention, the permeability ratio AB in the presence of the P-glycoprotein inhibitor ranges from about 0.6 to about 1.5.

In additional embodiments of JAK inhibitors in accordance with the present invention, the permeability coefficient BA is in the range from about 0.5 to about 5.

In additional embodiments of JAK inhibitors in accordance with the present invention, tPSA values range from about 100 to about 120.

Other embodiments of JAK inhibitors in accordance with the present invention have the following physicochemical properties of a JAK inhibitor: molar mass in the range from about 300 to about 500 ghmol ^-1 , the number of hydrogen bond donors in the range from about 2 to about 3, the number of hydrogen bond acceptors in the range from about 4 to about 5 and the number of rotatable bonds in the range from about 3 to about 6, in addition to plasma concentrations, cLogP values, permeability factors and tPSA values described above for JAK inhibitors in accordance with the present invention.

In additional embodiments of JAK inhibitors in accordance with the present invention, the molar weight ranges from about 350 to about 430 ghmol ^-1 .

In additional embodiments of the JAK inhibitors of the present invention, the number of rotatable bonds ranges from about 5 to about 6.

In the methods of treatment according to the invention, an effective amount of at least one active agent according to the invention is administered to a patient who has been diagnosed with a disease, disorder or medical condition or who is suffering from a disease, disorder or medical condition. The term "effective amount" means an amount or dosage that is sufficient to achieve a substantially desired therapeutic or prophylactic effect for patients in need of such treatment for a specified disease, disorder or medical condition. Effective amounts or dosages of the active substances of the present invention can be determined by standard methods, for example, modeling, dose escalation studies or clinical trials, taking into account standard factors such as the mode or route of administration or delivery of the drug, the pharmacokinetics of the substance, the severity and nature of the course of the disease. disorders or conditions, previous or current course of treatment of the object, the state of health and reaction of the object to medications, and the opinion of the attending physician. For a 70 kg human, an exemplary acceptable dosage is an amount of about 1 to 1000 mg / day taken one or more times.

Embodiments of the present invention are novel JAK inhibitors as active substances for preventing and / or reducing an excessive inflammatory response, the systemic effects of which are absent or reduced. Additional embodiments of the present invention are JAK inhibitors that have a local effect on gastrointestinal tissue for the treatment of conditions such as, but not limited to, IBD, the systemic effects of which are absent or reduced to an acceptable level.

Embodiments of the present invention are low permeability JAK inhibitors. Additional embodiments of the present invention are JAK inhibitors that are water soluble.

After improvement in the patient's condition, relief of symptoms of the disease or disorder, you can adjust the dose for prophylactic or maintenance treatment. For example, the dosage or frequency of administration, or both, can be reduced, depending on the symptoms, to a level that maintains the desired therapeutic or prophylactic effect. Of course, if the symptoms are reduced to an acceptable level, treatment can be discontinued. However, if symptoms recur, the patient may require long-term intermittent treatment.

In addition, the compounds of the present invention are intended to be used alone, in combination with one or more of the other compounds of the present invention, or in combination with additional active ingredients for the treatment of the conditions described below. Additional active ingredients can be administered as a separate dosage form together with at least one compound of the present invention, with the active agents of the invention, or incorporated into a pharmaceutical composition in accordance with the present invention. In one embodiment, the additional active ingredients are components with known or proven efficacy in the treatment of conditions, disorders or diseases mediated by JAK activity, such as another JAK inhibitor or a compound with activity against another target associated with a particular condition, disorder, or disease. Said combination of components may serve to increase efficacy (for example, by including in the combination a compound that enhances the efficacy or activity of an agent of the present invention), ameliorates one or more side effects, or reduces the required dosage of an active agent of the present invention.

In the context of inhibition of a target, an effective amount means an amount sufficient to alter the activity of at least one of the JAK family proteins. Measurement of target activity can be performed using standard analytical techniques.

The active agents of the present invention are provided for use alone or in combination with one or more additional active ingredients to prepare pharmaceutical compositions of the invention. A pharmaceutical composition for the purposes of the invention comprises an effective amount of at least one active agent according to the invention.

Pharmaceutically acceptable excipients commonly used in pharmaceutical compositions are substances that are non-toxic, biologically tolerable and otherwise biologically suitable for administration to a patient, such as an inert substance added to a pharmaceutical composition or otherwise used as a carrier, carrier or diluent to facilitate the administration of the agent, and also compatible with this agent. Examples of such excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Delivery forms of pharmaceutical compositions containing one or more dosage units of active agents can be prepared using pharmaceutically acceptable excipients and preparation techniques currently known or available to those skilled in the art in the future. The compositions can be administered using methods having the features of the invention, by an acceptable route of delivery, for example, orally, parenterally, rectally, topically, ophthalmic, or by inhalation.

The drug can be presented in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, reconstitution powders, liquid preparations or suppositories. The compositions can be prepared for any one of a variety of routes of administration such as intravenous infusion, subcutaneous injection, topical application, or oral administration. Preferably, the compositions can be prepared for oral administration.

For oral administration, the active agents of the invention may be provided in the form of tablets, capsules or granules, or in the form of a solution, emulsion or suspension. For the preparation of compositions for oral administration, the active agents can be mixed to form a dosage, for example, for a 70 kg human, from about 1 to about 1000 mg / day, to be administered one or multiple times as an illustrative range.

Tablets for oral administration may include the active ingredient (s) mixed with compatible pharmaceutically acceptable excipients such as diluents, tablet disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Acceptable excipients include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of liquid excipients for oral use include ethanol, glycerin, water, and the like. Examples of substances for improving the disintegration of tablets are starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose and alginic acid. Binding agents can include starch and gelatin. The lubricant, if present, can be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or they may be enteric coated. Additional coating that can be used includes coatings configured to release the compound or active agent depending on time, pH, or bacterial content.

Capsules for oral administration include hard and soft gelatin capsules or (hydroxypropyl) methylcellulose capsules. For the preparation of hard gelatine capsules, the active ingredients can be mixed with a solid, semi-solid or liquid diluent. Soft gelatin capsules can be prepared by mixing the active ingredient with an oil such as peanut or olive oil, liquid paraffin, a mixture of short chain mono- and diglycerides.

- 14 037347 fatty acids, polyethylene glycol 400 or propylene glycol. Oral fluids may be in the form of suspensions, solutions, emulsions, or syrups, or they may be lyophilized or dry for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain pharmaceutically acceptable excipients such as suspending agents (eg, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and the like); non-aqueous vehicles such as oil (eg almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol or water; preservatives (eg methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if necessary, flavoring or coloring agents.

The active agents of the present invention can also be administered non-orally. For example, the compositions can be formulated for rectal administration as a suppository, enema, or foam. For compositions for parenteral administration, including intravenous, intramuscular, intraperitoneal or subcutaneous administration, the agents of the invention can be formulated as sterile aqueous solutions or suspensions with the addition of appropriate buffers until the desired pH and isotonicity are obtained, or as a parenterally acceptable oil. Acceptable aqueous vehicles include Ringer's solution and isotonic sodium chloride solution. Such dosage forms can be formulated as a single dose form such as ampoules or disposable injection devices, as a multi-dose form, such as vials, from which the required amount of drug can be dispensed, or in solid or primary concentrate form, which can be used for the preparation of compositions for injection. Typical doses for infusion are in the range of about 1 to 1000 μg / kg / min of the substance as a mixture with a pharmaceutical carrier over a period of time from several minutes to several days.

For topical administration, the agents can be mixed with the pharmaceutical carrier at a concentration of about 0.01 to about 20% of the drug in the vehicle, preferably 0.1-10%. In another mode of administration, the agents of the invention may be administered as a patch with a transdermal delivery formulation.

Alternatively, in the methods of the invention, the active substances can be administered by inhalation, through the nose or mouth, for example in the form of a spray containing also a suitable carrier.

In a further embodiment, the invention relates to a method of treating a patient diagnosed with a disease, disorder or medical condition or suffering from a disease, disorder or medical condition mediated by JAK, comprising administering to a patient in need of such treatment an effective amount of an active agent.

In some embodiments of the method of the invention, the disease, disorder, or medical condition is inflammatory bowel disease such as Crohn's disease and ulcerative colitis.

In other embodiments, implementation of the present invention provides a method for modulating JAK activity, including when such a kinase is in a patient, comprising exposing JAK to an effective amount of at least one compound selected from the compounds of the invention.

The compounds of the invention can be used as JAK inhibitors, which are orally administered and distributed specifically to intestinal tissue while providing low level of systemic effects. Thus, the compounds of the invention differ from most of the known JAK inhibitors, which are orally administered and distributed to many tissues, due to the fact that they have extensive systemic effects.

Table 1a and 1b show the results of in vivo experiments. These results include plasma and colon tissue concentrations of fifteen compounds that were administered to mice as described in protocols 1, 2, or 3. Plasma and colon concentration measurements were obtained according to protocol 1 using dorsal metatarsal venipuncture for compounds (B), (C) and examples 6 and 11. The results of measuring the concentration in plasma and colon were obtained in accordance with protocol 2 using bleeding from the retroorbital sinus for compounds (A) and examples 1 and 3-5 and protocol 2 c using venipuncture of the dorsal metatarsal vein for examples 2, 7-10 and 12. The results of protocols 1 and 2 are presented in table. 1a. The results of measuring the concentration in plasma and colon were obtained in accordance with the following protocol 3 for examples 1, 3 and 4. The results of protocol 3 are presented in table. 1b. These protocols are described below in the In vivo studies section.

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Table 1a. Results of in vivo experiments after oral dosing, average ______________ concentration of test compounds _______________

Test compound Plasma concentration after oral dosing (ng / ml) Concentration in the colon after oral dosing (ng / g) Time = 0.5 h Time = 2 h Time = 4 h Time = 4 h Average * Standard deviation Average* Standard deviation Average* Standard deviation Average* Standard deviation BUT 347.0 78.5 69.1 40, 8 84.5 25.5 895.0 260.6 IN 352.7 85.7 66.3 26.2 11.3 3.7 6076.7 3125.8 FROM 547.0 71.4 130.2 63.7 16.7 5.9 7776.7 3500.2 Etc. one 13.4 1.5 6, 1 3.7 3.3 1,2 8591.7 10,245.7 Etc. 2 24.5 3.6 4.2 1.8 1,3 0.1 7600.0 983.6 Etc. 3 41.4 15, 1 3.9 0.7 1.5 ** * * * 2147.2 1821.6 Etc. four 12.9 1.6 7.5 2.8 3.3 1.5 4448.3 989.3 Etc. five 31.9 5.1 8.8 1.7 6, 0 1,2 5328.3 986, 0 Etc. 6 18.8 20, 6 3.0 0.9 1.7 # ## 11 706.7 11,305.2 Etc. 7 47.0 3.8 9.6 4.4 5.0 1,2 12,008.3 9461.1 Etc. eight 43.1 8.7 5.4 0.6 2.6 0.6 7396.7 3037.3 Etc. nine 15.1 1.8 6.2 4.5 3.9 0.9 7683.3 230.9 Etc. 10 26, 6 4.0 3.2 1.0 3.1 0.7 3005.0 1347.2 Etc. eleven 1.6 ** *** ^BUT A A BUT A A 4785.0 1059.9 Etc. 12 15.6 8.7 4.2 1.4 2,3 1, o 5885.0 3154.1

* Average calculated based on values obtained from three mice, unless otherwise indicated.

* * Average calculated based on the values obtained from two mice, since the values obtained from the third mouse were below the lower limit of quantification.

* ** Standard deviation was not calculated as the mean was derived from only two values.

# Average reported is the value obtained from one mouse because the values obtained from the second and third mice were below the lower limit of quantification.

## The standard deviation was not calculated for the reason stated in the Note # to this table.

l The mean was not calculated as the values for all three mice were below the lower limit of quantification.

LL The standard deviation was not calculated for the reason indicated in Note L to this table.

Table 1b. Results of in vivo experiments after i.e. dosing, average concentration __________________ test compounds ________________

Explore my connection Plasma concentration after i.w. dosing (ng / ml) Concentration in the colon after i.c. dosing (ng / g) Time = 0.5 h Time = 2 h Time = 4 h Time = 4 h Average* Standard deviation Average* Standard deviation Average* Standard deviation Average* Standard deviation Etc. one 2.5 # ## ΛΛ ΛΛ 681.0 437.0 Etc. 3 1.5 # ## ^Λ ΛΛ ^Λ ΛΛ 227.8 254.1 Etc. four 3.8 ** *** 2.5 # ## ^Λ ΛΛ 26, 1 # ##

* Average calculated based on values obtained from three mice, unless otherwise indicated.

** Average calculated based on the values obtained from two mice, since the values obtained from the third mouse were below the lower limit of quantification.

- 16 037347 *** Standard deviation was not calculated as the mean was derived from only two values.

# Average reported is the value obtained from one mouse because the values obtained from the second and third mice were below the lower limit of quantification.

## The standard deviation was not calculated for the reason stated in the Note # to this table.

^L The mean was not calculated because the values for all three mice were below the lower limit of quantification.

LL The standard deviation was not calculated for the reason indicated in Note L to this table.

Compounds (A) - (C) are the following reference compounds which have been described in WO 2013/007765 or WO 2011/086053 in connection with their use as Janus kinase inhibitors:

(A) (B) (C)

Connections pr. 1-12 in table. 1a and 1b are embodiments of the present invention given in the respective examples.

As shown in table. 1a, the concentrations in the colon of compounds pr. 1-12 were significantly higher than the corresponding concentrations in plasma, while the concentration ratios [colon (4 h)]: [plasma (0.5 h)] varied from about 52 to about 3000. In contrast, such ratios for compounds (A) - (C) varied from about 3 to about 17. In table. 1b also provides ancillary evidence that examples 1, 3 and 4 have weak systemic effects after i.e. dosing. The contrast between the properties of the embodiments of the present invention in relation to the reference compounds is much more pronounced when comparing the plasma concentration values after 4 hours. In this regard, the concentration ratios [colon (4 hours)]: [plasma (4 hours)] for compounds pr . 1-12 range from about 888 to about 6886. In contrast, such ratios for compounds (A) - (C) ranged from about 11 to about 538. These ratios of concentration in the colon to concentration in plasma show that the compounds of Ex 1 -12 have low systemic effects at any time after oral dosing, while compounds (A) - (C) have relatively high systemic effects. The presence of local LC effects for compounds pr. 1-12 is an unexpected result.

As shown in table. 4, the enzymatic activity of Ex 1-12 compounds was measured to determine the activity of each individual enzyme. For all tested compounds, inhibition of enzymatic activity was found, which indicates that these compounds are universal JAK inhibitors. The data shown in this table for compounds (A) - (C) also demonstrate the inhibition by these compounds of the enzymatic activity of all JAK proteins.

As shown in table. 5, the cellular activity of Ex 1-12 compounds was assessed in peripheral blood mononuclear cells (PBMCs) using the stimuli IL-2, IFN-α and GM-CSF and measuring the inhibition of the phosphorylation of STAT5, STAT4 and STAT5, respectively. For all tested compounds, the inhibition of STAT phosphorylation was measured using all three stimuli.

As shown in table. 6, the solubility values of Ex 1-12 compounds were measured in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). All compounds tested showed measurable solubility of over 400 μM in SGF and in the range of 81 to over 400 μM using SIF. As shown in the same table, the solubility data were comparable to the solubility values of compounds (A) - (C).

As shown in table. 7, the permeability of compounds (A) - (C), etc. 1-12 was measured using the MDCK-MDR1 cell line with and without the addition of elacridar (P-glycoprotein inhibitor). All compounds showed low permeability when measured apical-basolateral transport with and without the addition of a P-glycoprotein inhibitor (elacridar). The values of the permeability coefficient for compounds (A) - (C) and others 1-12 were low and comparable to the values of apical-basolateral transport without elacridar (for all such compounds) and with elacridar (for compounds (B) - (C) and compounds ex. 1-12) (columns 2 and 3 in the table), but the basolateral-apical permeability coefficients for compounds (A) - (C) were higher than for most compounds of ex. 112, as shown in column 4 of the same table. According to the data in columns 3 and 4 in table. 7, most of the compounds pr. 1-12 are characterized by low coefficients of apical-basolateral permeability, measured in the presence of elacridar (column 3), as well as low coefficients of basolateral-apical permeability (column 4). These two features indicate that such compounds are low permeability compounds. This cannot be said for compounds (A) - (C),

- 17 037347 whose basolateral-apical permeability coefficients (column 4) are higher than those of most compounds pr. 1-12. Outflow ratios given in the same table for compounds (A) - (C), etc.

1-12 show that all of these compounds are P-glycoprotein substrates.

There are no known concepts or assumptions indicating that a conclusion or prediction of a marked decrease in systemic effects for embodiments of the present invention compared to those of reference compounds (A) - (C) can be made based on structural comparisons or other characteristics of compounds (A) - (C), for example described with reference to table. 4, 6 and 7. This is the case even though some fragments of the reference compounds (A) - (C) have structural similarities to similar fragments of the embodiments of the present invention.

In addition, there are no known concepts or assumptions indicating that the conclusion or prediction of the low permeability of the embodiments of the present invention compared to the permeability of the reference compounds (A) - (C) can be made based on structural comparisons.

The following specific examples further illustrate the present invention and various embodiments.

Unless otherwise indicated, the following experimental and analytical protocols were used to prepare the compounds described in the examples below and the corresponding analytical data.

Unless otherwise indicated, the reaction mixtures were stirred on a magnetic stirrer at room temperature (room temp.). If the solutions have been dried, a drying agent such as Na ₂ SO ₄ or MgSO _{4 is} usually used for this. If mixtures, solutions and extracts were concentrated, they were usually concentrated on a rotary evaporator under reduced pressure.

Thin layer chromatography (TLC) was performed on silica gel-coated Merck 60 F ₂₅₄ 2.5x7.5 cm 250 μm or 5.0x10.0 cm 250 μm plates.

Normal phase flash column chromatography (CPC) was performed on silica gel columns (SiO ₂ ) eluted with 2 M NH ₃ in methanol / DCM, unless otherwise indicated.

Unless otherwise noted, mass spectra (MS) were acquired on an Agilent 1100 MSD analyzer using electrospray ionization (ESI) in positive mode. The calculated (calculated) mass corresponds to the exact mass.

Nuclear magnetic resonance (NMR) spectra were obtained on Bruker model DRX spectrometers. The 1H NMR data format is shown below: chemical shift in ppm downfield from the signal used as the tetramethylsilane standard (multiplicity, spin-spin coupling constant J in Hz, integral).

Chemical names were created using ChemDraw software (CambridgeSoft, Cambridge, MA) or ACD / Name version 9 (Advanced Chemistry Development, Toronto, Ontario, Canada). As an example, the notation (1r, 4r) refers to the transorientation around the cyclohexyl ring and was created using the Chemdraw Ultra Pro 14.0 naming feature.

For the sake of brevity of description, some of the quantitative expressions given in this document are not specified using the term about. It is understood that, regardless of whether the modifier is explicitly applied to or not, each numerical value given in this document refers to a specific recited value, as well as an approximation to this recited value, which can reasonably be estimated based on conventional knowledge in this area, including equivalents and approximations associated with the experimental and / or measurement conditions for such a reduced value.

When stated as a percentage, the yield refers to the mass of the substance for which the yield is indicated, in relation to the maximum achievable amount of that substance under specific stoichiometric conditions. Unless otherwise indicated, all percentages of reagent concentrations refer to weight ratios.

The following abbreviations and abbreviations are used in this document.

Abbreviations and Acronyms Used

Abbreviation Term AAS Accelerated aging conditions (40 ° C and 70% OV) ACN Acetonitrile aq. Water

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ush. Broadened cLogP Calculated logP DXM Dichloromethane DIPEA, DIEA, base Hünig Diisopropylethylamine DMA Dimethylacetamide DMF LDM-dimethylformamide DMPU 1,3-Dimethyl-3,4,5,6-tetrahydro-2 (1H) pyrimidinone DMSO Dimethyl sulfoxide EtOAc or EA Ethyl acetate EtOH Ethanol IER Electrospray ionization KFH Flash column chromatography with normal phase on silica gel r Gram (s) h Clock) HPLC High performance liquid chromatography HR-XRPD High Resolution Powder X-ray Diffractometry HT-XRPD High performance powder x-ray diffractometry I PA isopropanol i. c. inside the colon Hz Hertz LCMS Liquid chromatography with mass spectrometry M Molar m / z Mass to charge ratio MeOH Methanol MB Milligram (s) min Minute (s) ml Milliliter (s) μl mmol MTBE MC NMR by ppm PTFE RuVOR RuVgor OV Rt room pace. TEA Microliter (s) Millimel (s) Methyl tert-butyl ether Mass spectrometry Nuclear magnetic resonance, oral or oral PPM polytetrafluoroethylene Benzotriazol-1-yloxytripyrro lidinophosphonium hexafluorophosphate Bromotripyrrolidine phosphonium hexafluorophosphate Relative humidity Retention time Room temperature Trifluoroacetic acid THF Tetrahydrofuran those Thin layer chromatography tPSA Topological polar area surfaces

- 19 037347

Synthesis and description of the properties of intermediate compound 1: 2 - ((1d, 4d) -4 - ((5-nitro-1 (phenylsulfonyl) -1H-pyrrolo [2,3-b] pyridin-4-yl) amino) cyclohexyl) acetonitrile

Stage A: tert-butyl-N - [(1g, 4g) -4- (hydroxymethyl) cyclohexyl] carbamate

In a four-necked round-bottom flask with a volume of 20 L, in which an inert nitrogen atmosphere was maintained after purging, (1g, 4g) -4 - [[(tert-butoxy) carbonyl] amino] cyclohexane-1carboxylic acid (1066 g, 4.38 mol, 1.00 equiv.) And THF (10 L). Then BH ₃ -Me ₂ S (10 M, 660 ml) was added dropwise at -10 ° C over 1 hour. The resulting solution was stirred for 3 hours at 15 ° C. This reaction was carried out three times in parallel and the reaction mixtures were combined. Subsequently, the reaction mixture was quenched by the addition of methanol (2 L). The resulting mixture was concentrated in vacuo. This gave tert-butyl-N - [(1g, 4g) -4- (hydroxymethyl) cyclohexyl] carbamate (3000 g, 99.6%) as a white solid. MS (ESI): mass calculated for C ₁₂ H ₂₃ NO ₃ , 229.32; m / z obtained 215.2 [MtBu + MeCN + H] +;

¹ H NMR: (300 MHz, CDCl ₃ ): δ 4.40 (s, 1H), 3.45 (d, J = 6.3 Hz, 2H), 3.38 (s, 1H), 2.05 -2.02 (m, 2H), 1.841.81 (m, 2H), 1.44 (s, 11H), 1.17-1.01 (m, 4H).

Stage B: tert-butyl-N - [(1d, 4d) -4 - [(methanesulfonyloxy) methyl] cyclohexyl] carbamate.

In a four-necked round-bottom flask with a volume of 20 L, in which an inert nitrogen atmosphere was maintained after purging, tert-butyl-N - [(1g, 4g) -4- (hydroxymethyl) cyclohexyl] carbamate (1000 g, 4.36 mol, 1, 00 eq.), Dichloromethane (10 L), pyridine (1380 g, 17.5 mol, 4.00 eq.). Then MsCl (1000 g, 8.73 mol, 2.00 eq.) Was added dropwise at -15 ° C. The resulting solution was stirred overnight at 25 ° C. This reaction was carried out in parallel 3 times and the reaction mixtures were combined. Subsequently, the reaction was quenched by the addition of 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 (3x10 L), NaHCO ₃ (saturated aq. Solution) (2x10 L), water (1x10 L) and brine (1x10 L). The mixture was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. This gave tert-butyl-N - [(1g, 4g) 4 - [(methanesulfonyloxy) methyl] cyclohexyl] carbamate (3300 g, 82%) as a white solid. LCMS: MS (ESI): mass calculated for C ₁₃ H ₂₅ NO ₅ , 307.15; m / z obtained, 292.1 [M-tBu + MeCN + H] ⁺ ;

¹ H NMR: (300 MHz, CDCl ₃ ): δ 4.03 (d, J = 6.6 Hz, 2H), 3.38 (s, 1H), 3.00 (s, 3H), 2.07 -2.05 (m, 2H), 1.871.84 (m, 2H), 1.72-1.69 (m, 1H), 1.44 (s, 9H), 1.19-1.04 (m , 4H).

Stage C: tert-butyl-N - [(1d, 4d) -4- (cyanomethyl) cyclohexyl] carbamate

In a four-necked round-bottom flask with a volume of 10 L was placed tert-butyl-N - [(1g, 4g) -4 [(methanesulfonyloxy) methyl] cyclohexyl] carbamate (1100 g, 3.58 mol, 1.00 eq.), DMSO (5500 ml) and NaCN (406 g, 8.29 mol, 2.30 eq.). The resulting mixture was stirred for 5 h at 90 ° C. This reaction was carried out in parallel 3 times and the reaction mixtures were combined. Subsequently, the reaction mixture was quenched by the addition of 15 L of water / ice. The solids were collected by filtration. The solids were washed with water (3x10 L). This gave tert-butyl N - [(1g, 4g) -4- (cyanomethyl) cyclohexyl] carbamate (2480 g, 97%) as a white solid. MS (ESI): mass calculated for C ₁₃ H ₂₂ N ₂ O ₂ , 238.17; the resulting m / z 224 [M-tBu + MeCN + H] ⁺ ;

1H NMR: (300 MHz, CDCl ₃ ): δ 4.39 (s, 1H), 3.38 (s, 1H), 2.26 (d, J = 6.9 Hz, 2H), 2.08- 2.04 (m, 2H), 1.921.88 (m, 2H), 1.67-1.61 (m, 1H), 1.44 (s, 9H), 1.26-1.06 (m, 4H).

Stage D: 2 - [(1d, 4d) -4-aminocyclohexyl] acetonitrile hydrochloride.

Tert-butyl-N - [(1g, 4g) -4 (cyanomethyl) cyclohexyl] carbamate (620 g, 2.60 mol, 1.00 equiv.) And 1,4-dioxane (2 l). Then, a solution of HCl in 1,4-dioxane (5 L, 4M) was added dropwise with stirring at 10 ° C. The resulting solution was stirred overnight at 25 ° C. This reaction was carried out 4 times and the reaction mixtures were combined. The solids were collected by filtration. The solids were washed with 1,4-dioxane (3x3 L), ethyl acetate (3x3 L) and hexane (3x3 L). This gave 2 - [(1g, 4d) -4-aminocyclohexyl] acetonitrile hydrochloride (1753 g, 96%) as a white solid. MS (ESI): mass calculated for C ₈ H ₁₄ N ₂ , 138.12; m / z obtained 139.25, [M + H] ⁺ ;

1H NMR: (300 MHz, DMSO-d6): δ 8.14 (s, 3H), 2.96-2.84 (m, 1H), 2.46 (d, J = 6.3 Hz, 2H) , 1.98 (d, J = 11.1 Hz, 2H), 1.79 (d, J = 12.0 Hz, 2H), 1.64-1.49 (m, 1H), 1.42- 1.29 (m, 2H), 1.18-1.04 (m, 2H).

Stage E: 2 - ((1d, 4d) -4 - ((5-nitro-1- (phenylsulfonyl) -1H-pyrrolo [2,3-b] pyridin-4-yl) amino) cyclohexyl) acetonitrile

In a 1000 ml round bottom flask containing 2 - [(1g, 4g) -4-aminocyclohexyl] acetonitrile

- 20 037347 hydrochloride (29.10 g, 166.6 mmol), added DMA (400 ml). The resulting suspension was treated with 4chloro-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 N ₂ and kept at 80 ° C for 4 hours. The crude reaction mixture was cooled to room temperature and slowly poured into a 2 L vigorously stirred flask containing 1.6 L of water. The resulting slurry was stirred for 15 min at room temperature, then filtered and dried for 16 h in a vacuum oven at 70 ° C to afford the title compound (63.37 g, 95%) as a yellow solid. MS (ESI): mass calculated for C ₂₁ H ₂₁ N ₅ O ₄ S, 439.1; m / z obtained, 440.1 [M + H] ⁺ .

¹ H NMR (500 MHz, CDCl ₃ ): δ 9.10 (s, 1H), 8.99 (d, J = 7.8 Hz, 1H), 8.23-8.15 (m, 2H), 7.66-7.59 (m, 2H), 7.56-7.49 (m, 2H), 6.67 (d, J = 4.2 Hz, 1H), 3.95-3.79 ( m, 1H), 2.38 (d, J = 6.2 Hz, 2H), 2.32-2.21 (m, 2H), 2.081.98 (m, 2H), 1.88-1.76 (m, 1H), 1.60-1.32 (m, 4H).

Synthesis and description of properties of intermediate compound 2: 2 - ((1d, 4d) -4 - ((5-amino-1 (phenylsulfonyl) -1H-pyrrolo [2,3-b] pyridin-4-yl) amino) cyclohexyl) acetonitrile

Г Т% HN ^L

Al o

2 - ((1d, 4d) -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 Thales Nano H-Cube® at 10 ml / min with 100% hydrogen (atmospheric pressure, 80 ° C), after which the solution was concentrated to obtaining the product as a purple solid. The solid was triturated with EtOAc (400 ml) and subsequently triturated again with MeOH (200 ml), then filtered and dried under vacuum to give the title compound (50.2 g, 91.9% yield). MS (ESI): mass calculated for C ₂₁ H ₂₃ N ₅ O ₂ S, 409.2; obtained m / z, 410.2 [M + H] ⁺ .

1H NMR (400 MHz, CDCl3) δ 8.10-8.03 (m, 2H), 7.76 (s, 1H), 7.51-7.43 (m, 1H), 7.43-7, 34 (m, 3H), 6.44 (d, J = 4.2 Hz, 1H), 4.61 (d, J = 8.5 Hz, 1H), 3.65-3.51 (m, 1H ), 2.74 (s, 2H), 2.26 (d, J = 6.4 Hz, 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).

Synthesis and description of properties of intermediate compound 3: ethyl 2- (1 - ((1d, 4d) -4 (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2 , 3-b] pyridine-2yl) acetate

Into a 1 L round bottom flask containing a magnetic anchor and 2 - ((1g, 4g) -4 - ((5-amino-1 (phenylsulfonyl) -1H-pyrrolo [2,3-b] pyridin-4-yl) amino ) cyclohexyl) acetonitrile (intermediate 2, 58.31 g, 142.4 mmol) was added ethyl 3-ethoxy-3-iminopropanoate (60.51 g, 309.3 mmol) followed by the addition of EtOH (600 ml, dried over molecular sieve 3A for 48 h). A reflux condenser was attached to the reaction flask, the reaction mixture was purged with N ₂ and kept at 90 ° C for 9 h. The reaction mixture was cooled to room temperature and left for 30 h, during which the product crystallized as brown needles. The solids were broken up with a spatula and the reaction mixture was transferred to a 2 L flask. Water (1.4 L) was added slowly through a separatory funnel with vigorous stirring. After completing the addition of water, the slurry was stirred for 30 minutes. The brown needles were isolated by filtration and then dried by pulling air through the filter for 1 hour. The product was transferred to a 500 ml flask and treated with EtOAc (200 ml). A small amount of seed crystals were added, which induced the formation of a white solid precipitate. The suspension was stirred for 30 min at room temperature, filtered, washed with EtOAc (25 ml) and dried in vacuo to give the product as a white solid (48.65 g, 68% yield). MS (ESI): mass calculated for C ₂₆ H ₂₇ N ₅ O ₄ S, 505.2; obtained m / z, 506.2 [M + H] ⁺ .

1H NMR (400 MHz, CDCl ₃ ) δ 8.85 (s, 1H), 8.28-8.19 (m, 2H), 7.84 (d, J = 4.0 Hz, 1H), 7, 61-7.53 (m, 1H), 7.52-7.43 (m, 2H), 6.84 (d, J = 4.1 Hz, 1H), 4.32 (s, 1H), 4 , 20 (q, J = 7.1 Hz, 2H), 4.09 (s, 2H), 2.44 (d, J = 6.2 Hz, 2H), 2.40-2.27 (m, 2H), 2.16 (d, J = 13.3 Hz, 2H), 2.12-1.96 (m, 3H), 1.54-1.38 (m, 2H), 1.27 (t , J = 7.1 Hz, 3H).

Synthesis and description of the properties of intermediate compound 4: sodium 2- (1 - ((1d, 4d) -4 (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridine- 2-yl) acetate

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To a solution of ethyl 2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) acetate (intermediate 3, 9.50 g, 18.8 mmol) in MeOH (30 ml) and THF (30 ml) was added an aqueous solution of sodium hydroxide (56.4 ml, 56.4 mmol, 1M) and stirred at room temperature for 14 hours. The solvent was removed under reduced pressure at room temperature to give the title compound (7 g) as a brown solid which was used in the next step without further purification. MS (ESI): mass calculated for

C ₁₈ H ₁₈ N ₅ NaO ₂ , 359.1; m / z obtained, 337.9 [M + H-Na] +.

¹ H NMR (400 MHz, CD3OD) δ 8.52-8.47 (m, 1H), 7.85-7.81 (m, 2H), 7.46-7.41 (m, 4H), 6 , 85-6.81 (m, 1H), 4.60-4.46 (m, 1H), 3.96 (s, 2H), 2.59-2.49 (m, 4H), 2.19 -2.05 (m, 6H), 1.56-1.43 (m, 2H) (1: 1 mixture of the title compound and benzenesulfonic acid).

Synthesis and description of the properties of example 1: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl ) -N- (2-hydroxy-2-methylpropyl) acetamide

Stage A: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridine-2 -yl) -N- (2-hydroxy-2-methylpropyl) acetamide

To obtain a dry starting material, ethyl 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6 (phenylsulfonyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3- b] pyridin-2-yl) acetate (intermediate compound 3) before the reaction was kept in a vacuum at 50 ° C for 18 h. In a 1 L flask ethyl2- (1 - ((1 r, 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 mixture was kept at 110 ° C for 45 min and then at 125 ° C for 5 h. The reaction mixture was cooled to room temperature and diluted with EtOAc (800 ml). The organic layer was extracted three times with water / brine solution, the solution consisted of 1 L of water and 50 ml of brine. The aqueous layers were again extracted with EtOAc (2x600 ml). The combined organic layers were dried over anhydrous MgSO ₄ , concentrated to dryness, and then dried for 3 days in vacuo to afford the title compound (65.9 g, 98% yield) as a yellow foam. The product was used in the next step without further purification. MS (ESI): mass calculated for C ₂₈ H ₃₂ N ₆ O ₄ S, 548.22; m / z obtained, 549.2 [M + H] ⁺ .

¹ H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H), 8.26-8.19 (m, 2H), 7.84 (d, J = 4.1 Hz, 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 = 5.9 Hz, 2H), 2.41 (d, J = 6.5 Hz , 2H), 2.38-2.25 (m, 2H), 2.23-2.12 (m, 2H), 2.09-1.94 (m, 4H), 1.48 (cd, J = 13.6, 4.0 Hz, 2H), 1.21 (s, 6H).

Stage B: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) -N- (2-hydroxy-2-methylpropyl) acetamide

2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyridine-2- yl) -N- (2-hydroxy-2-methylpropyl) acetamide (65.90 g, 102.1 mmol) was added to a 1 L flask containing a magnetic armature. 1,4-dioxane (300 ml) was added followed by aq. KOH (3M, 150 ml). The reaction mixture was kept at 80 ° C for 2 hours. The reaction mixture was cooled to room temperature and the volume of the solvent was reduced to about 200 ml on a rotary evaporator. The residue was treated with water / brine solution (100 ml / 100 ml) and subsequently extracted with 10% MeOH in CH ₂ Cl ₂ (2x1 L). The combined layers were dried over anhydrous MgSO ₄ and concentrated to dryness to give a yellow solid. The solid was suspended in CH2Cl2 (200 ml), stirred vigorously for 30 min, and then collected by filtration. The solid was washed with CH ₂ Cl ₂ (100 ml), dried by pulling air through the filter and subsequently dried further in vacuo at room temperature for 16 h to give the title compound (41.59 g, 89% yield) as white solid. MS (ESI): mass calculated for C ₂₂ H ₂₈ N ₆ O ₂ , 408.23; m / z obtained, 409.2 [M + H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ ): δ 11.85 (s, 1H), 8.50 (s, 1H), 8.21-8.10 (m, 1H), 7.49-7 , 43 (m, 1H), 6.746.65 (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),

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2.09-1.92 (m, 5H), 1.42-1.31 (m, 2H), 1.09 (s, 6H).

The synthesis and determination of the properties of the active compound for each of the embodiments of the present invention are presented herein by way of examples. Due to the crystal structure of some embodiments of the present invention, polymorphic screening can be performed to determine additional properties of specific forms of any such compound. This is shown as a non-limiting example for the compound Ex. 1 under the indication of polymorphic screening.

Synthesis and description of the properties of example 2: 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3b] pyridine-1 (6H) -yl) cyclohexyl) acetonitrile _HN ^ _N ⁿ io ^N H (ex. 2)

Stage A: 2 - ((1 r, 4r) -4- (2- (1 H-imidazol-4-yl) -6- (phenylsulfonyl) imidazo [4,5-d] pyrrolo [2,3b] pyridine- 1 (6H) -yl) cyclohexyl) acetonitrile.

2 - ((1r, 4r) -4 - ((5-nitro-1- (phenylsulfonyl) -III-pyrrolo [2,3-b] pyridin-4-yl) amino) cyclohexyl) acetonitrile (intermediate 1, 23 , 3 g, 53.0 mmol) was added to a 1 L round bottom flask containing a magnetic armature followed by the addition of DMSO (200 mL) and methanol (200 mL). 1Himidazole-4-carbaldehyde (8.56 g, 89.1 mmol) was added as a solid followed by sodium hydrogen sulfite (32.7 g, 188 mmol) as a solution in water (100 mL). The reaction vessel was equipped with a reflux condenser and kept at 90 ° C in a heating block for 15 hours. Subsequently, the reaction mixture was cooled to room temperature and added to a flask containing water (2000 ml) with stirring, resulting in the formation of a white precipitate. The mixture was stirred for 30 min and the solids were collected by filtration. The solids were dried by pulling air through the filter for 6 h and then further dried in a vacuum oven at 60 ° C for 3 days to afford the title compound (22.7 g, 88% yield) as a yellow solid. MS (ESI): mass calculated for C ₂₅ H ₂₃ N ₇ O ₂ S, 485.16; m / z obtained, 486.1 [M + H] ⁺ .

¹ H NMR (400 MHz, CDC ^): δ 8.74 (s, 1H), 8.29 (s, 1H), 8.20-8.11 (m, 2H), 8.04-7.96 (m, 2H), 7.76-7.68 (m, 1H), 7.68-7.60 (m, 2H), 7.19 (d, J = 4.2Hz, 1H), 5, 56 (s, 1H), 2.58 (d, J = 6.3 Hz, 2H), 2.38-2.24 (m, 2H), 2.07 (s, 1H), 1.98 (d , J = 10.8 Hz, 5H), 1.35 (q, J = 12.3 Hz, 2H).

Stage B: 2 - ((1 r, 4r) -4- (2- (1 H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3 F] pyridine-1 (6H) - yl) cyclohexyl) acetonitrile

The title compound was prepared using conditions similar to Example 1 (Step B) using 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) -6- (phenylsulfonyl) imidazo [4,5d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile (222 mg, 0.46 mmol) instead of 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, and the residue was purified by flash column chromatography (0-15% 2H NH ₃ -MeOH / EA) to afford the title compound (97 mg, 69% yield). MS (ESI): mass calculated for C ₁₉ H ₁₉ N ₇ , 345.17; m / z obtained, 346.0 [M + H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ): δ 12.61 (s, 1H), 11.86 (s, 1H), 8.55 (s, 1H), 7.92 (d, J = 1, 3 Hz, 1H), 7.83 (s, 1H), 7.48 (t, J = 3.0 Hz, 1H), 6.76 (dd, J = 3.5, 1.8 Hz, 1H) , 5.85 (s, 1H), 2.60 (d, J = 6.0 Hz, 2H), 2.57-2.41 (m, 2H), 2.14-1.88 (m, 5H ), 1.45-1.27 (m, 2H).

Synthesis and description of the properties of example 3: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl ) -N- (cyclopropylmethyl) acetamide

Stage A: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridine-2 -yl) -N- (cyclopropylmethyl) acetamide

A mixture of ethyl 2- (1 - ((1 r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridine- 2-yl) acetate (intermediate 3, 555 mg, 1.06 mmol) and cyclopropylmethylamine (1.87 ml, 21.1 mmol) were kept at 125 ° C for 1 h in a microwave reactor. The residue was taken up in water and then extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, passed through a pad of silica gel and concentrated to dryness with a rotary evaporator to give the title compound (642 mg). MS (ESI): mass calculated for C ₂₈ H ₃₀ N ₆ O ₃ S, 530.21; obtained m / z, 531.2 [M + H] ⁺ .

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1H NMR (400 MHz, CD3OD): δ 8.64 (s, 1H), 8.19-8.11 (m, 2H), 7.95 (d, J = 4.1Hz, 1H), 7, 66-7.57 (m, 1H),

7.57-7.48 (m, 2H), 7.11 (d, J = 4.1 Hz, 1H), 4.53 (s, 1H), 4.08 (s, 2H), 3.07 (d, J = 7.1 Hz, 2H), 2.53 (d, J = 5.9 Hz,

2H), 2.45-2.30 (m, 2H), 2.14-2.03 (m, 5H), 1.55-1.42 (m, 2H), 1.02-0.94 ( m, 1H), 0.54-0.47 (m, 2H), 0.24-0.18 (m, 2H).

Stage B: 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-0] pyrrolo [2,3b] pyridin-2-yl) -N - (cyclopropylmethyl) acetamide

To a mixture of 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridine-2- yl) -N- (cyclopropylmethyl) acetamide (560 mg, 1.05 mmol) in 1,4-dioxane (4.22 ml) was added 3 N. KOH (2.81 ml). The mixture was kept at 80 ° C for 1 h, then purified using alkaline HPLC: Xbridge Prep OBD C ₁₈ 50x100 mm, 5 μm column (eluent 0-100% aqueous NH4OH / ACN solution (10 min)) to obtain the specified title compound (187 mg, 46% yield). MS (ESI): mass calculated for C ₂₂ H ₂₆ N ₆ O, 390.22; m / z obtained, 391.2 [M + H] +.

1H NMR (400 MHz, CD3OD): δ 8.57 (s, 1H), 7.50 (d, J = 3.5 Hz, 1H), 6.86 (d, J = 3.5 Hz, 1H) , 4.57 (s, 1H), 4.11 (s, 2H), 3.13 (d, J = 7.0 Hz, 2H), 2.67-2.52 (m, 4H), 2, 20-2.03 (m, 5H), 1.58-1.44 (m, 2H), 1.12-0.97 (m, 1H), 0.60-0.48 (m, 2H), 0.31-0.22 (m, 2H).

Synthesis and description of the properties of example 4:

N- (2-cyanoethyl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3b] pyridine-2- yl) acetamide

To a solution of sodium 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) acetate (intermediate compound 4, 400 mg, 1.11 mmol) and 3-aminopropanenitrile (320 mg, 2.24 mmol) in DMF (5 ml), PyBOP (870 mg, 1.67 mmol) and DIPEA (0.60 ml, 3.5 mmol). The reaction mixture was stirred at room temperature for 40 hours. After removal of DMF in vacuo, the residue was purified by flash column chromatography using 50-100% ethyl acetate in heptane. The collected fractions were concentrated in vacuo to low volume and the white solid that precipitated was filtered, washed with 10% MeOH in CH ₂ Cl ₂ and dried to give the title compound (75 mg, 17% yield). The filtrate was concentrated to dryness and purified by reverse phase HPLC using a Varian Pursuit XR _s 5 Diphenyl 100x30 mm column (eluent 10-90% CH ₃ CN in water, 0.1% TFA) to give a clear oil. The material was dissolved in 10% MeOH in CH ₂ Cl ₂ , passed through three columns of 500 mg SILICYCLE SPER66030B-03P Carbonate (cartridge for solid phase extraction and absorption of SiliaBond acids) to remove TFA, and eluted with 10% MeOH in CH ₂ Cl ₂ to obtain additional fractions of the title compound (88 mg, 20% yield). The two fractions were combined to give the final product (163 mg, 37% yield) as a white solid. MS (ESI): mass calculated for C ₂₁ H ₂₃ N ₇ O, 389.20; m / z obtained, 390.3 [M + H] ⁺ .

1H NMR (400 MHz, CD3OD): δ 8.53 (s, 1H), 7.48 (d, J = 3.0 Hz, 1H), 6.84 (d, J = 3.5 Hz, 1H) , 4.51 (br s, 1H), 4.12 (s, 2H), 3.50 (t, J = 6.6 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 2.45-2.63 (m, 4H), 2.03-2.19 (m, 5H), 1.441.57 (m, 2H).

Synthesis and description of the properties of example 5: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl ) -N- (tetrahydro-2H-pyran-4-yl) acetamide

A mixture of ethyl 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridine-2 -yl) acetate (intermediate 3, 309 mg, 0.61 mmol) and 4-aminotetrahydropyran (195 mg, 1.93 mmol) in 1,4-dioxane (0.5 ml) were heated in a microwave reactor at 180 ° C for 1 hour Then the reaction mixture was diluted with 1,4-dioxane (1.5 ml), treated with aq. 3 n. KOH (2 ml) and kept at 80 ° C for 1.5 h. Subsequently, the residue was treated with water (10 ml) and extracted with CH ₂ Cl ₂ (3x50 ml). The organic layers were combined, dried with MgSO ₄ and concentrated in vacuo. The crude material was purified by flash column chromatography (5-10% MeOH / CH ₂ Cl ₂ ) to afford the title compound (146 mg, 57% yield). MS (ESI): mass calculated for C ₂₃ H ₂₈ N ₆ O ₂ , 420.23; m / z obtained, 421.2 [M + H] ⁺ .

1H NMR (600 MHz, DMSO-d6): δ 11.84 (s, 1H), 8.49 (s, 1H), 8.36 (d, J = 7.5 Hz, 1H), 7.46 ( t, J = 3.0 Hz, 1H), 6.73-6.67 (m, 1H), 4.54-4.41 (m, 1H), 3.98 (s, 2H), 3.86 -3.81 (m, 2H), 3.81-3.74 (m, 1H), 3.40-3.34 (m, 2H), 2.60 (d, J = 6.0 Hz, 2H ), 2.41-2.29 (m, 2H), 2.10-2.01 (m, 1H), 2.00-1.93 (m, 4H), 1.77-1.70 (m , 2H), 1.49-1.33 (m, 4H).

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Synthesis and description of the properties of example 6: 2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl ) -N - ((tetrahydro-2H-pyran-4-yl) methyl) acetamide

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, 300 mg, 0.593 mmol) and 4-aminomethyltetrahydropyran (683 mg, 5.93 mmol). The resulting solution was stirred at 125 ° C for 1 h. Then dioxane (2.37 ml) and KOH (3 M in water, 1.58 ml, 4.75 mmol) were added and the reaction mixture was stirred in a microwave oven at 80 ° C for 1 h. The reaction mixture was purified by alkaline HPLC using a Waters Xbridge Prep OBD C ₁₈ 150x30 mm, 5 μm column (eluent 0-100% water (0.05% NH ₄ OH) / ACN (10 min)) with obtaining the title compound (97 mg, 38% yield). MS (ESI): mass calculated for C24H30N6O2, 434.24; m / z obtained, 435.2 [M + H] ⁺ .

1H NMR (500 MHz, CD3OD): δ 8.43 (s, 1H), 7.38 (d, J = 3.5 Hz, 1H), 6.74 (d, J = 3.5 Hz, 1H) , 4.43 (s, 1H), 4.03-3.96 (m, 2H), 3.89-3.79 (m, 2H), 3.34-3.25 (m, 2H), 3 , 04 (d, J = 6.8 Hz, 2H), 2.54-2.38 (m, 4H), 2.08-1.96 (m, 5H), 1.74-1.63 (m , 1H), 1.59-1.54 (m, 2H), 1.46-1.33 (m, 2H), 1.25-1.14 (m, 2H).

Synthesis and description of the properties of example 7: N- (2-cyano-2-methylpropyl) -2- (1 - ((1r, 4r) -4 (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) acetamide

A solution of sodium 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) - 1,6-dihydroimidazo [4.5-0] pyrrolo [2,3b] pyridin-2-yl) acetate (intermediate compound 4, 300 mg, 0.835 mmol), 3-amino-2,2 dimethylpropanenitrile (82.0 mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol) and DMF (5 ml) were stirred at 0 ° C for 1 h. Then PyBrOP (467 mg, 1.00 mmol) was added and the reaction mixture was stirred overnight at room temperature. The mixture was quenched with 10 ml water and purified by preparative HPLC using a Waters Xbridge Prep OBD C ₁₈ 150x30 mm 5 μm column (eluent: 28% water (0.05% ammonium hydroxide v / v) - ACN to give the title compound (58 mg, 16% yield) as a white solid MS (ESI): mass calculated for C ₂₃ H ₂₇ N ₇ O, 417.23; obtained m / z, 418.2 [M + H] +.

1H NMR (400 MHz, DMSO-d6): δ 11.86 (br s, 1H), 8.71-8.66 (m, 1H), 8.50 (s, 1H), 7.48-7 , 45 (m, 1H), 6.73-6.69 (m, 1H), 4.53-4.42 (m, 1H), 4.10 (s, 2H), 3.33-3.31 (m, 1H), 2.57 (d, J = 5.6Hz, 2H), 2.41-2.27 (m, 2H), 2.05-1.93 (m, 5H), 1, 45-1.26 (m, 9H).

Synthesis and description of the properties of example 8: 2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl ) -N - ((1-hydroxycyclobutyl) methyl) acetamide

A solution of sodium 2- (1 - ((1 r, 4r) -4- (cyanomethyl) cyclohexyl) - 1,6-dihydroimidazo [4,5-d] pyrrolo [2,3 b] pyridin-2-yl) acetate ( intermediate 4, 300 mg, 0.835 mmol), 1 (aminomethyl) cyclobutanol (84.4 mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol) and DMF (5 ml) were stirred at 0 ° C for 1 h. Then PyBrOP (467 mg, 1.00 mmol) was added and the reaction mixture was stirred overnight at room temperature, after which it was quenched with 10 ml of water. The reaction mixture was purified by preparative alkaline HPLC using a Kromasil 150x25 mm, 10 μm column (eluent: water (0.05% ammonium hydroxide v / v) - ACN 9% to 39%, v / v) to give the title compound (90 mg, 26% yield) as a white solid. MS (ESI): mass calculated for C ₂₃ H ₂₈ N ₆ O ₂ , 420.23; m / z obtained, 421.2 [M + H] ⁺ .

1H NMR (400 MHz, DMSO-d6): δ 11.58 (br s, 1H), 8.50 (s, 1H), 7.94-7.85 (m, 1H), 7.46-7 , 40 (m, 1H), 6.75-6.70 (m, 1H), 4.89 (br s, 1H), 4.57-4.47 (m, 1H), 4.04 (s , 2H), 3.27 (d, J = 6.0 Hz, 2H), 2.55 (d, J = 6.0 Hz, 2H), 2.45-2.31 (m, 2H), 2 , 10-1.88 (m, 9H), 1.71-1.60 (m, 1H), 1.54-1.35 (m, 3H).

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Synthesis and description of the properties of example 9: 2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3 -b] pyridin-2-yl ) -N- (1-methyl-1 H-pyrazol-4-yl) acetamide ° / L ^N А Υχ5 ^Ν Η (ex. 9)

To a solution of sodium 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) acetate (intermediate compound 4, 100 mg, 0.278 mmol) and 1-methyl-1H-pyrazole-4amine (54.0 mg, 0.557 mmol) in DMF (0.8 ml) were added PyBrOP (217 mg, 0.417 mmol) and DIPEA (0.144 ml , 0.835 mmol) and the mixture was stirred at room temperature overnight. DMF was removed under reduced pressure and the residue was purified by flash column chromatography (50-100% EtOAc / heptanes, then 10% MeOH / DCM) followed by reverse phase HPLC using a Varian Pursuit XR _s 5 Diphenyl 100x30 mm column (eluent 10-90% CH ₃ CN in water, 0.1% TFA) to give the product as a TFA salt. This material was dissolved in 10% MeOH in CH ₂ Cl ₂ and passed through a 500 mg SILICYCLE SPE-R66030B-03P Carbonate column (SiliaBond solid phase extraction and acid uptake cartridge) to remove the TFA to give the title compound (34 mg, yield 29%) as a white solid. MS (ESI): mass calculated for C ₂₂ H ₂₄ N ₈ O, 416.21; m / z obtained, 417.3 [M + H] ⁺ .

1H NMR (400 MHz, CDCl ₃ ) δ = 12.77 (s br, 1H), 11.24 (s br, 1H), 7.94 (s, 1H), 7.89 (s br, 1H), 7.46 (s, 1H), 7.29-7.20 (m, 1H), 6.74 (s br, 1H), 4.85-4.65 (m, 1H), 4 , 23 (s, 2H), 3.85 (s, 3H), 2.80-2.55 (m, 1H), 2.45 (d, J = 6.6 Hz, 2H), 2.32- 1.98 (m, 6H), 1.62-1.44 (m, 2H).

Synthesis and description of the properties of example 10: N- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1 - ((1r, 4r) 4- (cyanomethyl) cyclohexyl) -1.6 -dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) acetamide / - = ^N

HN \ Y

N A oe n (pr. 10)

Stage A: dimethylcyclopentane-1,3-dicarboxylate

A solution of cyclopentane-1,3-dicarboxylic acid (70.0 g, 443 mmol) and anhydrous methanol (300 ml) was cooled to 0 ° C in an ice water bath. Concentrated sulfuric acid (14 ml) was added dropwise keeping the temperature <15 ° C. After the addition, the reaction mixture was heated to 90 ° C and stirred overnight. The resulting reaction mixture was cooled to room temperature and concentrated to dryness. The residue was treated with MTBE (500 ml) and H2O (100 ml). The aqueous layer was separated and extracted with MTBE (2x100 ml). The combined organic extracts were washed with saturated sodium bicarbonate solution (2x100 ml), brine (100 ml), dried with anhydrous MgSO ₄ , filtered and concentrated to dryness to give the title compound (72.5 g, 88%) as a pale yellow oil ...

1H NMR (400 MHz, CDCl ₃ ) δ 3.65 (s, 6H), 2.84-2.72 (m, 2H), 2.26-2.17 (m, 1H), 2.11-2 02 (m, 1H), 1.961.88 (m, 4H).

Stage B: dimethylbicyclo [2.2.1] heptane-1,4-dicarboxylate n-butyllithium (2.5 M in hexane, 419.0 ml, 1048 mmol) was slowly added to a solution of diisopropylamine (152 ml, 1090 mmol) and anhydrous THF (1000 ml) at -78 ° C (dry ice / acetone) under N2. Thereafter, the reaction mixture was stirred for 0.5 h at 0 ° C, and then cooled to -78 ° C. DMPU (404 mL, 3350 mmol) was added via an addition funnel. Subsequently, a solution of dimethylcyclopentane-1,3-dicarboxylate (78.0 g, 419 mmol) and anhydrous THF (300 ml) was slowly added via an addition funnel. The reaction mixture was warmed to 0 ° C and stirred for 30 min, then cooled to -78 ° C and treated with a solution of 1-bromo-2-chloroethane (59.0 ml, 712 mmol) and anhydrous THF (200 ml). The reaction mixture was allowed to slowly warm to room temperature and stirred for 12 hours at room temperature. Subsequently, the reaction mixture was quenched with saturated aqueous ammonium chloride solution (400 ml). The reaction mixture was diluted with ethyl acetate (500 ml), the organic layer was separated and the aqueous layer was additionally extracted with ethyl acetate (2x500 ml). The combined organic extracts were washed with brine (2x300 ml), dried over anhydrous MgSO ₄ , filtered and concentrated to dryness. The residue was filtered through a pad of silica gel and washed with ethyl acetate (2000 ml). The filtrate was concentrated to dryness and the residue purified by flash column chromatography (petroleum ether / ethyl acetate, gradient elution 30: 1 to 20: 1) to afford the title compound (48.5 g, 54%) as a white solid.

1H NMR (400 MHz, CDCl ₃ ): δ 3.69 (s, 6H), 2.08-1.99 (m, 4H), 1.91 (s, 2H), 1.73-1.63 ( m, 4H).

Step C: 4- (methoxycarbonyl) bicyclo [2.2.1] heptane-1-carboxylic acid.

- 26 037347

A solution of sodium hydroxide (5.145 g, 128.6 mmol) in methanol (80 ml) was slowly added to a solution of dimethylbicyclo [2.2.1] heptane-1,4-dicarboxylate (27.3 g, 129 mmol) and THF (700 ml) at 0 ° C and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness and the residue was triturated with MTBE (15 ml). The precipitate was collected by filtration, washed with MTBE (5 ml) and dissolved in 100 ml H ₂ O. The solution was acidified with 2M HCl until pH = 4. The precipitate was collected by filtration and dried in vacuo to afford the title compound (13.0 g, 51.0%) as a white solid. The filtrate was extracted with ethyl acetate (3x75 ml) and the combined organic extracts were washed with brine (50 ml), dried over anhydrous MgSO ₄ , filtered and concentrated to dryness to give the second fraction of the title compound (8.0 g, 31%) as a white solid. substances.

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.21 (br s, 1H), 3.59 (s, 3H), 1.94-1.86 (m, 4H), 1.74 (s , 2H), 1.611.54 (m, 4H).

Stage D: methyl 4 - (((benzyloxy) carbonyl) amino) bicyclo [2.2.1] heptane-1 -carboxylate

Diphenylphosphoryl azide (17.1 ml, 78.6 mmol) was added to a solution of 4- (methoxycarbonyl) bicyclo [2.2.1] -heptane-1-carboxylic acid (13.0 g, 65.6 mmol), DIPEA (22.8 ml, 131 mmol) and anhydrous toluene (200 ml) and the reaction mixture was stirred at 110 ° C for 2 h.The reaction mixture was cooled to 50 ° C and benzyl alcohol (13.6 ml, 131 mmol) was added and the reaction mixture was stirred at a temperature of 110 ° C during the night. The reaction mixture was concentrated to dryness, dissolved in MTBE (250 ml) and washed with H ₂ O (150 ml). The organic layer was separated and the aqueous layer was extracted with MTBE (2x100 ml). The combined organic extracts were washed with brine (100 ml), dried with anhydrous MgSO ₄ , filtered and concentrated to dryness. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate, gradient elution 10: 1 to 5: 1) to give the crude product (28.5 g) as a pale yellow oil. The product was further purified by preparative acidic HPLC using a Phenomenex Synergi Max-RP 250x50 mm x 10 μm column (eluent: 38 to 68% (v / v) CH ₃ CN and H2O with 0.1% TFA). The pure fractions were pooled and the volatiles were removed in vacuo. The residue was diluted with H ₂ O (80 ml), the pH of the solution was adjusted to pH = 8 with saturated aqueous NaHCO 3 and the resulting solution was extracted with CH ₂ Cl ₂ (3x100 ml). The combined organic extracts were washed with brine (75 ml), dried over anhydrous MgSO4, filtered and concentrated to dryness to give the title compound (17.3 g, 85%) as a colorless oil. MS (ESI): mass calculated for C ₁₇ H ₂₁ NO ₄ , 303.2; m / z obtained, 303.9 [M + H] +.

1H NMR (400 MHz, DMSO-d ₆ ) δ 7.56 (br s, 1H), 7.37-7.30 (m, 5H), 4.97 (s, 2H), 3.58 (s , 3H), 1.90-1.80 (m, 6H), 1.65-1.59 (m, 4H).

Stage E: methyl 4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptane-1-carboxylate

A mixture of methyl 4 - (((benzyloxy) carbonyl) amino) bicyclo [2.2.1] heptane-1-carboxylate (17 g, 56 mmol), di-tert-butyl dicarbonate (18.35 g, 84.06 mmol), MeOH (200 ml) and wet Pd / C (4 g, 10 wt%, 50% H ₂ O) were added to a 500 ml round bottom flask with a hydrogen balloon (13 psi) and stirred at room temperature for 72 h. The catalyst was filtered and the filtrate was concentrated to dryness. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate, gradient elution 20: 1 to 1: 1) to afford the title compound (12.0 g, 79.5%) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 7.04 (br s, 1H), 3.57 (s, 3H), 1.93-1.78 (m, 4H), 1.77 (s , 2H), 1.63-1.53 (m, 4H), 1.35 (s, 9H).

Stage F: 4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptane-1-carboxylic acid

To a solution of methyl 4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptane-1-carboxylate (5.0 g, 19 mmol), THF (40 ml) and MeOH (20 ml) was added aqueous sodium hydroxide (1.0M, 46.4 ml, 46.4 mmol) at room temperature and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was concentrated to dryness and the residue was diluted with H ₂ O (20 ml), acidified to pH = 4 -5 with 2M HCl to give a precipitate. The precipitate was dissolved in 150 ml ethyl acetate, washed with brine (45 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness to give the title compound (4.74 g, 100% yield) as a white solid which was used directly to the next stage.

1H NMR (400 MHz, DMSO-d6) δ 12.06 (s br, 1H), 7.00 (s br, 1H), 1.87-1.73 (m, 6H), 1.58- 1.50 (m, 4H), 1.35 (s, 9H).

Stage G: tert-butyl- (4- (hydroxymethyl) bicyclo [2.2.1] -heptane-1-yl) carbamate

A solution of borane-tetrahydrofuran complex (1.0 M, 37.1 ml, 37.1 mmol) was slowly added to a solution of 4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptane-1-carboxylic acid (4.74 d, 18.6 mmol) and anhydrous THF (50 ml) at 0 ° C under nitrogen atmosphere. After the addition was complete, the reaction mixture was stirred at room temperature overnight. Water (30 ml) was added slowly to the mixture and stirred for an additional 30 minutes. The reaction mixture was concentrated to dryness, the residue was diluted with ethyl acetate (50 ml), washed with H ₂ O (15 ml) and brine (10 ml), dried with anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate 2: 1) to afford the title compound (4.0 g, 89% yield) as a white solid. TLC (petroleum ether / ethyl acetate, 2: 1) Rf = 0.5.

1H NMR (400 MHz, DMSO-d6): 6.88 (br.s, 1H), 4.38 (t, J = 5.4 Hz, 1H), 3.36 (d, J = 5.4 Hz , 2H), 1.73 (s, 2H), 1.64-1.49 (m, 4H), 1.42 (s, 2H), 1.39-1.33 (m, 9H), 1.25-1.16 (m, 2H).

Step H: (4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptan-1-yl) methyl methanesulfonate.

Pyridine (2.7 ml, 33 mmol) was added to a solution of tert-butyl- (4- (hydroxymethyl) bicyclo [2.2.1] heptan-1-yl) carbamate (2.0 g, 8.3 mmol) and anhydrous CH ₂ Cl ₂ (30 ml). The reaction mixture was cooled to 0 ° C and methanesulfonyl chloride (2.0 ml, 25.0 mmol) was added and the mixture was stirred for 3 h at room temperature. The reaction mixture was diluted with CH2Cl2 (50 ml) and water (30 ml). The organic layer was separated, washed with brine (15 ml), dried over MgSO ₄ , filtered and concentrated to dryness. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate, gradient elution 5: 1 to 1: 1) to afford the title compound (2.58 g, 97%) as a white solid. TLC (petroleum ether / ethyl acetate, 1: 1) Rf = 0.85.

¹ H NMR (400 MHz, CDCb) 4.73 (s br, 1H), 4.21 (s, 2H), 2.98 (s, 3H), 1.93-1.90 (m, 2H) , 1.78-1.62 (m, 6H), 1.53-1.34 (m, 11H).

Stage I: tert-butyl- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) carbamate.

To a solution of (4 - ((tert-butoxycarbonyl) amino) bicyclo [2.2.1] heptan-1-yl) methyl methanesulfonate (2.58 g, 8.07 mmol) and DMSO (25 ml) was added sodium cyanide (1.20 d, 24.5 mmol). The reaction mixture was heated to 100 ° C and stirred for 24 hours. The reaction mixture was diluted with 50 ml of water and was extracted with ethyl acetate (3x40 ml). The combined organic extracts were washed with brine (20 ml), dried with anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate 5: 1) to afford the title compound (1.8 g, 89% yield) as a white solid.

¹ H NMR (400 MHz, DMSO-d6): δ 7.00 (br s, 1H), 2.67 (s, 2H), 1.82 (br s, 2H), 1.69-1, 52 (m, 6H), 1.47-1.40 (m, 2H), 1.37 (s, 9H).

Step J: 2- (4-Aminobicyclo [2.2.1] heptan-1-yl) acetonitrile hydrochloride.

To a suspension of tert-butyl- (4- (cyanomethyl) bicyclo [2.2.1] -heptan-1-yl) carbamate (850 mg, 3.40 mmol) and ethyl acetate (2 ml) at 0 ° C was added a solution of HCl in ethyl acetate (4.0 M, 10 ml, 40 mmol). After stirring at room temperature for 2 hours, the mixture was concentrated under reduced pressure to dryness. The residue was triturated with MTBE (5 ml) and the suspension was separated by filtration. The filter cake was washed with MTBE (1 ml) and dried under reduced pressure to give the title compound (450 mg, 71%) as a white solid. MS (ESI): mass calculated for C ₉ H ₁₄ N ₂ 150.12, obtained m / z, 151.1 [M + H] +.

1H NMR (400 MHz, DMSO-d6) δ 8.59 (br s, 3H), 2.78 (s, 2H), 1.90-1.77 (m, 2H), 1.74-1, 62 (m, 4H), 1.60 (s, 2H), 1.56-1.46 (m, 2H).

Stage K: N- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) 1,6-dihydroimidazo [4 , 5-d] pyrrolo [2,3-b] pyridin-2-yl) acetamide.

To a solution of sodium 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) acetate (intermediate compound 4, 300 mg, 0.835 mmol), 2- (4aminobicyclo [2.2.1] heptan-1-yl) acetonitrile hydrochloride (138 mg, 0.918 mmol) and DIPEA (0.2 91 ml, 1.67 mmol) in dry DMF (6 ml) was added PyBrOP (428 mg, 0.918 mmol) at 0 ° C. The reaction mixture was stirred at room temperature for 12 hours. The mixture was quenched with 10 ml of water and extracted with EtOAc (3x20 ml). The combined organic phases were washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to dryness. The residue was purified by preparative HPLC (using Xtimate C ₁₈ column 150x25 mm x5 μm (eluent: 23 to 33% (v / v) CH3CN and H2O with 10 mM NH4HCO3) and preparative TLC (dichloromethane: methanol = 15: 1 ) to give the title compound (36.6 mg, 9% yield) as a white solid MS (ESI): mass calculated for C ₂₇ H ₃₁ N ₇ O 469.3; obtained m / z, 470 , 2 [M + H] +.

¹ H NMR (400 MHz, CD3OD): δ 8.53 (s, 1H), 7.48 (d, J = 4.0 Hz, 1H), 6.84 (d, J = 4.0 Hz, 1H ), 4.47 (br s, 1H), 4.07-4.02 (m, 2H), 2.63 (s, 2H), 2.61-2.50 (m, 4H), 2, 18-1.98 (m, 7H), 1.94-1.84 (m, 2H), 1.82 (s, 2H), 1.791.68 (m, 2H), 1.62-1.43 ( m, 4H).

Synthesis and description of the properties of example 11: 2- (1 - ((1 r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridine-2- yl) -N- (1 H-pyrazol-3-yl) acetamide

Stage A: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4.5-28 037347

d] pyrrolo [2,3-b] pyridin-2-yl) -N- (1H-pyrazol-3-yl) acetamide.

The title compound (383 mg, 70%) was prepared in a similar manner to Example 1 (Step A) using 1H-pyrazol-3-amine (465 mg, 5.48 mmol) instead of 1-amino-2methylpropane-2 -ola. MS (ESI): mass calculated for C ₂₇ H ₂₆ N ₈ O ₃ S, 542.2; m / z obtained 543.2

[M + H] ⁺ .

1H NMR (500 MHz, DMSO-d6) δ 12.36 (s, 1H), 11.36 (s, 1H), 10.81 (s, 1H), 8.65 (s, 1H), 8.14 -8.10 (m, 2H), 7.96 (d, J = 4.1 Hz, 1H), 7.72-7.68 (m, 1H), 7.63-7.60 (m, 2H ), 7.30 (s, 1H), 7.13 (d, J = 4.2 Hz, 1H), 6.446.41 (m, 1H), 5.41 (s, 1H), 4.57 (s , 1H), 4.44 (s, 2H), 4.24 (s, 2H), 2.56 (d, J = 6.2 Hz, 2H), 2.23-2.13 (m, 2H) , 2.02-1.95 (m, 1H), 1.41-1.32 (m, 2H).

Stage B: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) - N- (1 H-pyrazol-3-yl) acetamide.

The title compound was prepared in a similar manner to Example 1 (Step B) using 2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6 dihydroimidazo [4, 5-d] pyrrolo [2,3-b] pyridin-2-yl) -N- (1H-pyrazol-3-yl) acetamide (270 mg, 0.500 mmol) instead of 2- (1 - ((1g, 4d) -4- (cyanomethyl) cyclohexyl) -6- (phenylsulfonyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridin-2-yl) -N- (2-hydroxy-2-methylpropyl ) acetamide and purified by alkaline HPLC using Xbridge Prep OBD C ₁₈ 150x30 mm, 5 μm column, eluent 5% ACN / NH4OH (aq) (10 min) to afford the title compound (15 mg, 7%). MS (ESI): mass calculated for C ₂₁ H ₂₂ N ₈ O, 402.5; m / z obtained, 403.2 [M + H] ⁺ .

1H NMR (500 MHz, DMSO-d6): δ 12.35 (s, 1H), 11.85 (s, 1H), 10.84 (s, 1H), 8.50 (s, 1H), 7, 58 (d, J = 2.3 Hz, 1H), 7.46 (d, J = 3.4 Hz, 1H), 6.72 (d, J = 3.5 Hz, 1H), 6.47- 6.39 (m, 1H), 4.64-4.46 (m, 1H), 4.21 (s, 2H), 2.57 (d, J = 6.1 Hz, 2H), 2.45 -2.26 (m, 2H), 2.08-1.88 (m, 5H), 1.39 (q, J = 11.7 Hz, 2H).

Synthesis and description of the properties of example 12: 2- (1 - ((1 r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6 dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridine-2- yl) -N - ((1-hydroxycyclopropyl) methyl) acetamide

Sodium 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridin-2-yl) acetate solution (intermediate 4, 300 mg, 0.835 mmol), 1 (aminomethyl) cyclopropanol (72.7 mg, 0.835 mmol), DIPEA (216 mg, 1.67 mmol) and DMF (5 ml) were stirred at 0 ° C for 1 h. Subsequently, PyBrOP (467 mg, 1.00 mmol) was added and the reaction mixture was stirred overnight at room temperature. The mixture was quenched with 10 ml of water and purified by preparative alkaline HPLC using a Kromasil 150x25 mm column, 10 μm (eluent: water (0.05% ammonium hydroxide v / v) - ACN 5 to 35%, v / v. ) to afford the title compound (84 mg, 25% yield) as a white solid. MS (ESI): mass calculated for C ₂₂ H ₂₆ N ₆ O ₂ , 406.21; m / z obtained, 407.2 [M + H] ⁺ . 1H NMR (400 MHz, DMSO-d ₆ ): δ 11.53 (br s, 1H), 8.50 (s, 1H), 8.03-7.93 (m, 1H), 7.44- 7.40 (m, 1H), 6.74-6.71 (m, 1H), 5.04 (s, 1H), 4.58-4.49 (m, 1H), 4.02 (s, 2H), 3.29 (d, J = 6.0 Hz, 2H), 2.55 (d, J = 6.0 Hz, 2H), 2.45-2.31 (m, 2H), 2, 10-1.97 (m, 5H), 1.50-1.37 (m, 2H), 0.62-0.55 (m, 2H), 0.55-0.48 (m, 2H).

An example of polymorphic screening

Certain free base embodiments of the compounds of the present invention include a variety of crystal configurations that have complex solid-state characteristics, some of which may in turn have differing characteristics due to different amounts of solvent included. Some embodiments of compounds according to the present invention are in the form of pseudopolymers, which are embodiments of the same compound that have differences in crystal lattice structure due to different amounts of solvent in the crystal lattice itself. In addition, in some embodiments of the crystal structure of the compounds of the present invention, channel solvation may also occur, in which the solvent is incorporated into channels or voids present in the crystal lattice. For example, in table. 2 shows the various configurations of the crystal structure for the compound of ex. 1. Because of these features, non-stoichiometric solvates were often observed, as shown in table. 2. Moreover, the presence of such channels or voids in the crystal structure of some embodiments in accordance with the present invention provides water and / or solvent molecules that are retained within the crystal structure with varying degrees of bond strength. Therefore, changes in specific environmental conditions can easily lead to some decrease or increase in the number of water molecules and / or solvent molecules in some embodiments of the implementation in accordance with the present invention. It should be understood that solvation (third column in Table 2) for each of the embodiments listed in

- 29 037347 tab. 2, is a solvation by the formula and that the actual definition of it in the form of a stoichiometric number (fourth column in Table 2) may differ slightly from the solvation of the formula, depending on the actual environmental conditions in the experimental determination. For example, if about half of the water molecules in one embodiment can be present in the crystal lattice in a hydrogen bonded active state, and about the other half of the water molecules can be in channels or voids in the crystal lattice, then changes in environmental conditions can change the amount such unbound water molecules in voids or channels and, therefore, can lead to a small difference between solvation according to the formula, which is established in accordance with, for example, single crystal diffraction, and stoichiometry, which is determined, for example, by thermogravimetric analysis performed in conjunction with mass spectroscopy.

Table 2. Embodiments of the crystalline forms of the compound of ex. 1

Embodiment Crystallization solvent Solvation Stoichiometry 1s monohydrate 0.8 H ₂ O la Water monohydrate 1.3 H ₂ O lb Toluene Toluene solvate 0.4 toluene 1s Ethyl acetate / 1,4 dioxane monohydrate 1.1 H ₂ O Id Acetonitrile / Chloroform 1.7 hydrate 1.7 H ₂ O le Ethyl acetate / 1,4 dioxane monohydrate 1 H ₂ O If p-xylene p-xylene solvate 0.3 p-xylene If Kumen Kumen solvate 0.3 cumene 1 g Anisol Anisole solvate 0.3 anisole Ih p-xylene p-xylene solvate 0.2 p-xylene 2 1,4-dioxane 1,4-dioxane solvate 1.2 1.4- dioxane 3b Cyclohexanone Cyclohexanone solvate 0.3 cyclohexanone 3c 1,4-dioxane 1,4-dioxane solvate 0.5 1.4- dioxane 3d THF THF solvate 0.4 THF 3e Isobutanol Isobutanol solvate 0.7 isobutanol lb + 4 Water / methanol Mixture hydrate / methanol solvate five Chloroform Chloroform solvate 0.5 chloroform 6 Acetonitrile Anhydrous 0.2 acetonitrile ls + 7 Heptane Heptane solvate 0.1 heptane 7 - Not solvated - eight - Not solvated - nine - Not solvated - 10 dihydrate 1.8 H ₂ O

The compound obtained as described in example 1 was additionally crystallized by preparing a suspension in DCM (1: 3, for example, 10 g of the compound in 30 ml of DCM), which was stirred at 40 ° C for 4 h and further stirred for 14 h at 25 ° C, then heptane was slowly added (1: 2, for example, 20 ml of heptane to the compound / suspension DCM / solution mixture) at 25 ° C, stirred at 40 ° C for 4 h, cooled to 25 ° C and stirred for another 14 hours at 25 ° C. Subsequent filtration afforded Ex 1 as an off-white solid which, when authenticated, was the monohydrate (Embodiment 1s).

Embodiments 1-10 in table. 2 and FIG. 2 are crystalline. Embodiments 1s and 1a-1h are isostructural. Embodiment 1s crystallizes into the centrally symmetric triclinic space group P-1. The term embodiment 1 in co

30 037347 refers to isostructural embodiments 1s and 1a-1h. Any of such embodiments 1s and 1a-1h are sometimes referred to as the isostructural element of Embodiment 1, or simply the element of Embodiment 1. Embodiments 3b, 3c, 3d and 3e are isostructural and crystallize into a monoclinic system (space group C 2 / c). The term Embodiment 3 collectively refers to isostructural Embodiments 3b, 3c, 3d, and 3e. Any of such embodiments 3b, 3c, 3d, and 3e are sometimes referred to as the isostructural element of Embodiment 3, or simply the element of Embodiment 3. The isostructural embodiments are such that they have similar crystal structure properties (same symmetry and similar unit cell parameters and crystal packing ) and at the same time have different chemical compositions (i.e. different numbers of solvent and / or water molecules embedded in the crystal lattice). The unit cell parameters in isostructural embodiments may differ slightly depending on the composition (solvent or water embedded in the crystal structure). The options for implementation mentioned in table. 2 have been obtained and / or mutually transformed as schematically shown in FIG. 2 and is described in more detail in the following screening procedures.

The screening included crystallization protocols such as equilibration of the solvent in pure solvents, evaporative crystallization, hot filtration cooling crystallization, quench crystallization with an anti-solvent, and thermal cycling crystallization. Solids were analyzed using HT-XRPD. If applicable, the mother liquors were completely evaporated and the remaining solids were also analyzed by HT-XRPD. The determined predominant solid form was the embodiment of the starting material 1s as monohydrate.

Equilibration of solvent at 25 and 50 ° C

Long term suspension experiments were carried out by suspending embodiment 1s in twenty pure solvents and stirring at room temperature for two weeks and at 50 ° C for one week. At the end of the equilibration time, residual solids were separated from the mother liquors. The solids were dried at ambient conditions and dried under vacuum (5 mbar) and then analyzed by HT-XRPD. The solids were then subjected to accelerated aging conditions (40 ° C / 70% relative humidity) for two days and again analyzed by HT-XRPD.

From most crystallization solvents, the starting material was prepared as Embodiment 1s. Crystals were obtained from several crystallization solvents whose HTXRPD diffraction patterns were similar to those of the original embodiment 1s. Most of the diffractogram data showed peak offsets and / or additional peaks. Each of these diffractograms corresponded to an embodiment that was designated one of Embodiments 1a-1h, and based on the similarity of HT-XRPD diffractograms for such embodiments, they are represented as embodiments being isostructural elements of Embodiment 1. All isostructural elements embodiment 1 were converted to embodiment 1a after exposure to 40 ° C and 75% RH for two days.

Embodiment 1s was converted to hydrated Embodiment 10 after exposure to 100% RH at 25 ° C. However, embodiment 10 was physically unstable under ambient conditions. Although embodiment 1s crystallized into a triclinic system (space group P-1), it was found that embodiment 10 crystallized into a monoclinic system (space group C 2 / c). Embodiment 10 had limited physical stability under environmental conditions and was converted to another embodiment such as 1s or 1a. This behavior is explained by the unevenly strong binding of all molecules that provide hydration / solvation. In this case, embodiment 10 would have a less strongly bound second water molecule that would be lost to ambient conditions. More specifically, the physical stability of embodiment 1s was tested in climatic chambers by exposing a 20 mg sample of this embodiment to a temperature of 40 ° C and 70% relative humidity for four days, and another 20 mg sample of the same embodiment also for four days. days were exposed to a temperature of 25 ° C and a relative humidity of 100%. Four days later, various samples of solids were analyzed by HR-XRPD, crystal cell parameters were determined and diffraction patterns were interpreted. The diffractograms are shown in FIG. 6. From bottom to top: the first diffractogram in FIG. 6 corresponds to embodiment 1s as starting material and the second corresponds to the same shape after 4 days of exposure to 40 ° C and 70% RH, marked in this figure as 1s 70 OB. This analysis indicated that the original embodiment 1s was recovered, albeit with a small amount of the second crystalline form, which may have been another hydrated embodiment with a higher water content. The classification of this form turned out to be impossible due to the small number in which it was present. The third diffractogram corresponded to embodiment 1s after 4 days of exposure to 25 ° C and a relative humidity of 100% and is marked on the same figure with the number 10. These conditions led to the conversion of embodiment 1s to embodiment 10 with a small content of the original embodiment implementation of 1s and solvation, as shown in table. 2. After dehydration, both embodiments

1s and 10 were recrystallized to anhydrous form with a melting point of 148 ° C.

Equilibration of the solvent at room temperature resulted in embodiment 1b from toluene as crystallization solvent and embodiment 1f from p-xylene as crystallization solvent.

Three additional solid embodiments were identified and designated as Embodiments 2, 3 and 7. Embodiment 2 was determined in a solvent equilibration experiment conducted at room temperature in 1,4-dioxane, while Embodiment 7 was found as a mixture with embodiment 1s in one solvent equilibration experiment at 50 ° C from heptane. Several similar but not identical diffraction patterns were found which were grouped as Embodiments 3b, 3c, 3d and 3e, being isostructural elements of Embodiment 3. Isostructural elements of Embodiment 3 were found mixed with elements of Embodiment 1. Mixtures containing elements of Embodiment implementation 3, in some cases converted to embodiment 1a or a mixture of embodiments 1a and 3e. Embodiment 7 was found to be physically stable, but Embodiment 2 changed to Embodiment 3e after being exposed to AAC for two days.

Evaporative crystallization

The mother liquors from the solvent equilibration experiments conducted at room temperature were used for the slow evaporative crystallization experiments. The mother liquors were filtered to remove any particulate matter and allowed to slowly evaporate at ambient conditions. The resulting solids were analyzed by HT-XRPD and repeated after exposure to AAC for two days.

Due to the low solubility of the compound of Ex. 1 in some solvents, no solid particles were recovered during their use. In experiments in which precipitation of solids occurred, the amorphous residue or isostructural elements of Embodiments 1 or 3 were recovered. During the stability study, various elements of Embodiment 1 were converted to Embodiment 1a, while the sample of Embodiment 3 appeared physically stable. Amorphous solids in some cases remained amorphous after stability studies, became soluble, or showed some signs of crystallization.

Cooling crystallization

Stock solutions from solvent equilibration experiments were run at 50 ° C, filtered at 50 ° C to remove any particulate matter. Suspensions at 50 ° C were filtered using PTFE filters with a pore size of 0.2 μm, and the solutions were placed under a temperature of 5 ° C and held for 72 h. After precipitation due to aging, the solids were separated from the liquid, dried under ambient conditions. media and in vacuum and analyzed using HT-XRPD. The remaining mother liquors were allowed to evaporate slowly and the remaining solids were analyzed by HT-XRPD. Samples that did not precipitate were placed under vacuum and dried solids analyzed by HT-XRPD. All solids were then exposed to AAS (2 days at 40 ° C / 70% RH) and re-analyzed by HT-XRPD.

In some solutions, solids did not precipitate on cooling, in which case the solutions were evaporated at ambient conditions. Due to the poor solubility of the compound Ex. 1 in some solvents, no solids were obtained from some solutions.

A precipitate formed from four solvents (2-propanol, 2-butanone, acetonitrile and methanol). Embodiment 6 was detected after evaporation of a solution from a single cooling crystallization experiment in the milliliter range in 800 μl of acetonitrile (concentration 25 mg / ml). Embodiment 6 looked like a stable solid form after 2 days of AAC and was an unsolvated embodiment.

Cooling / Evaporative Crystallization in the Microliter Range

Cooling / evaporative crystallization experiments in the microliter range were performed on a 96 well plate using 12 pure solvents and 12 solvent mixtures and using four temperature profiles. Approximately 4 mg of embodiment 1s was dosed into each well as a solid. Thereafter, crystallization solvents (80 μl) and solvent mixtures were added to achieve a concentration of 50 mg / ml, and the plate with individually sealed each well was subsequently exposed to one of four temperature profiles. After the completion of the temperature treatment, the solvents were left to evaporate at low ambient pressure (24 h) and the remaining solids were analyzed by HT-XRPD before and after exposure to AAC for 2 days (40 ° C / 70% RH).

Elements of Embodiments 1 and 3 have been found in most solvent systems and

- 32 037347 temperature profiles. In this case, a certain dependence of the solid form on the temperature profile was observed. Embodiment 1b was mainly found when exposed to short temperature profiles (3 hours aging). However, with a long aging time, the same solvent systems led to the discovery of Embodiment If, elements of Embodiment 3 or mixtures of elements of Embodiments 1 and 3. Embodiment 3c was prepared using 1,4-dioxane as crystallization solvent and the following temperature profile : an initial temperature of 50 ° C maintained for 60 minutes and then cooling at a rate of 1 ° C / hr to a final temperature of 20 ° C held for 48 hours; Embodiment 3d was prepared using tetrahydrofuran as crystallization solvent and the same temperature profile as Embodiment 3c.

Embodiment 4 was determined in experiments carried out in methanol / water (50/50, v / v), THF and DCM / IPA (50/50, v / v) using fast aging conditions. Embodiment 4 was prepared by treating Embodiment 1s with a 50/50 mixture of water and methanol and using the following temperature profile: an initial temperature of 50 ° C, maintained for 60 min, and then cooling at a rate of 20 ° C / h to a final temperature 5 ° C, which was maintained for 3 hours, resulting in an embodiment 4 together with an embodiment 1b. Embodiment 4, together with Embodiment 1b, was also prepared by treating 1s with a 50/50 mixture of water and methanol and using the following temperature profile: an initial temperature of 50 ° C, maintained for 60 minutes, and then cooling at a rate of 20 ° C / h to a final temperature of 20 ° C, which was maintained for 3 h. Embodiment 4 was not physically stable under ambient conditions. In cooling crystallization experiments, Embodiment 1c was prepared from ethyl acetate / 1,4-dioxane (50/50, v / v) as crystallization solvent using the following temperature profile: initial temperature of 50 ° C maintained for 60 minutes and then cooling at a rate of 1 ° C / h to a final temperature of 5 ° C, which was maintained for 48 h; embodiment Id from acetonitrile / chloroform (50/50, v / v) as a solvent for crystallization using the following temperature profile: an initial temperature of 50 ° C maintained for 60 min and then cooling at a rate of 1 ° C / h to a final temperature of 5 ° C, which was maintained for 48 h; and embodiment 1e of ethyl acetate / 1,4-dioxane (50/50, v / v) as a solvent for crystallization using the following temperature profile: an initial temperature of 50 ° C maintained for 60 minutes, and then cooling with rate from 1 ° C / h to a final temperature of 20 ° C, which was maintained for 48 h.

Embodiment 5 was determined in experiments carried out in chloroform as a crystallization solvent and with the following temperature profile: an initial temperature of 50 ° C maintained for 60 min and then cooling at a rate of 1 ° C / h to a final temperature of 20 ° C. which was maintained for 48 hours.

Similar transformations were observed during the stability study, as previously noted with other crystallization methods. In most cases, all solid forms are converted to embodiment la or to mixtures containing embodiment la.

Evaporative crystallization from solid mixtures

Evaporative crystallization using solvent / antisolvent mixtures produces clear solutions of the compound, from which the solvent first evaporates (high vapor pressure), resulting in the compound precipitating somewhat in the form of crystals. Subsequently, these crystals act as seeds for the evaporation of the anti-solvent (lower vapor pressure).

Compound pr. L did not completely dissolve in each of the solvent systems. For this reason, all experiments included filtration before evaporation.

The results of analysis using HT-XRPD showed that the compound ex. L crystallized predominantly as embodiment 1s after evaporation of the solvent mixtures. This was observed for the following solvent / anti-solvent systems: tetrahydrofuran / water, acetonitrile / water, chloroform / ethanol, methanol / ethyl acetate, 2-butanone / isopropanol, and heptane / acetone. From the two systems (acetone / cumene and 1,4-dioxane / ethylformat), isostructural embodiments 3b and 3e were obtained, which after exposure to AAS were transformed into different mixtures of embodiments la and 3d, and 1s and 3e, respectively.

Crystallization using an anti-solvent

Saturated solutions of the compound pr. L were obtained in pure solvents. The anti-solvent was added by direct and reverse addition. When added directly to the compound solution, the anti-solvent was added in three aliquots. Reverse addition was performed by adding a volume of compound solution to a large excess of anti-solvent (20 ml).

After settling, the solids were separated from the liquid, dried at ambient conditions and dried under vacuum (5 mbar) and then analyzed by HT-XRPD. Solutions from experiments in which no precipitation occurred after the addition of solvent was stored at 5 ° С for 48 h.

- 33 037347 to induce deposition. Thereafter, the precipitated solids were separated and analyzed by HT-XRPD. In the absence of solids, the solutions were mildly evaporated and the remaining solids analyzed by HT-XRPD. All solids were exposed to AAS (2 days at 40 ° C / 70% RH) and re-analyzed by HT-XRPD.

In all cases, the formation of a precipitate was observed during direct crystallization using an anti-solvent. All solids can be classified as isostructural elements (1s, 1b, 1j, 1f) of embodiment 1 or embodiment 3 (3b, 3d, 3f). After exposure to AAS, all solid samples were converted to Embodiment 1a except one, which was converted to a mixture of Embodiments 1a and 3e.

Anti-solvent reverse crystallization experiments performed in DMSO as solvent resulted in different solid forms depending on the anti-solvent used. When using dichloromethane or p-xylene, the isostructural elements (1s and lb) of embodiment 1 were determined, while using MTBE, an amorphous residue was obtained. Evaporation of the two solutions with heptane and water as anti-solvents, in which no precipitation was observed upon addition of the anti-solvent, resulted in an oil. Conversions in accordance with embodiment 1a were observed after exposure to AAS, and the amorphous residues became soluble.

Hot Filtration Experiments

Experiments on cooling crystallization with hot filtration were carried out from supersaturated solutions of the compound of Ex. 1 obtained at 50 ° C in various mixtures of solvents. Filtered hot solutions were cooled for 48 hours. The vials, in which the solids precipitated after heat treatment, were centrifuged and the solids were separated from the liquid and analyzed by HT-XRPD (after vacuum drying). If no solids precipitated, the solutions were evaporated in vacuo and the solids analyzed by HT-XRPD. All solids were exposed to AAC (2 days at 40 ° C / 70% RH) and re-analyzed by HT-XRPD. In half of the hot filtration experiments, no precipitation was observed and no sufficient solids were found after evaporation of the solvents due to the low solubility of Ex 1 in these solvent systems. In three experiments, an amorphous residue was reduced, which after exposure to AAC crystallized into a mixture of elements of embodiment 1 (1s or 1a) and 3 (3e) or became soluble. Embodiment 5 was determined experimentally in acetone / chloroform (50/50, v / v). This embodiment was found to be physically unstable because after exposure to AAC, conversion to embodiment 1a took place.

Thermal cycling experiments

Suspensions of about 6 mg of embodiment 1s were prepared in 10 solvents at room temperature. The suspensions were subjected to cycling in the range from 5 to 50 ° C. After completion of thermal cycling, the solids were centrifuged and dried under ambient conditions and under vacuum (5 mbar) and then analyzed by HT-XRPD. Subsequently, all solids were exposed to AAS for two days and again analyzed by HT-XRPD. Thermal cycling experiments tend to produce a more stable polymorphic form. Except for the experiment performed in cyclohexanone, all vials after heat treatment contained solids. The cyclohexanone solution was slowly evaporated under light vacuum. The elements of embodiments 1, 3, or mixtures thereof, were detected predominantly in wet solids. After drying, these solids were converted to Embodiment 1s. Embodiments 3b and 3e were prepared by thermal cycling in 300 μl of cyclohexanone at a concentration of 51 mg / ml (3b) and in 400 μl of isobutanol at a concentration of 37.3 mg / ml (3e). Embodiment 5 was prepared by thermal cycling in 800 μl of chloroform at a concentration of 18.6 mg / ml.

FIG. 3, 4 and 5 show an overlay of HT-XRPD patterns of the embodiments listed in Table. 2 as well as those mentioned in the above screening.

Embodiment 1s was recovered in most crystallization experiments. It is a channel hydrate having a variable number of embedded water molecules and / or other solvents, depending on environmental conditions. Conversion to embodiment 1a was observed. This form contained slightly more water (1.3 water molecules). All isostructural elements of embodiment 1 were converted to embodiment 1a after exposure to 40 ° C and 75% RH for two days. The displacement of some of the diffraction peaks in the XRPD patterns of the elements of Embodiment 1 may be due to the fact that a different number of solvent or water molecules were included in the crystal lattice. FIG. 4 shows an overlay of HT-XRPD patterns of elements of Embodiment 1. Diffraction pattern 1s corresponds to compound Ex 1 as starting material in the form of Embodiment 1s. The diffractogram 1a corresponds to embodiment 1a obtained after exposing several samples of embodiment 1s to AAS. Diffractogram 1b corresponds to embodiment 1b obtained in a solvent equilibration experiment at rt. pace. in toluene. Diffractogram

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1c corresponds to embodiment 1c obtained in a cooling crystallization experiment in the microliter range in ethyl acetate / 1,4-dioxane (50/50 v / v). Diffractogram 1c corresponds to embodiment 1d obtained in a cooling crystallization experiment in the microliter range in acetonitrile / chloroform (50/50 v / v). Diffractogram 1e corresponds to embodiment 1e obtained in a cooling crystallization experiment in the microliter range in ethyl acetate / 1,4-dioxane (50/50 v / v). Diffractogram 1f corresponds to embodiment 1f obtained in a solvent equilibration experiment at rt. temp, in p-xylene. Diffractogram lg corresponds to embodiment 1g obtained in a solvent equilibration experiment at 50 ° C. in anisole. Diffractogram 1n corresponds to embodiment 1n obtained in a microliter range freeze crystallization experiment in p-xylene.

Diffraction patterns for elements of Embodiment 3 are shown in FIG. 5. The displacements observed in the different HT-XRPD patterns are most likely related to the different number of solvent molecules included in the crystal lattice. Embodiment 3 was prepared by heating Embodiment 2 to 40 ° C at 70% RH for 4 days. Embodiments 3b-3e were solvated forms containing a non-stoichiometric amount of solvent that varied depending on the solvent included in the crystal structure (0.3-0.7 molecules). Mixtures containing the elements of Embodiment 3 were unstable when exposed to AAS and, in some cases, converted to Embodiment 1a or in a mixture of Embodiments 1a and 3e. Conversion to Embodiment 1a is due to the replacement of solvent molecules by water molecules when exposed to high relative humidity and recrystallization to hydrated Embodiment 1a.

Embodiment 9 was obtained by heating Embodiment 2 to a temperature of about 200 ° C, followed by cooling to 25 ° C, and also by differential scanning calorimetry (DSC) 25-200-25-300 ° C.

Embodiment 8 was prepared by heating Embodiment 5 to a temperature of about 175 ° C.

Embodiments of the present invention include the compound of Ex. 1 in the form of 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. Embodiments of the present invention include compounds of the present invention in the form of pharmaceutically acceptable co-crystals.

Examples 1-12 are JAK inhibitors and were tested in enzymatic and cellular assays. The results of the enzymatic analysis are presented in table. 4 titled Results of Enzymatic Inhibition Assays. Examples 1-12 were also investigated in three cellular assays: IL-2 pSTAT5 (JAK1 / JAK3), IFNa pSTAT4 (JAK1 / TYK2) and GM-CSF pSTAT5 (JAK2 / JAK2), the results of which are presented in table. 5 titled Cellular Analysis Data. The following is a description of the method for performing the enzymatic analysis, including the materials used in the analysis (under the heading Materials), the progress of the analysis (under the heading Analysis protocol), and the method used to analyze the data (under the heading High performance mass spectrometry (HTMS) method).

Enzymatic Inhibition Assay Materials

Substrate (NH2-KGGEEEEYFELVKK-CO2), internal standard peptide (NH2SWGAIETDKEYYTVKD-CO2) and final peptide (standard curve only) (NH2-KGGEEEEYPi-FELVKK-CO2) were purchased from AnaSpec (Fremont, CA, USA). JAK1JH1JH2 (574-1154 with His-GST tag and C-terminal TEV cleavage site (ENLYFQ-G)), JAK3JH1JH2 (512-1124 with GST tag and C-terminal TEV cleavage site (ENLYFQ-G)) and Tyk2-JH1JH2 8H_tev_580-1182-C936A-C1142A with a C-terminal TEV cleavage site (ENLYFQ-G)) was purified in-house. JAK2-JH1JH2 (532-1132 labeled with GST and C-terminal TEV cleavage site (ENLYFQ-G)) was purchased from Invitrogen. LC / MS grade water and acetonitrile (ACN) were purchased from HoneyWell, Burdick & Jackson (Muskegon, Michigan, USA). Dimethyl sulfoxide 99.8% (DMSO) and trifluoroacetic acid 99.5% (TFA) were purchased from EMD Chemical (Gibstown, NJ, USA). Adenosine triphosphate (ATP), 4-morpholine propanesulfonic acid (MOPS), magnesium chloride (MgCl ₂ ), ethylenediamine tetraacetic acid (EDTA), dithiothreitol (DTT),> 95% formic acid (FA) and Tween-20 (purchased from Sig) Saint Louis, Missouri, USA). 384-well polypropylene plates (p / n 781280) were purchased from Greiner, Monroe, NC, RapidFireTM cassette, C4 column (Agilent Technologies, Santa Clara, CA).

Experiments using HTMS were performed in positive ionization mode on a Rapidfire 300 instrument (Agilent Technologies, Santa Clara, Calif.) In combination with an ABSiex QTrap 4000 ionization system with an electrospray source (RF-MS) (Concord, Ontario, Canada).

The RapidFire system was run using 3 Agilent 1200 series isocratic pumps from Agilent Technologies (Santa Clara, Calif.) And one model ISM832C peristaltic pump from Ismatec (Wertheim, Germany). The entire system was controlled using Rapidfire software interfaced with Analyst mass spectrometer software.

Analysis protocol

For each compound, a series of 11 samples were prepared by serial dilution of 1: 3 or 1: 4 in DMSO, with sample 12 being the control (DMSO). From serial dilution plates, sample was transferred to a 384-well assay plate (# 781280, Greiner, Monroe, NC) using Labcyte Echo (Sunnyvale, CA) or Biosero ATS (San Diego, CA) ... Compounds were tested in duplicate. Column 12 was used for positive control and column 24 contained negative controls without enzyme addition. A compound from our internal collection with inhibitory activity against JAK isoforms was used as a reference compound. The final DMSO concentration was <0.25% in a 20 μL reaction mixture. The analysis conditions for each of the proteins are shown in table. 3. The enzymatic reaction was initiated by adding 10 μl of the enzyme and a mixture of ATP to 10 μl of a substrate solution in the reaction buffer (50 mM MOPS, pH 7.5, 10 mM MgCl ₂ , 1 mM EDTA, 2 mM DTT, 0.002% Tween20). The Tyk2 enzyme was preincubated with 2 mM ATP for 30 min, after which the reaction was initiated. Immediately after adding the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 min and incubated at 25 ° C for 45 min in the case of JAK3 and for 90 min in the case of JAK1, JAK2 and Tyk2. The reaction was suppressed by the addition of 20 μl 0.5% TFA containing 0.15 μM internal standard peptide using a Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, MA). For the standard product curve, typically several wells from column 24 were used. After quenching, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (p / n 06644-001, Agilent) using PlateLoc (Agilent Technologies , Santa Clara, California). Subsequently, the plates were transferred to RapidFire for MS analysis. Compound inhibition was assessed by the decrease in phosphorylated product concentrations in the test wells compared to uninhibited enzymatic reaction. The conditions for the above analyzes are given in table. 3, and the results of the study of pr. 1-12 in these analyzes are shown in table. four.

Table 3. Conditions for the analysis of the JAK * family enzyme

Enzyme [enzyme], nM [ATP], μM [Substrate], μM [IS], nm JAK1-JH1JH2 8.0 12.5 200 100 JAK2-JH1JH2 7.0 or 3.6 thirty 40 100 JAK3-JH1JH2 2.0 150 40 100 Tyk2-JH1JH2 2 5 or 14.7 fifty 200 100

* Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl ₂ , 1 mM EDTA, 2 mM DTT, 0.002% Tween20; IS means internal standard peptide; substrate means peptide.

High performance mass spectrometry (HTMS) method

Sample analysis on a RapidFire instrument was carried out using the A1 mobile phase consisting of water / TFA / FA (100: 0.01: 0.1, v / v / v), the B1 mobile phase consisting of ACN / water / TFA / FA (80: 20: 0.01: 0.1, v / v / v). The following test parameters were used: state 1 (aspiration), 250 ms; state 2 (loading / flushing), 3000 ms; state 3 (elution), 4000 ms; state 4 (recalibration), 1000 ms with a flow rate of 1.25 ml / min. Samples were aspirated directly from a 384-well assay plate and loaded onto a C4 solid phase extraction cassette in the microliter range (type A) for RF-MS. Unwanted component such as salt, cofactor, detergent and large proteins were washed out and the remaining analytes (substrate, product and IS) were co-eluted directly onto the ABSiex Qtrap 4000 system. Quantification of peptide (substrate), phosphopeptide (product) and internal reference peptide (IS) was performed by MRM using transitions 562 ^ 136.0, 589.2 ^ 215.7 and 953.2 ^ 158.8 (or 974.2 ^ 158.8), respectively.

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Table 4. Results of Enzymatic Inhibition Assays

Test compound JAK1_JH1JH 2 1C ₅₀ (nM) JAK2_I_JH1JH 2 1C ₅₀ (nM) JAK3_I_JH1JH 2 1C ₅₀ (nM) Tyk2_I_JHlJH 2 1C ₅₀ (nM) BUT <0.2 <0.2 12.4 0, 9 IN <0.2 <0.2 13.4 <0.2 FROM <0.2 0, 6 49, 7 0.2 Etc. one 0,4 8, 6 92.2 7.4 Etc. 2 0.2 1.0 33, 9 1.5 Etc. 3 0.2 6, 2 82.8 11, 6 Etc. four 0, 1 6, 6 96.2 2.2 Etc. five 0.3 2.1 23, 1 4.1 Etc. 6 0, 1 1.4 28.2 1.1 Etc. 7 0.2 5, 6 98.4 4.8 Etc. eight 0,4 7.0 75, 6 6, 6 Etc. nine 0.2 6, 6 79, 9 4.7 Etc. 10 1.0 6, 5 87.5 9, 0 Etc. eleven 0,4 sixteen 30.3 1.5 Etc. 12 0, 9 9, 4 101.3 8.7

Cellular analyzes

IL-2 pSTAT5 Cellular Assay (JAK1 / JAK3)

The AlphaLISA assay (based on PerkinElmer's Alpha technology) was performed by plating freshly thawed PBMCs (Biological Specialty Corporation) into 384-well plates at 30,000 cells per 4 μl per well in Hanks Balanced Salt Solution (HBSS) containing 0.1% - bovine serum albumin (BSA), without immunoglobulin G (IgG) and protease (Jackson ImmunoResearch, cat. No. 001-000-161). Subsequently, the cells were treated with compounds in a volume of 2 μl / well, diluted in DMSO at semi-log titrated concentrations (the highest experimental concentration was 10 μM, and the final concentration of DMSO was 0.5%) for 30 min at 37 ° C. Subsequently, the cells were stimulated with 2 μl / well IL-2 (R&D systems, cat. # 202-IL-050) at a concentration of 5 ng / ml for 30 min at 37 ° C. Cellular reactions were terminated by the addition of 2 μl / well of lysis buffer (PerkinElmer, cat # ALSU-PST5-A10K) followed by incubation for 5 min at room temperature. 5 μl / well of acceptor mixture (PerkinElmer, cat. # ALSU-PST5-A10K) was added to the cells and incubated in the dark for one hour at room temperature. Subsequently, 5 μl / well of the donor mixture (PerkinElmer, cat. # ALSUPST5-A10K) was added to the cells and incubated in the dark overnight at room temperature. Ultimately, the plates were read on a PerkinElmer Envision instrument to detect the time resolved fluorescence signal. The percentage of IL-2-dependent inhibition of pSTAT5 was determined at the test compound concentrations and a dose-response curve was plotted for each compound and the IC _{50 was} calculated. Compound IC _{50 was} calculated by non-linear regression, i. E. by analyzing the sigmoidal dose response based on a semi-log titration curve representing the dependence of the Alpha signal on the concentration of the compound. Alpha stands for Homogeneous Approach Luminescence Enhancement Analysis; The alpha signal is a luminescent / fluorescent signal.

IFNa pSTAT4 Cellular Assay (JAK1 / TYK2)

The AlphaLISA assay (based on PerkinElmer's Alpha technology) was performed by plating freshly thawed PBMCs (Biological Specialty Corporation) into 384-well plates at a density of 100,000 cells per 6 μl per well in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% - fetal bovine serum (FBS) and 1000 IU / ml of penicillin and 1000 μg / ml of streptomycin. Subsequently, the cells were treated with compounds in a volume of 2 μl / well, diluted in DMSO at semi-log titrated concentrations (the highest experimental concentration was 10 μM, and the final concentration of DMSO was 0.5%) for 30 min at 37 ° C. The cells were then stimulated with 2 μl / well IFNa (PBL Assay Science, Cat. # 11101-2) at a concentration of 4 ng / ml for 30 min at 37 ° C. Cellular reactions were terminated by the addition of 2 μl / well of lysis buffer (PerkinElmer, cat # ALSU-PST4-A10K) followed by incubation for five minutes at room temperature. To the cells was added 4 μl / well of an acceptor mixture (PerkinElmer, cat # ALSU-PST4-A10K) and incubated in the dark for one hour at room temperature. Subsequently, 4 μl / well of the donor mixture (PerkinElmer, cat. # ALSU-PST4-A10K) was added to the cells and incubated in the dark overnight at room temperature. Ultimately, the plates were read on a PerkinElmer Envision instrument to detect the fluorescence signal with a resolution of

- 37 037347 time. The percentage of IFNa-dependent inhibition of pSTAT4 was determined at the test compound concentrations and for each compound a dose-response curve was constructed and the IC50 calculated. Compound IC50 was calculated by non-linear regression, i. E. by analyzing the sigmoidal dose response based on a semi-log titration curve representing the dependence of the Alpha signal on the concentration of the compound. The term Alpha is defined in the immediately preceding description of cell assay.

PSTAT5 GM-CSF Cellular Assay (JAK2 / JAK2)

AlphaLISA assay (based on PerkinElmer's Alpha technology) was performed by plating freshly thawed PBMCs (Biological Specialty Corporation) into 384-well plates at 30,000 cells per 4 μl per well in HBSS containing 0.1% BSA, IgG and protease free (Jackson ImmunoResearch Cat # 001-000-161). Subsequently, the cells were treated with compounds in a volume of 2 μl / well, diluted in DMSO at semi-log titrated concentrations (the highest experimental concentration was 10 μM, and the final concentration of DMSO was 0.5%) for 30 min at 37 ° C. Cells were then stimulated with 2 μl / well GM-CSF (R&D systems, cat. # 215-GM-050) at a concentration of 11 pg / ml for 15 min at 37 ° C. Cellular reactions were terminated by the addition of 2 μl / well of lysis buffer (PerkinElmer, cat # ALSU-PST5-A10K) followed by incubation for five minutes at room temperature. 5 μl / well of acceptor mixture (PerkinElmer, cat. # ALSU-PST5-A10K) was added to the cells and incubated in the dark for one hour at room temperature. Subsequently, 5 μl / well of the donor mixture (PerkinElmer, cat. # ALSU-PST5-A10K) was added to the cells and incubated in the dark overnight at room temperature. Ultimately, the plates were read on a PerkinElmer Envision instrument to detect the time resolved fluorescence signal. The percentage of GM-CSF-dependent inhibition of pSTAT5 was determined at the test compound concentrations and a dose-response curve was plotted for each compound and the IC _{50 was} calculated. Compound IC _{50 was} calculated by non-linear regression, i. E. by analyzing the sigmoidal dose response based on a semi-log titration curve representing the dependence of the Alpha signal on the concentration of the compound. The term Alpha is defined in the pSTAT5 IL-2 Cell Assay (JAK1 / JAK3).

Table 5. Cellular analysis data

Test compound IL-2 pSTAT5 (JAK1 / JAK3) 1C ₅₀ (nM) IFNa pSTAT4 (JAK1 / TYK2) IC ₅₀ (nM) GM-CSF pSTAT5 (JAK2 / JAK2) IC ₅₀ (nM) Etc. one 21, 6 59, 5 83.9 Etc. 2 9, 0 20, 8 61.0 Etc. 3 6, 4 10, 1 21.9 Etc. four 35, 5 64.7 119, 4 Etc. five 6, 4 38.1 28.9 Etc. 6 6, 7 39, 4 25, 1 Etc. 7 9, 7 38.5 67.3 Etc. eight 20, 6 42.8 33.8 Etc. nine 11.2 35, 9 26, 9 Etc. 10 16, 8 40.4 44.6 Etc. eleven 49, 1 96, 5 201.5 Etc. 12 13, 9 81.4 75, 4

Examples 1-12 were investigated in solubility and permeability assays. The results of the analysis of solubility are presented in table. 6, entitled Solubility analysis data, and the results of the permeability analysis are presented in Table. 7, entitled Permeability Data MDCK-MDR1. These solubility and permeability analyzes are described below under the headings Solubility Assays and Permeability Assays, respectively.

Solubility analyzes

Solubility measurements were performed in the following solubility measurement media: simulated gastric juice (34.2 mM sodium chloride and 100 mM hydrochloric acid) or simulated intestinal juice (fasting [pH 6.5]: 3 mM sodium taurocholate, 0.75 mM lecithin, 28.4 mM sodium phosphate monobasic, 8.7 mM sodium hydroxide and 105.9 mM sodium chloride). The test compounds were dissolved in DMSO at a concentration of 10 mM. Test compounds were dosed (20 μl) into Nunc 1-mL-96-DeepWell-PP plates and DMSO was vaporized by flushing with nitrogen from the TurboVap 96 for 6 hours or until a dry residue was obtained. Subsequently, 400 μl of dissolution medium was added to the well containing the dry solid. The Pre-Slit Well Cap was tightly closed the well plate assembly and the samples were vigorously mixed for 2-5 days at ambient temperature. At the end of the incubation period, 38,037347 samples were filtered through an AcroPrep l-mL-96-Filter filter plate into a new 2-mL96-Deep-Well-PP plate and the supernatants were quantified by UV-HPLC using a 3-point calibration curve in in the range of 0.004-0.55 mM. The solubility for each compound was calculated using the following equation:

_p _ Sample peak area

Average response rate from 3 standards

Solubility values were in the range of 4-400 μM. Values outside this range were reported as <4 μM or> 400 μM. Solubility values were recorded until the test compound was stable enough to perform an appropriate solubility determination.

_____ Table 6. Dissolution analysis data

Test compound Solubility SGF (μM) Solubility SIF (μM) BUT > 400 > 400 IN > 400 75 FROM > 400 > 400 Etc. one > 400 387 Etc. 2 > 400 > 400 Etc. 3 > 400 > 400 Etc. four > 400 > 400 Etc. five > 400 198 Etc. 6 > 400 > 400 Etc. 7 > 400 81 Etc. eight > 400 > 400 Etc. nine > 400 > 400 Etc. 10 > 400 359 Etc. eleven > 400 > 400 Etc. 12 > 400 > 400

Permeability analyzes

Permeability measurements were performed according to the Cyprotex protocol using the MDCK-MDR1 cell line obtained from NIH (Rockville, Maryland, USA). Cells between passages 6-30 were seeded on a Multiscreen plate ™ (Millipore) with a cell density of 3.4x10 ⁵ cells / cm ² and cultured for three days, after which permeability studies were performed. The cells in this assay form a cohesive sheet of one cell layer filling the surface area of the culture dish, also known as a confluent monolayer, and on the fourth day, test compound is added to the apical side of the membrane and transport of the compound through the monolayer is monitored for 60 min.

In a simple and basic way of introducing the terms A and B, often used in such analyzes, the apical (A) side or compartment of an object is the side of such an object that is open to the lumen or the external environment, and the basolateral (B) side or compartment is the side or a compartment of such an object, which is open, as a rule, to the internal environment, covering the opposite side. For example, if such an object is by way of example an intestinal epithelial cell, then the apical side of such intestinal cell would be the side of the cell open into the intestinal lumen, and the basolateral side would be the side open into the bloodstream.

The test compounds were dissolved in DMSO at a concentration of 10 mM. Dosing solutions were prepared by diluting test compound with assay buffer (Hanks Balanced Salt Solution), pH 7.4, to a final concentration of 5 μM. To assess apical-basolateral (AB) permeability, the buffer solution was removed from the apical compartment and replaced with a solution for dosing the test compound with or without a glycoprotein permeability inhibitor (P-glycoprotein, PgP, P-gP, Pgp or P-gp) elacridar (2 μM) ... To assess the basolateral-apical (BA) permeability, the buffer solution was removed from the additional plate and replaced with a solution for dosing the test compound. Incubations were performed in duplicate at 37 ° C in an atmosphere of 5% CO ₂ with a relative humidity of 95%. Each assay included reference markers such as propranolol (high permeability) and prazosin (PgP substrate). After incubation for 60 minutes, apical and basolateral samples were diluted and test compounds were quantified by LC / MS / MS using an 8-point calibration curve ranging from 0.0039 to 3 μM with an appropriate sample dilution (coefficient

- 39 037347 acceptor dilutions = 1; donor dilution factor and C0 = 10). The permeability coefficient (Papp) for each compound was calculated using the following equation: P _app = (dQ / dt) / (C ₀ xS), where dQ / dt is the rate of passage of the drug through the cells, C0 is the concentration in the donor compartment at zero time stamp, and S is the area of the cell monolayer.

The percentage recovery was measured for all incubation conditions. These measurements did not reveal inappropriate compound / plate binding or compound accumulation in the cell monolayer.

In the second and third columns of the table. 7 shows the P _app ( _AB ) values for apical-basolateral transport of the compound with and without a P-glycoprotein inhibitor (third column, designated P ^e app (AB)), which was elacridar, and without it (second column). Papp (AB) shows the extent of cell penetration in this assay, representing a model of transcellular transport through Pgp-expressing cells such as Pgp-expressing cells of the gastrointestinal tract. The P ^e app ( _AB ) (P _app ( _AB ) values in the presence of a P-glycoprotein inhibitor) given in column 3 are determined to confirm the role of P-glycoprotein in compound efflux. In the fourth column of the table. 7 shows the P _app ( _BA ) values for basolateral-apical junction transport. The outflow ratios of the test compound are shown in the fifth column of the table. 7 as P _app ( _BA ) / P _app ( _AB ) using the corresponding values of the permeability coefficient from the fourth and second columns of the same table. Outflow ratios (fifth column, Table 7) in all cases exceed 2 for compounds (A) - (C), as well as for compounds of pr. 1-12, which indicates the presence of outflow for all such compounds.

The P _app ( _AB ) values in column 2 are generally low and comparable to the values for reference compounds (A) - (C) as well as Ex 1-12 compounds. These low values indicate a low permeability for all such compounds, which is due to the action of P-glycoprotein, since all such compounds are substrates of P-glycoprotein, as indicated by the values given in column 5 (all above 2). To infer low permeability, the values given in the third and fourth columns (P ^e app (AB) and Papp (BA), respectively) must be low. However, these data show that the P _app ( _BA ) values for compounds (A) - (C) are higher than the corresponding values for compounds ex. 1-12.

The integrity of each monolayer was monitored by measuring the permeability to Lucifer yellow using fluorimetric analysis. This study showed that the cells in this assay retained a satisfactory confluent monolayer.

Table 7. Data on permeability MDCK-MDR1

Test compound ** MDCK-MDR1 Papp (A-B) (10 ⁶ cm / sec) at 5 (uM) MDCK-MDR1 P ⁱⁿ app (A-B) (10 ~ ⁶ cm / s) at 5 (μM) MDCK-MDR1 Papp (B-A) (10 ⁶ cm / sec) at 5 (uM) Rarr (B-A) / Rarr (A-B) BUT 1,3 22 55.3 43 IN 0, 4 1.7 23.5 59 FROM 0.5 2.5 23, 1 46 Etc. one <0.5; 0,4 <0.5; 1.1 0.9; 1.1 1.9; 3.3 Etc. 2 * eleven sixteen 4, 8 4.4 Etc. 3 <0.4; <0.5 2.3; 1.6 17; sixteen > 41; > 33 Etc. four 0, 1 0.5 1,3 8.7 Etc. five <0.4; <0.5 0.7; 0.5 1.8; 2.1 > 4.8; > 4.5 Etc. 6 <0.3; <0.3 0.9; 1.1 2.5; 2.6 > 8.4; > 8.7 Etc. 7 <0.4 1,2 3, 6 > 9 Etc. eight 0, 1 0.5 1.7 11.5 Etc. nine <0.4 0, 6 eighteen > 4.2 Etc. 10 <0.4 eleven 7.1 > 16, 9 Etc. eleven <0.4 0, 8 eleven > 2.9 Etc. 12 <0.5 0, 6 eleven > 2.2

* Initial concentration was> 7 μM for conditions A-B, A-B (with elacridar) and B-A.

** Unless otherwise indicated, compounds (A) - (C), etc. 1-12 were tested at a concentration of 5 μM. For data shown in cells with two data points, compounds were examined twice.

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In vivo studies Oral dosing protocol 1

Three non-fasting female C57BL / 6 mice were orally administered the test compound at a dose of 25 mg / kg orally as a solution in 20% hydroxypropyl beta-cyclodextrin (HPeCD) at a dose of 5 ml / kg. Blood samples were collected at 0.5, 2 and 4 hours after dosing by retroorbital sinus bleeding or dorsal metatarsal vein venipuncture. Blood samples were collected in tubes containing an anticoagulant (sodium heparin) and placed in liquid ice. The plasma fraction was separated by centrifugation and frozen at -20 ° C for up to 4 h and -80 ° C after 4 h prior to analysis shortly after sample collection. Colon samples were collected 4 hours after dosing. From the beginning of the cecum, a 4-6 cm sample of the colon was excised, cut along the longitudinal axis, and the solid contents were removed by washing with 2 ml of saline. The colon was further washed by placing it in 5 ml of saline and shaking for 5 s. Subsequently, the colon sample was blotted dry, weighed and homogenized as a mixture of 1 part tissue (g) and up to 4 parts HPLC water (ml). Plasma and colon homogenate concentrations of the compound were determined by quantitative liquid chromatography with triple quadrupole mass spectrometry (LC / MS). This protocol was used to evaluate the following test compounds: compounds (B) and (C) and examples 6 and 11.

Oral dosing protocol 2

Three non-fasting female C57BL / 6 mice were orally administered the test compound at a dose of 25 mg / kg orally as a solution in 20% HPeCD at a dose of 5 ml / kg. Blood samples were collected at 0.5, 2 and 4 hours after dosing by retroorbital sinus bleeding or dorsal metatarsal vein venipuncture. Blood samples were collected in tubes containing an anticoagulant (sodium heparin) and placed in liquid ice. The plasma fraction was separated by centrifugation and frozen at -20 ° C for up to 4 h and -80 ° C after 4 h prior to analysis shortly after sample collection. Colon samples were collected 4 hours after dosing. After 2 cm from the beginning of the cecum, a 4 cm sample of the colon was excised, cut along the longitudinal axis, and the solid content was removed by washing with 2 ml of saline. The colon was further washed by placing it in 5 ml of saline and shaking for 5 s. Subsequently, the colon sample was blotted dry, weighed and homogenized as a mixture of 1 part tissue (g) and up to 4 parts HPLC water (ml). Plasma and colon homogenate concentrations of the compound were determined by quantitative liquid chromatography with triple quadrupole mass spectrometry (LC / MS). This protocol was used to evaluate the following test compounds: Compound (A) and Examples 1-5, 7-10 and 12. Protocol 3, Intra-Colonic Dosing Intracolonic (i.e.) dosing group. Following inhalation anesthesia with isoflurane, three female C57BL / 6 mice, on an empty stomach, were injected into the colon through a small incision in the abdominal wall using a syringe and a needle at a dose of 5 mg / kg as a solution in 20% HPeCD in a dose volume of 1 ml / kg. Blood samples were collected at 0.5, 2 and 4 hours after dosing by bleeding from the retroorbital sinus. Blood samples were collected in tubes containing an anticoagulant (sodium heparin) and placed in liquid ice. The plasma fraction was separated by centrifugation and frozen at -20 ° C for up to 4 h and -80 ° C after 4 h prior to analysis shortly after sample collection. Colon samples were collected 4 hours after dosing. After 2 cm from the beginning of the cecum, a 4 cm sample of the colon was excised, cut along the longitudinal axis, and the solid content was removed by washing with 2 ml of saline. The colon was further washed by placing it in 5 ml of saline and shaking for 5 seconds. Subsequently, the colon sample was blotted dry, weighed and homogenized as a mixture of 1 part tissue (g) and up to 4 parts HPLC water (ml). Plasma and colon homogenate concentrations of the compound were determined by quantitative liquid chromatography with triple quadrupole mass spectrometry (LC / MS). This protocol was used to evaluate intracolic dosing of the following test compounds: Examples 1, 3 and 4.

Compounds pr. 1-12 were additionally evaluated for the physicochemical properties given in table. 8. The cLogP and tPSA values were calculated using ChemBioDraw Ultra 14.0, where P is the n-octanol / water partition coefficient. The topological polar surface area (tPSA) was calculated as the sum of the surfaces of all polar atoms, mainly oxygen and nitrogen, also including the hydrogen atoms attached to them.

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Table 8. Some physical and chemical properties of compounds pr. 1-12

Test compound cLog P tPSA # hydrogen bond donors # hydrogen bond acceptors # rotatable links Etc. one 0, 94 113.11 3 five 6 Etc. 2 2.31 88.17 2 four 3 Etc. 3 1.58 92.88 2 four 6 Etc. four 0.54 116.67 2 five 6 Etc. five 0.24 102.11 2 five five Etc. 6 0.86 102.11 2 five 6 Etc. 7 1.25 116.67 2 five 6 Etc. eight 1, 13 113.11 3 five 6 Etc. nine 1, 14 108.48 2 five five Etc. 10 1.35 116.67 2 five 6 Etc. eleven 1.50 117.27 3 five five Etc. 12 0.57 113.11 3 five 6

CLAIM

Claims (21)

CLAIM 1. A compound selected from the group consisting of the following compounds: 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( 2-hydroxy-2-methylpropyl) acetamide; 2 - ((1 g, 4d) -4- (2- (1 H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( cyclopropylmethyl) acetamide; N- (2-cyanoethyl) -2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridine-2 - yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( tetrahydro-2H-pyran-4-yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( (tetrahydro-2H-pyran-4-yl) methyl) acetamide; N- (2-cyano-2-methylpropyl) -2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( (1-hydroxycyclobutyl) methyl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( 1-methyl-1 H-pyrazol-4-yl) acetamide; N- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5 -d] pyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( 1 H-pyrazol-3-yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( (1-hydroxycyclopropyl) methyl) acetamide; and pharmaceutically acceptable salts thereof. 2. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( 2-hydroxy-2-methylpropyl) acetamide; 2 - ((1 g, 4d) -4- (2- (1 H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3 -b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( cyclopropylmethyl) acetamide; N- (2-cyanoethyl) -2- (1 - ((1d, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridine-2 - yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( tetrahydro-2H-pyran-4-yl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- ( (1-hydroxycyclobutyl) methyl) acetamide; 2- (1 - ((1 g, 4d) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) - 42 037347 N - ((1-hydroxycyclopropyl) methyl) acetamide; and their pharmaceutically acceptable salts. 3. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (2 -hydroxy-2-methylpropyl) acetamide; 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (cyclopropylmethyl ) αcetamide; N- (2-cyanoethyl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] pyridine-2- yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (tetrahydro -2H-pyran-4-yl) acetamide; and their pharmaceutically acceptable salts. 4. A compound according to claim 1, wherein said compound is 2- (1 - ((1r, 4r) -4 (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3- b] pyridin-2-yl) -N- (2-hydroxy-2methylpropyl) acetamide. 5. A compound according to claim 1, wherein said compound is 2 - ((1r, 4r) -4- (2- (1H-imidαzol-4yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile. 6. A compound according to claim 1, wherein said compound is 2- (1 - ((1r, 4r) -4 (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3- b] pyridin-2-yl) -N - ((1-hydroxycyclobutyl) methyl) acetamide. 7. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (2 -hydroxy-2-methylnropyl) acetamide; 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidαzo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1r, 4r) -4- (cyαnomethyl) cyclohexyl) -1,6-dihydroimudαzo [4,5-d] nirrolo [2,3-b] nyrudine-2-ul) N - (( 1-hydroxycyclobutyl) methyl) acetamide, and pharmaceutically acceptable salts thereof. 8. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N - (( tetrahydro-2H-pyran-4-yl) methyl) acetamide; N- (2-cyano-2-methylnpropyl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5d] nyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyαnomethyl) cyclohexyl) -1,6-dihydroimudαzo [4,5-d] nirrolo [2,3-b] nyrudine-2-ul) N - (( 1-hydroxycyclobutyl) methyl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimudazo [4,5-d] pyrrolo [2,3-b] pyrudin-2-ul) N- (1 -methyl-1H-π-pyrazol-4-yl) acetamide; N- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5 -d] pyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (1H -pyrazol-3-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] nirrolo [2,3-b] nyrudin-2-ul) N - (( 1-hydroxycyclopropyl) methyl) acetamide; and their pharmaceutically acceptable salts. 9. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] nyridin-2-yl) N- (2 -hydroxy-2-methylpropyl) acetamide; 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (cyclonropylmethyl ) acetamide; N- (2-cyanoethyl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3b] nyridine-2- yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (tetrahydro -2H-pyran-4-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] nirrolo [2,3-b] puridin-2-ul) - 43 037347 N - ((1-hydroxycyclobutyl) methyl) acetamide; and their pharmaceutically acceptable salts. 10. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N - (( tetrahydro-2H-pyran-4-yl) methyl) acetamide; N- (2-cyano-2-methylpropyl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5d] pyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyαnomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyrudin-2-ul) N - (( 1-hydroxycyclobutyl) methyl) acetamide; and their pharmaceutically acceptable salts. 11. A compound according to claim 1, wherein said compound is selected from the group consisting of the following compounds: 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; N- (4- (cyanomethyl) bicyclo [2.2.1] heptan-1-yl) -2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5 -d] pyrrolo [2,3-b] pyridin-2-yl) acetamide; 2- (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2,3-b] pyridin-2-yl) N- (1H -pyrazol-3-yl) acetamide; and their pharmaceutically acceptable salts. 12. A pharmaceutical composition having the ability to inhibit JAK kinases, comprising a therapeutically effective amount of at least one compound of claim 1 and pharmaceutically acceptable excipients. 13. The compound of claim 4, wherein said compound is in the 1s form having a diffractogram substantially as shown in FIG. 3. 14. A compound selected from the group consisting of the following compounds: 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 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1r, 4r) -4- (cyαnomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyrudin-2-ul) N - (( 1-hydroxycyclopropyl) methyl) acetamide; and their pharmaceutically acceptable salts. 15. A compound according to claim 14, wherein said compound is selected from the group consisting of the following compounds: 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 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; and their pharmaceutically acceptable salts. 16. A compound according to claim 14, wherein said compound is selected from the group consisting of the following compounds: 2 - ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3-b] pyridin-1 (6H) -yl) cyclohexyl) acetonitrile; 2- (1 - ((1r, 4r) -4- (cyαnomethyl) cyclohexyl) -1,6-dihydromidαzo [4,5-d] pyrrolo [2,3-b] pyrudin-2-ul) N - (( 1-hydroxycyclopropyl) methyl) acetamide; and their pharmaceutically acceptable salts. 17. A compound according to claim 14, wherein said compound is selected from the group consisting of the following compounds: 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- (cyαnomethyl) cyclohexyl) -1,6-dihydroimidαzo [4,5-d] pyrrolo [2,3-b] pyrudin-2-ul) N - (( 1-hydroxycyclopropyl) methyl) acetamide; and their pharmaceutically acceptable salts. 18. A pharmaceutical composition having the ability to inhibit JAK kinases, comprising a therapeutically effective amount of a compound according to claim 14 and pharmaceutically acceptable excipients. 19. A pharmaceutical composition according to claim 18, wherein said compound is 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. 20. A pharmaceutical composition according to claim 18, wherein said compound is 2 ((1r, 4r) -4- (2- (1H-imidazol-4-yl) imidazo [4,5-d] pyrrolo [2,3 -b] pyridin-1 (6H) -yl) cyclohexyl) acetonyryl. - 44 037347 21. A pharmaceutical composition according to claim 18, wherein said compound is 2 (1 - ((1r, 4r) -4- (cyanomethyl) cyclohexyl) -1,6-dihydroimidazo [4,5-d] pyrrolo [2, 3-b] pyridin-2-yl) -N - ((1hydroxycyclopropyl) methyl) acetamide.