US20200017498A1 - Small Molecule Inhibitors of the JAK Family of Kinases - Google Patents

Small Molecule Inhibitors of the JAK Family of Kinases Download PDF

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US20200017498A1
US20200017498A1 US16/581,081 US201916581081A US2020017498A1 US 20200017498 A1 US20200017498 A1 US 20200017498A1 US 201916581081 A US201916581081 A US 201916581081A US 2020017498 A1 US2020017498 A1 US 2020017498A1
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yielded
suspension
diffractogram
compound
jak
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Philippe Fernandes
Paul J. Krawczuk
Mark S. Tichenor
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Priority to US16/776,243 priority patent/US11066406B2/en
Priority to US17/377,249 priority patent/US11787807B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to certain imidazopyrrolopyridine compounds, pharmaceutical compositions containing them, methods of making them, and methods of using them as JAK inhibitors and for the treatment of disease states, disorders, and conditions mediated by JAK.
  • autoimmune disease autoimmune disease
  • autoimmune disease does not exclude conditions whose causes comprise external factors or agents, such as environmental or bacterial factors, and internal factors such as genetic susceptibility.
  • a condition such as Crohn's disease is referred to herein as an autoimmune disease, regardless of whether it is triggered by the body itself or by external factors.
  • CD Crohn's disease
  • autoimmune diseases Among the various adverse effects caused by autoimmune diseases, at least one of the following is typically observed: Damage to, and sometimes destruction of, tissues, and organ alteration that can impact organ growth and organ function. Examples of autoimmune diseases affect most major organs, endocrine and exocrine glands, the blood and muscles, and a plurality of systems, such as the digestive, vascular, connective and nervous systems. Immunosuppressive treatments are often adopted to treat autoimmune diseases.
  • autoimmune diseases arise, some focusing on endogenous factors and others also including exogenous factors.
  • the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is considered to play an important role in transmitting information from extracellular chemical signals to the cell nucleus resulting in regulation of genes that are involved in cellular activities such as immunity.
  • Cytokines are an example of an extracellular molecule that plays an important role in cell 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 central to the intracellular signaling of type I and type II cytokine receptors.
  • JAK refers to either JAK1, JAK2, JAK3 or Tyk2, or any combination thereof.
  • Each JAK selectively associates with receptor subunits which dimerize (or multimerize) to form functional receptors.
  • J. D. Clark, et al. Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases, J. Med. Chem.
  • the activation step occurs when a cytokine binds to its receptor, inducing a multimerization (dimerization or higher order complexes) of receptor subunits.
  • STAT signal transducers and activators of transcription
  • a phosphorylated STAT dimer then translocates to the nucleus of the cell where it binds to target genes modulating their expression.” Once in the nucleus, STATs regulate gene transcription of numerous mediators in the inflammatory process via binding to specific recognition sites on DNA. See, for example, J. Med. Chem.
  • 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 diseases including Crohn's disease and ulcerative colitis (UC) are characterized by recurrent intestinal inflammation, disruption of the epithelial barrier and microbial dysbiosis.
  • the excessive inflammatory response in the gastrointestinal tract is mediated by several pro-inflammatory cytokines including TNF ⁇ , IFN- ⁇ , IL-1, IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-21, and IL-23 that exert their effects on cells of the innate and adaptive immune system including T and B lymphocytes, epithelial cells, macrophages and dendritic cells (DC).
  • T and B lymphocytes including T and B lymphocytes, epithelial cells, macrophages and dendritic cells (DC).
  • pan-JAK inhibitors JAK inhibitors that inhibit a plurality of such JAK proteins, are referred to here as pan-JAK inhibitors. Examples of therapeutic benefits of such prevention or control have been seen with tofacitinib, an orally bioavailable pan-JAK inhibitor approved in the United States for the treatment of rheumatoid arthritis and currently in clinical development for ulcerative colitis.
  • ANC absolute neutrophil counts
  • RA rheumatoid arthritis
  • an orally administered medication can in principle follow the gastro-intestinal tract from the mouth to the esophagus ( 1 ), to the stomach ( 2 ) through the duodenum ( 3 ) to the jejunum ( 4 ), then to the ileum ( 5 ), and then to the colon ( 6 ).
  • the relative absorption areas for such various parts are approximately 60% for the jejunum ( 4 ), approximately 26% for the ileum ( 5 ), and approximately 13% for the colon ( 6 ).
  • Absorption through these various gastro-intestinal regions can lead to the onset of systemic distribution that in turn could lead to undesirable side-effects.
  • the gastro-intestinal tract has a very large surface area. See, for example, H. F.
  • FIG. 1 as permeating through the colon walls for simplified illustrative purposes, but such distribution is not limited to the colon walls, for it also can take place through the walls of other parts of the gastrointestinal tract shown in FIG. 1 , such as those of the small intestine.
  • the dashed arrow lines in FIG. 1 represent systemic distribution beyond the gastrointestinal track as such systemic distribution is known to take place in reference to the gastrointestinal track physiology, and that such dashed line arrows simply refer in a schematic illustrative manner to such systemic distribution. See, for example, Current Topics in Medicinal Chemistry 13, 777-80 (2013), cited above, for a description of intestinal tissue, transport across the same, and metabolism.
  • Tissue-selective modulation of targets in the gastrointestinal tissue with compounds achieving limited systemic exposures can potentially improve the therapeutic index of such compounds for the treatment of diseases of the gastrointestinal tract including ulcerative colitis and Crohn's disease.
  • diseases of the gastrointestinal tract including ulcerative colitis and Crohn's disease.
  • systemic effects is used herein to refer to systemic exposure and the effects of any such systemic exposure, even though they are not always the same.
  • JAK inhibitors Because some known JAK inhibitors have adverse effects that are associated with their systemic effects, it is desirable to find new JAK inhibitors as active substances for the prevention and/or control of excessive inflammatory response and whose systemic effects are eliminated or reduced. It is furthermore desireable to find JAK inhibitors with local effects on gastro-intestinal tissues for the treatment of conditions such as, but not limited to IBD, with reduced systemic effects. Because of the role played by the various JAK proteins, it is furthermore desirable to find pan-JAK inhibitors.
  • Intestinal tissue targeting can in principle be pursued according to multiple strategies. See, for example, Current Topics in Medicinal Chemistry 13, at 780-95 (2013), cited above, referring to approaches that include physicochemical property approaches, transport-mediated approaches, prodrug approaches, and formulation and technology approaches. It is acknowledged, however, that a “number of challenges and pitfalls exist that are endemic to tissue targeting programs” and in particular to intestinally targeted compounds, as described in Current Topics in Medicinal Chemistry 13, at 795 (2013), cited above.
  • IBD conditions can extend to multiple parts of the gastrointestinal tract. Even though for simplified illustrative purposes only a colonic disease site ( 10 ) is shown in the descending colon in FIG. 1 , inflammatory bowel disease may affect any part of the gastrointestinal tract as is the case with Crohn's disease, or in the rectum and colon, as with 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.
  • 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.
  • IBD disease sites can be, for example, ileal (ileum-located), ileocolic (affecting portions of the ileum and colon), and colonic (located in the colon, as illustratively shown in the descending colon in FIG. 1 ). So, in certain disease scenarios, a drug delivery along the entire or a large portion of the intestinal tract may be desirable. In other disease scenarios, it may be desirable to increase local concentration at any given portion of the gastrointestinal tract. Still in other scenarios, a combination of these two forms of delivery at different sites in the intestinal tract could be desirable.
  • JAK inhibitors are envisaged as treatment candidates for other diseases. They are envisaged for use in the treatment of ocular conditions including dry eye (B. Colligris, et al., Recent developments on dry eye disease treatment compounds, Saudi J. Ophthalmol. 28(1), 19-30 (2014)), myeloproliferative neoplasms, myeloproliferative diseases (E. J. Baxter, et al., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders, Lancet 365, 1054-1061 (2005); C.
  • type III hypersensitivity reactions type IV hypersensitivity, inflammation of the aorta, iridocyclitis/uveitis/optic neuritis, juvenile spinal muscular atrophy, diabetic retinopathy, diabetic kidney disease including diabetic nephropathy
  • type III hypersensitivity reactions type IV hypersensitivity
  • inflammation of the aorta iridocyclitis/uveitis/optic neuritis
  • juvenile spinal muscular atrophy diabetic retinopathy
  • diabetic kidney disease including diabetic nephropathy
  • F. C. Brosius, et al. JAK inhibition in the treatment of diabetic kidney disease, Diabetologia 59(8), 1624-7, (2016)
  • C. C. 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); E. N.
  • psoriasis type 1 psoriasis type 2
  • plaque psoriasis moderate to severe chronic plaque psoriasis
  • autoimmune neutropaenia sperm autoimmunity
  • multiple sclerosis all subtypes, B. M. Segal, et al., Repeated subcutaneous injections of IL 12/23 p40 neutralising antibody, ustekinumab, in patients with relapsing-remitting multiple sclerosis: a phase II, double-blind, placebo-controlled, randomised, dose-ranging study, Lancet Neurol. 7, 796-804 (2008); Z.
  • JAK inhibitors have been reported as having therapeutic applications in cancer treatment in addition to inflammatory diseases.
  • S. J. 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.
  • JAK inhibitors could be useful in the prevention of colorectal cancer because inflammation reduction in the colon could lead to cancer prevention in such organ.
  • This invention relates to the following compounds:
  • Embodiments of the present invention relate to compounds, pharmaceutical compositions containing them, methods of making and purifying them, methods of using them as JAK inhibitors and methods for using them in the treatment of disease states, disorders, and conditions mediated by JAK.
  • Embodiments of this invention exhibit pan-JAK inhibition effects with local GI effects and low or negligible systemic effects. Furthermore, embodiments of this invention with such features can be orally administered.
  • An additional embodiment of the invention is a method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by JAK using compounds of the invention or active agents of the invention.
  • FIG. 1 A first figure.
  • the duodenum ( 3 ), jejunum ( 4 ), and ileum ( 5 ) form the small intestine after the stomach ( 2 ) and esophagus ( 1 ).
  • the large intestine comprises the colon ( 6 ), in turn including the cecum ( 7 ) and appendix (not shown), ascending colon, transverse colon, descending colon, sigmoid colon (loop in the same not shown), and rectum ( 11 ).
  • the transverse colon is the portion comprised between the right ( 8 ) and left ( 9 ) colonic flexures, the ascending colon extends from the cecum ( 7 ) to the right colonic flexure ( 8 ), and the descending colon extends from the left colonic flexure ( 9 ) to the rectum ( 11 ).
  • Various distribution patterns are illustrated in reference to the colon for convenience, but they can also refer to other parts of the gastrointestinal tract. Systemic distribution is represented by dashed line arrows in FIG. 1 as permeating through the colon walls for simplified illustrative purposes, but such distribution is not limited to the colon walls, for it also can take place through the walls of other parts of the gastrointestinal tract shown in FIG. 1 , such as those of the small intestine.
  • a disease site is illustratively shown as a colonic disease site ( 10 ) in the descending colon.
  • Embodiments 19-36, 38 and 39 were obtained from embodiment 1s, and embodiments 37 and 40-53 were obtained from embodiment 19, as symbolized in this figure by the dashed line arrow and the legend “19-53” in the box shown in the same.
  • HT-XRPD high throughput X-ray powder diffraction
  • HT-XRPD high throughput X-ray powder diffraction
  • XRPD X-ray powder diffraction
  • XRPD X-ray powder diffraction
  • XRPD X-ray powder diffraction
  • X-ray powder diffraction (XRPD) pattern of embodiment 11b is X-ray powder diffraction (XRPD) pattern of embodiment 11b.
  • Modulated DSC (“mDSC”) profile for embodiment 19 showing a glass transition point (T g ) at 115.3° C. (“Rev” in the ordinate axis label refers to “reversible”).
  • TGA thermogravimeteric analysis
  • DSC differential scanning calorimetry of embodiment 18 showing an endotherm of 52.8 J/g between 45° C. and 90° C., an endotherm of 31.0 J/g at 140.6° C., an exotherm of 24.3 J/g at 168.8° C., and an endotherm of 31.3 J/g at 200.0° C.
  • FIG. 12A is a diagrammatic representation of FIG. 12A
  • TGA of embodiment 17 showing a 4.2% w/w loss between 30° C. and 100° C.
  • DSC of embodiment 17 showing an endotherm of 90.3 J/g between 45° C. and 100° C., an endotherm of 35.5 J/g at 143.8° C., an endotherm of 1.6 J/g at 168.3° C., an exotherm of 3.8 J/g at 178° C., and an endotherm of 9.2 J/g at 200.0° C.
  • XRPD X-ray powder diffraction
  • XRPD X-ray powder diffraction
  • XRPD X-ray powder diffraction
  • TGA of embodiment 11 showing a 4.7% w/w loss between 155° C. and 185° C.
  • DSC of embodiment 11 showing a first endotherm of 57.8 J/g at 167.8° C. due to solvent loss and a second endotherm of 90.8 J/g at 194.5° C. due to sample melt.
  • Gravimetric Vapor Sorption isotherm plot of embodiment 11 showing a mass change of 0.66% between 0-90% RH.
  • the mass change on the ordinate axis is in reference to the mass of the starting sample.
  • TGA of embodiment 6 showing weight loss at temperatures above 260° C., which weight loss is interpreted as being associated with sample degradation.
  • DSC of embodiment 6 showing an endotherm of 95.8 J/g at 194.4° C. due to sample melt.
  • FIG. 20A is a diagrammatic representation of FIG. 20A
  • TGA of embodiment 8 showing a 1.4% w/w loss between 40° C. and 240° C., which corresponds to a loss of 0.07 mol of 1,4-dioxane.
  • DSC of embodiment 8 showing an endotherm of 58.6 J/g at 199.7° C. due to sample melt.
  • FIG. 21A is a diagrammatic representation of FIG. 21A
  • TGA of embodiment 2 showing a 7.4% w/w loss between 75° C. and 110° C., a 11.9% w/w loss between 110° C. and 130° C., a 2.0% w/w loss between 130° C. and 165° C., and a 2.5% w/w loss between 165° C. and 210° C.
  • DSC of embodiment 2 showing an endotherm of 86.2 J/g at 92.8° C., an endotherm of 11.1 J/g at 111.5° C., an endotherm of 45.5 J/g at 149.0° C., an exotherm of 20.6 J/g at 165.2° C., an endotherm of 3.7 J/g at 177.1° C., an endotherm of 43.0 J/g at 200.2° C., and an endotherm of 29.3 J/g at 220.6° C.
  • FIG. 22A is a diagrammatic representation of FIG. 22A
  • DSC of embodiment 9 showing an endotherm of 104.4 J/g at 221.8° C.
  • TGA of embodiment 16 showing a 5.2% w/w loss between 30° C. and 105° C.
  • DSC of embodiment 16 showing an endotherm of 48.4 J/g between 35° C. and 90° C., an endotherm of 41.8 J/g at 147.0° C., an endotherm of 1.0 J/g at 166.6° C., an exotherm of 4.4 J/g at 180.7° C., and an endotherm of 7.7 J/g at 201.1° C.
  • X-ray powder diffraction (XRPD) of embodiment 11 (labeled “11”) and X-ray powder diffraction (XRPD) of embodiment 11 after the variable temperature (VT)-XRPD experiment (labeled “11 post VT”), and X-ray powder diffraction (XRPD) of embodiment 6.
  • Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or forms. Certain structures may exist as tautomers. Additionally, an amorphous form, hydrates, solvates, polymorphs and pseudopolymorphs of such compounds of this invention, and mixtures thereof, are also envisaged as parts of this invention. Embodiments of this invention are in a solvent-free form or in any one of hydrated and/or solvated forms as illustrated herein.
  • references to a compound herein stands for a reference to any one of: (a) the actually recited form of such compound, and (b) any of the forms of such compound in the medium in which the compound is being considered when named.
  • reference herein to a compound such as R—COOH encompasses reference to any one of, for example, R—COOH (s) , R—COOH (sol) , and R—COO ⁇ (sol) .
  • R—COOH (s) refers to the solid compound, as it could be for example in a tablet or some other solid pharmaceutical composition or preparation
  • R—COOH (sol) refers to the undissociated form of the compound in a solvent
  • R—COO ⁇ (sol) refers to the dissociated form of the compound in a solvent, such as the dissociated form of the compound in an aqueous environment, whether such dissociated form derives from R—COOH, from a salt thereof, or from any other entity that yields R—COO ⁇ upon dissociation in the medium being considered.
  • an expression such as “exposing an entity to compound of formula R—COOH” refers to the exposure of such entity to the form, or forms, of the compound R—COOH that exists, or exist, in the medium in which such exposure takes place.
  • an expression such as “reacting an entity with a compound of formula R—COOH” refers to the reacting of (a) such entity in the chemically relevant form, or forms, of such entity that exists, or exist, in the medium in which such reacting takes place, with (b) the chemically relevant form, or forms, of the compound R—COOH that exists, or exist, in the medium in which such reacting takes place.
  • any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.
  • Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number in an enriched form.
  • isotopes that can be incorporated into compounds of the invention in a form that exceeds natural abundances 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 are useful in metabolic studies (preferably with 14 C), reaction kinetic studies (with, for example deuterium (i.e., D or 2 H); or tritium (i.e., T or 3 H)), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F or 11 C labeled compound may be particularly preferred for PET or SPECT studies.
  • substitution with heavier isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased local in vivo half-life or reduced dosage requirements.
  • Isotopically labeled compounds of this invention can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • Tautomers refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures that have an H member in different positions may be in equilibrium while satisfying valency rules. For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base.
  • embodiments of this invention comprise the various groupings that can be made from the listed assignments, taken independently, and equivalents thereof.
  • substituent S example is one of S 1 , S 2 , and S 3
  • this listing refers to embodiments of this invention for which S example is S 1 ; S example is S 2 ; S example is S 3 ; S example is one of S 1 and S 2 ; S example is one of S 1 and S 3 ; S example is one of S 2 and S 3 ; S example is one of S 1 , S 2 and S 3 ; and S example is any equivalent of each one of these choices.
  • Additional embodiments of the invention are pharmaceutically acceptable salts of compounds given above.
  • compositions each comprising an effective amount of at least one of the compounds given above 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 suitable for administration to the subject. See, generally, 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. Compounds of the invention may possess a sufficiently acidic group, a sufficiently basic group, or both types of functional groups, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phen
  • the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, and phosphoric acid, or with 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, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as mandelic acid, citric acid, or tartaric acid, an amino acid, such
  • an inorganic acid such as hydrochlor
  • the compounds of the invention including their pharmaceutically acceptable salts, whether alone or in combination, (collectively, “active agent” or “active agents”) are useful as JAK inhibitors in the methods of the invention.
  • Such methods for modulating JAK activity comprise exposing JAK to an effective amount of at least one chemical compound of the invention.
  • the JAK inhibitor is used in a subject diagnosed with or suffering from a disease, disorder, or medical condition mediated through JAK activity, such as those described herein. Symptoms or disease states are intended to be included within the scope of “diseases, disorders or medical conditions.”
  • the invention relates to methods of using the active agents described herein to treat subjects diagnosed with or suffering from a disease, disorder, or medical condition mediated through JAK.
  • treat or “treating” as used herein is intended to refer to administration of an active agent or composition of the invention to a subject for the purpose of affecting a therapeutic or prophylactic benefit through modulation of JAK. Treating includes reversing, ameliorating, alleviating, inhibiting the progress of, lessening the severity of, reducing, or preventing a disease, disorder, or condition, or one or more symptoms of such disease, disorder or condition mediated through modulation of JAK activity.
  • subject refers to a mammalian patient in need of such treatment, such as a human.
  • inhibitortors or “inhibitor” refers to compounds that decrease, prevent, inactivate, desensitize or down-regulate JAK expression or activity.
  • Embodiments of this invention provide JAK inhibitors for the prevention and/or control of excessive inflammatory response.
  • Embodiments of JAK inhibitors according to this invention are pan-JAK inhibitors.
  • JAK inhibitor physico-chemical properties refers to the corresponding named properties as follows:
  • Embodiments of this invention provide methods of inhibiting JAK, comprising exposing a JAK receptor to a JAK inhibitor that is characterized by having the following JAK inhibitor physico-chemical properties: a plasma concentration in the range from about 0.1 ng/mL to about 60 ng/mL, c Log P in the range from about 0.1 to about 2.8, A-B permeability coefficients in the presence of a P-gp inhibitor in the range from about 0.1 to about 2.5, B-A permeability coefficients in the range from about 0.5 to about 20, tPSA in the range from about 85 to about 120.
  • the plasma concentration is in the range from about 10 ng/mL to about 20 ng/mL.
  • c Log P is in the range from about 0.8 to about 1.4.
  • the A-B permeability coefficient in the presence of a P-gp inhibitor is in the range from about 0.6 to about 1.5.
  • the B-A permeability coefficient is in the range from about 0.5 to about 5.
  • the tPSA is in the range from about 100 to about 120.
  • the molar mass is in the range from about 340 g mol ⁇ 1 to about 430 g mol ⁇ 1 .
  • the number of rotatable bonds is in the range from about 5 to about 6.
  • Embodiments of this invention provide methods for treating inflammation in the gastrointestinal tract of a subject, comprising administering to a subject a pharmaceutically effective amount of a JAK inhibitor that is characterized by having the following JAK inhibitor physico-chemical properties: A plasma concentration in the range from about 0.1 ng/mL to about 60 ng/mL, c Log P in the range from about 0.1 to about 2.8, A-B permeability coefficients in the presence of a P-gp inhibitor in the range from about 0.1 to about 2.5, B-A permeability coefficients in the range from about 0.5 to about 20, tPSA in the range from about 85 to about 120.
  • a JAK inhibitor that is characterized by having the following JAK inhibitor physico-chemical properties: A plasma concentration in the range from about 0.1 ng/mL to about 60 ng/mL, c Log P in the range from about 0.1 to about 2.8, A-B permeability coefficients in the presence of a P-gp inhibitor in the range from about
  • the plasma concentration is in the range from about 10 ng/mL to about 20 ng/mL.
  • c Log P is in the range from about 0.8 to about 1.4.
  • the A-B permeability coefficient is in the presence of a P-gp inhibitor is in the range from about 0.6 to about 1.5.
  • the B-A permeability coefficient is in the range from about 0.5 to about 5.
  • the tPSA is in the range from about 100 to about 120.
  • JAK inhibitor physico-chemical properties are further characterized by having the following JAK inhibitor physico-chemical properties: A molar mass in the range from about 300 g mol 1 to about 500 g mol ⁇ 1 , a number of hydrogen bond donors in the range from about 2 to about 3, a number of hydrogen bond acceptors in the range from about 4 to about 5, and a number of rotatable bonds in the range from about 3 to about 6, in addition to the plasma concentrations, c Log P values, permeability coefficients, and tPSA values described above for methodologies of treating inflammation according to this invention.
  • the molar mass is in the range from about 350 g mol ⁇ 1 to about 430 g mol ⁇ 1 .
  • the number of rotatable bonds is in the range from about 5 to about 6.
  • Embodiments of JAK inhibitors according to this invention have the following JAK physico-chemical properties: a plasma concentration in the range from about 0.1 ng/mL to about 60 ng/mL, a c Log P in the range from 0.1 to about 2.8, an A-B permeability coefficient in the presence of a P-gp inhibitor in the range from about 0.1 to about 2.5, a B-A permeability coefficient in the range from about 0.5 to about 20, and a tPSA in the range from about 85 to about 120.
  • JAK inhibitors according to this invention have a plasma concentration is in the range from about 10 ng/mL to about 20 ng/mL.
  • JAK inhibitors according to this invention have c Log P values in the range from about 0.8 to about 1.4.
  • JAK inhibitors according to this invention have A-B permeability coefficient in the presence of a P-gp inhibitor in the range from about 0.6 to about 1.5.
  • JAK inhibitors according to this invention have B-A permeability coefficient in the range from about 0.5 to about 5.
  • JAK inhibitors according to this invention have tPSA values in the range from about 100 to about 120.
  • JAK inhibitors according to this invention have the following JAK inhibitor physico-chemical properties: A molar mass in the range from about 300 g mol ⁇ 1 to about 500 g mol ⁇ 1 , a number of hydrogen bond donors in the range from about 2 to about 3, a number of hydrogen bond acceptors in the range from about 4 to about 5, and a number of rotatable bonds in the range from about 3 to about 6 in addition to the plasma concentrations, c Log P values, permeability coefficients, and tPSA values described above for JAK inhibitors according to this invention.
  • JAK inhibitors according to this invention have a molar mass is in the range from about 350 g mol ⁇ 1 to about 430 g mol ⁇ 1 .
  • JAK inhibitors according to this invention have a number of rotatable bonds is in the range from about 5 to about 6.
  • an effective amount of at least one active agent according to the invention is administered to a subject suffering from or diagnosed as having such a disease, disorder, or medical condition.
  • An “effective amount” means an amount or dose sufficient to generally bring about the desired therapeutic or prophylactic benefit in patients in need of such treatment for the designated disease, disorder, or medical condition.
  • Effective amounts or doses of the active agents of the present invention may be ascertained by methods such as modeling, dose escalation studies or clinical trials, and by taking into consideration factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician.
  • an illustrative range for a suitable dosage amount is from about 1 to 1000 mg/day in single or multiple dosage units.
  • Embodiments of this invention are new JAK inhibitors as active substances for the prevention and/or control of excessive inflammatory response and whose systemic effects are eliminated or reduced. Further embodiments of this invention are JAK inhibitors with local effects on gastro-intestinal tissues for the treatment of conditions such as, but not limited to, IBD, without causing systemic effects or with such systemic effects acceptably reduced.
  • Embodiments of this invention are low permeability JAK inhibitors. Further embodiments of this invention are JAK inhibitors that have aqueous solubility.
  • the dose may be adjusted for preventive or maintenance treatment.
  • the dosage or the frequency of administration, or both may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained.
  • treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.
  • the compounds of the invention are envisaged for use alone, in combination with one or more of other compounds of this invention, or in combination with additional active ingredients in the treatment of the conditions discussed below.
  • the additional active ingredients may be co-administered separately with at least one compound of the invention, with active agents of the invention or included with such an agent in a pharmaceutical composition according to the invention.
  • additional active ingredients are those that are known or discovered to be effective in the treatment of conditions, disorders, or diseases mediated by JAK activity, such as another JAK inhibitor or a compound active against another target associated with the particular condition, disorder, or disease.
  • the combination may serve to increase efficacy (e.g., by including in the combination a compound potentiating the potency or effectiveness of an agent according to the invention), decrease one or more side effects, or decrease the required dose of the active agent according to the invention.
  • an “effective amount” means an amount sufficient to affect the activity of at least one of the JAK family of proteins. Measuring the activity of the target may be performed by analytical methods.
  • the active agents of the invention are envisaged for use, alone or in combination with one or more additional active ingredients, to formulate pharmaceutical compositions of the invention.
  • a pharmaceutical composition of the invention comprises an effective amount of at least one active agent in accordance with the invention.
  • compositions commonly used in pharmaceutical compositions are substances that are non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • compositions containing one or more dosage units of the active agents may be prepared using pharmaceutically acceptable excipients and compounding techniques known or that become available to those of ordinary skill in the art.
  • the compositions may be administered in the inventive methods by a suitable route of delivery, e.g., oral, parenteral, rectal, topical, or ocular routes, or by inhalation.
  • the preparation may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories.
  • the compositions may be formulated for any one of a plurality of administration routes, such as intravenous infusion, subcutaneous injection, topical administration, or oral administration.
  • the compositions may be formulated for oral administration.
  • the active agents of the invention can be provided in the form of tablets, capsules, or beads, or as a solution, emulsion, or suspension.
  • the active agents may be formulated to yield a dosage of, e.g., for a 70-kg human, from about 1 to 1000 mg/day in single or multiple dosage units as an illustrative range.
  • Oral tablets may include the active ingredient(s) mixed with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents.
  • suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like.
  • Liquid oral excipients include ethanol, glycerol, water, and the like.
  • Starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are disintegrating agents.
  • Binding agents may include starch and gelatin.
  • the lubricating agent if present, may 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 may be coated with an enteric coating. Additional coating that may be used include coatings that are designed to release the compound or active agent as a function of time, pH or bacterial content.
  • Capsules for oral administration include hard and soft gelatin or (hydroxypropyl)methyl cellulose capsules.
  • active ingredient(s) may be mixed with a solid, semi-solid, or liquid diluent.
  • Soft gelatin capsules may be prepared by mixing the active ingredient with an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.
  • Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.
  • suspending agents for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like
  • non-aqueous vehicles e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water
  • compositions may be formulated for rectal administration as a suppository, enema or foam.
  • parenteral use including intravenous, intramuscular, intraperitoneal, or subcutaneous routes, the agents of the invention may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil.
  • Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride.
  • Such forms may be presented in unit-dose form such as ampules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation.
  • Illustrative infusion doses range from about 1 to 1000 ⁇ g/kg/minute of agent admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.
  • the agents may be mixed with a pharmaceutical carrier at a concentration of about 0.01% to about 20% of drug to vehicle, preferably 0.1% to 10%.
  • a pharmaceutical carrier for topical administration, may be mixed with a pharmaceutical carrier at a concentration of about 0.01% to about 20% of drug to vehicle, preferably 0.1% to 10%.
  • Another mode of administering the agents of the invention may utilize a patch formulation to effect transdermal delivery.
  • Active agents may alternatively be administered in methods of this invention by inhalation, via the nasal or oral routes, e.g., in a spray formulation also containing a suitable carrier.
  • the invention is directed to a method of treating a subject suffering from or diagnosed with a disease, disorder, or medical condition mediated by JAK, comprising administering to the subject in need of such treatment an effective amount of the active agent.
  • the disease, disorder, or medical condition is an inflammatory bowel disease, such as Crohn's disease and ulcerative colitis.
  • inventions provide for a method for modulating JAK activity, including when such kinase is in a subject, comprising exposing JAK to an effective amount of at least one compound selected from compounds of the invention.
  • the compounds of the invention are useful as JAK inhibitors that can be dosed orally and specifically distribute to intestinal tissue while maintaining low systemic exposures. This is in contrast to most known JAK inhibitors which are dosed orally and distribute to many tissues due to the fact that they have extensive systemic exposure.
  • Table 1a and Table 1b show results of in vivo experiments. These results comprise plasma and colon tissue concentrations for fifteen compounds that had been administered to mice as described in Protocols 1, 2 or 3. Plasma and colon concentration results were obtained by following Protocol 1 using venipuncture of dorsal metatarsal vein bleed for Compounds (B), (C), and Examples 6 and 11. Plasma and colon concentration results were obtained by following Protocol 2 using retro-orbital bleed for Compounds (A), and Examples 1, and 3-5 and Protocol 2 using venipuncture of the dorsal metatarsal vein for Examples 2, 7-10, and 12. The results of Protocols 1 and 2 are shown in Table 1a. Plasma and colon concentration results were obtained by following Protocol 3 for Examples 1, 3 and 4. The results of Protocol 3 are shown in Table 1b. These protocols are described below under the heading In vivo Studies.
  • Compounds (A)-(C) are the following reference compounds that have been disclosed in WO2013/007765 or WO2011/086053 for their use as inhibitors of Janus kinases:
  • solubilities of compounds Ex. 1-12 were measured in simulated gastric fluid (“SGF”) and simulated intestinal fluid (“SIF”). All compounds tested showed measurable solubility above 400 ⁇ M in SGF, and in the range of 81 ⁇ M to above 400 ⁇ M with SIF. As shown in the same table, these solubility data were comparable to the solubilities of compounds (A)-(C).
  • the permeability of compounds (A)-(C) and Ex. 1-12 was measured using MDCK-MDR1 cell line with and without elacridar, a P-gp inhibitor. All compounds demonstrated low permeability for apical-to-basolateral transport measurements, with and without P-gp inhibitor (elacridar).
  • the permeability coefficient values for compounds (A)-(C) and Ex. 1-12 were low and comparable for apical-to-basolateral transport without elacridar (for all such compounds) and with elacridar (for compounds (B)-(C) and compounds Ex.
  • reaction mixtures were magnetically stirred at room temperature (rt). Where solutions are “dried,” they are generally dried over a drying agent such as Na 2 SO 4 or MgSO 4 . Where mixtures, solutions, and extracts were “concentrated”, they were typically concentrated on a rotary evaporator under reduced pressure.
  • Thin-layer chromatography was performed using Merck silica gel 60 F 254 2.5 cm ⁇ 7.5 cm, 250 ⁇ m or 5.0 cm ⁇ 10.0 cm, 250 ⁇ m pre-coated silica gel plates.
  • Mass spectra were obtained on an Agilent series 1100 MSD using electrospray ionization (ESI) in positive mode unless otherwise indicated. Calculated (calcd.) mass corresponds to the exact mass.
  • Nuclear magnetic resonance (NMR) spectra were obtained on Bruker model DRX spectrometers.
  • the format of the 1 H NMR data below is: chemical shift in ppm downfield of the tetramethylsilane reference (multiplicity, coupling constant J in Hz, integration).
  • yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity that could be obtained under the particular stoichiometric conditions.
  • Reagent concentrations that are given as percentages refer to mass ratios, unless indicated differently.
  • the TGA plots for individual embodiments are shown in terms of temperature in ° C. on the X-axis and weight loss in % on the Y-axis.
  • the DSC plots for individual embodiments are shown in terms of temperature in ° C. on the X-axis and heat flow in W/g on the Y-axis.
  • the DSC heating rate was 10° C./min. Integrations of endothermic and exothermic events provide the energy absorbed (for an endothermic event) or energy released (for an exothermic event) in J/g. Dashed lines shown going across the trace represent the area that was integrated.
  • diffractograms have been presented in an overlay arrangement of diffractograms that are separated by spacings to allow visualization.
  • Each of the diffractograms is referenced to a zero relative intensity that is the intersection of each of such diffractograms with the ordinate axis or to the lowest relative intensity reading of each of such diffractograms.
  • Figures that display a plurality of XPRD patterns for any single embodiment reflect different patterns obtained for samples of such embodiment that were nevertheless prepared with the same method in different solvents.
  • Step A tert-butyl N-[(1r,4r)-4-(Hydroxymethyl)cyclohexyl]carbamate.
  • the resulting solution was stirred for 3 h at 15° C.
  • Step B tert-butyl N-[(1r,4r)-4-[(Methanesulfonyloxy)methyl]cyclohexyl]carbamate.
  • tert-butyl N-[(1r,4r)-4-(hydroxymethyl)cyclohexyl]carbamate 1000 g, 4.36 mol, 1.00 equiv.
  • dichloromethane 10 L
  • pyridine (1380 g, 17.5 mol, 4.00 equiv.).
  • Step C tert-butyl N-[(1r,4r)-4-(Cyanomethyl)cyclohexyl]carbamate.
  • tert-butyl N-[(1r,4r)-4-[(methanesulfonyloxy)methyl]cyclohexyl]carbamate (1100 g, 3.58 mol, 1.00 equiv.)
  • DMSO 5500 mL
  • NaCN 406 g, 8.29 mol, 2.30 equiv.
  • Step D 2-[(1r,4r)-4-Aminocyclohexyl]acetonitrile hydrochloride.
  • tert-butyl N-[(1r,4r)-4-(cyanomethyl)cyclohexyl]carbamate 620 g, 2.60 mol, 1.00 equiv.
  • 1,4-dioxane 2 L
  • the resulting solution was stirred overnight at 25° C. This reaction was performed for 4 times and the reaction mixtures were combined.
  • the solids were collected by filtration.
  • Step E 2-((1r,4r)-4-((5-Nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)cyclohexyl)acetonitrile.
  • 2-[(1r,4r)-4-aminocyclohexyl]acetonitrile hydrochloride 29.10 g, 166.6 mmol
  • DMA 400 mL
  • a reflux condenser was attached to the reaction flask, the reaction was purged with N 2 , and was heated at 90° C. for 9 h.
  • the reaction mixture was cooled to room temperature and left to stand for 30 h where the product crystallized out 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 via separatory funnel with vigorous stirring. After addition of the water was complete, the suspension was stirred for 30 minutes.
  • the brown needles were isolated by filtration and then dried by pulling air through the filter for 1 h.
  • the product was transferred to a 500 mL flask and treated with EtOAc (200 mL).
  • Step A 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide.
  • Step B 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide.
  • the reaction was cooled to room temperature and the solvent volume was reduced to about 200 mL on a rotovap.
  • the residue was treated with a solution of water/brine (100 mL/100 mL), then extracted with 10% MeOH in CH 2 Cl 2 (2 ⁇ 1 L).
  • the organic layers were combined, dried over anhydrous MgSO 4 , and concentrated to dryness to provide a yellow solid.
  • the solid was suspended in CH 2 Cl 2 (200 mL), stirred vigorously for 30 minutes, and then collected by filtration.
  • the solid was rinsed with CH 2 Cl 2 (100 mL), dried by pulling air through the filter, and then further dried under vacuum at room temperature for 16 h to provide the title compound (41.59 g, 89% yield) as a white solid.
  • Step A 2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)-6-(phenylsulfonyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1 (6H)-yl)cyclohexyl)acetonitrile.
  • 1H-Imidazole-4-carbaldehyde (8.56 g, 89.1 mmol) was added as a solid, followed by the addition of sodium hydrosulfite (32.7 g, 188 mmol) as a solution in water (100 mL).
  • the reaction vessel was equipped with a reflux condenser and heated to 90° C. in a heating block for 15 h.
  • the reaction mixture was then cooled to room temperature and added to a flask containing water (2000 mL) with stirring, which resulted in formation of a white precipitate.
  • the mixture was stirred for 30 minutes 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 heating at 60° C.
  • Step B 2-((1r,4r)-4-(2-(1H-Imidazol-4-yl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)cyclohexyl)acetonitrile.
  • Step A 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide.
  • Step B 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(cyclopropylmethyl)acetamide.
  • the DMF was removed under reduced pressure and the residue was purified by flash column chromatography (50-100% EtOAc/heptanes, then 10% MeOH/DCM) and the subsequently by reverse phase HPLC using a Varian Pursuit XR s 5 Diphenyl 100 mm ⁇ 30 mm column (eluent 10-90% CH 3 CN in water, 0.1% TFA) to provide the product as the TFA salt.
  • This material was dissolved in 10% MeOH in CH 2 Cl 2 and passed through a 500 mg column of SILICYCLE SPE-R66030B-03P Carbonate (SiliaBond acid scavenger solid phase extraction cartridge) to remove the TFA to provide the title compound (34 mg, 29% yield) as a white solid.
  • Step A Dimethyl cyclopentane-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, maintaining the temperature at ⁇ 15° C. After the addition, the reaction was heated to 90° C. and stirred overnight. The reaction was cooled to room temperature and concentrated to dryness. The residue was treated with MTBE (500 mL) and H 2 O (100 mL). The aqueous layer was separated and extracted with MTBE (2 ⁇ 100 mL).
  • Step B Dimethyl bicyclo[2.2.1]heptane-1,4-dicarboxylate.
  • n-Butyllithium 2.5 M in hexane, 419.0 mL, 1048 mmol
  • diisopropylamine 152 mL, 1090 mmol
  • anhydrous THF 1000 mL
  • DMPU 404 mL, 3350 mmol
  • Step C 4-(Methoxycarbonyl)bicyclo[2.2.1]heptane-1-carboxylic acid.
  • a methanol (80 mL) solution of sodium hydroxide (5.145 g, 128.6 mmol) was added slowly to a solution of dimethyl bicyclo[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 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 of H 2 O.
  • the precipitate was collected by filtration and dried under vacuum to provide the title compound (13.0 g, 51.0%) as white solid.
  • the filtrate was extracted with ethyl acetate (3 ⁇ 75 mL) and the combined organic extracts were washed with brine (50 mL), dried over anhydrous MgSO 4 , filtered and concentrated to dryness to provide a second fraction of the title compound (8.0 g, 31%) as a white solid.
  • Step 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 hours. The reaction was cooled to 50° C.
  • Step E Methyl 4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylate.
  • Step F 4-((tert-Butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylic acid.
  • THF 40 mL
  • MeOH 20 mL
  • aqueous sodium hydroxide 1.0 M, 46.4 mL, 46.4 mmol
  • Step G tert-Butyl (4-(hydroxymethyl)bicyclo[2.2.1]heptan-1-yl)carbamate.
  • a solution of borane-tetrahydrofuran complex (1.0 M, 37.1 mL, 37.1 mmol) was added slowly to a solution of 4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptane-1-carboxylic acid (4.74 g, 18.6 mmol) and anhydrous THF (50 mL) at 0° C. under a nitrogen atmosphere. After the addition was complete, the reaction was stirred at room temperature overnight. Water (30 mL) was added to the mixture slowly and it was stirred for additional 30 minutes.
  • 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 was cooled to 0° C. and methansulfonyl chloride (2.0 mL, 25.0 mmol) was added and the mixture was stirred for 3 hours at room temperature.
  • Step I tert-Butyl (4-(cyanomethyl)bicyclo[2.2.1]heptan-1-yl)carbamate.
  • (4-((tert-butoxycarbonyl)amino)bicyclo[2.2.1]heptan-1-yl)methyl methanesulfonate (2.58 g, 8.07 mmol)
  • DMSO 25 mL
  • sodium cyanide (1.20 g, 24.5 mmol.
  • the reaction was heated to 100° C. and stirred for 24 hours.
  • the reaction was diluted with 50 mL of water and extracted with ethyl acetate (3 ⁇ 40 mL).
  • Step J 2-(4-Aminobicyclo[2.2.1]heptan-1-yl)acetonitrile hydrochloride.
  • 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 isolated via filtration.
  • Step 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.
  • Step A 2-(1-((1r,4r)-4-(Cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(1H-pyrazol-3-yl)acetamide.
  • the title compound (383 mg, 70%) was prepared in a manner analogous to that described in Example 1, Step A using 1H-pyrazol-3-amine (465 mg, 5.48 mmol) instead of 1-amino-2-methylpropan-2-ol.
  • Step B 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.
  • Some embodiments of compounds according to this invention as free bases present multiple crystalline configurations that have a complex solid-state behavior, some of which in turn can present distinguishing features among themselves due to different amounts of incorporated solvent.
  • Some embodiments of compounds according to this invention are in the form of pseudopolymorphs, which are embodiments of the same compound that present crystal lattice compositional differences due to different amounts of solvent in the crystal lattice itself.
  • channel solvation can also be present in some crystalline embodiments of compounds according to this invention, in which solvent is incorporated within channels or voids that are present in the crystal lattice. For example, various crystalline configurations including those given in Table 2, were found for compound Ex. 1.
  • Embodi- Crystallization ment solvent Solvation Stoichiometry 1s — monohydrate 0.8 H 2 O 1a Water monohydrate 1.3 H 2 O 1b Toluene Toluene solvate 0.4 toluene 1c Ethyl acetate/ monohydrate 1.1 H 2 O 1,4-dioxane 1d Acetonitrile/ 1.7 hydrate 1.7 H 2 O chloroform 1e Ethyl acetate/ monohydrate 1 H 2 O 1,4-dioxane 1f p-xylene p-xylene solvate 0.3 p-xylene 1f Cumene Cumene solvate 0.3 cumene 1g Anisole Anisole solvate 0.3 anisole 1h p-xylene p-xylene solvate 0.2 p-xylene 2 1,4-dioxane 1,4-dioxane solvate 1.2 1,4-
  • Example 1 The compound that was obtained as described in Example 1 was further crystallized by preparing a slurry in DCM (1:3, for example 10 g of compound in 30 ml DCM) that was stirred at 40° C. for 4 hours, and further stirred for 14 hours at 25° C., then heptane was slowly added (1:2, for example 20 ml of heptane into the compound/DCM slurry/solution) at 25° C., stirred at 40° C. for 4 hours, cooled to 25° C. and stirred for further 14 hours at 25° C. Subsequent filtration lead to compound Ex. 1 in the form of an off-white solid, that was identified as a monohydrate, a 1 s embodiment.
  • An amorphous form of compound Ex. 1, embodiment 19, was prepared as follows. Embodiment is (1 g) was dissolved in t-butanol (40 vol) and stirred at 50° C. Pre-dried molecular sieves were added to the solution and stirred for 10 min. The solution was filtered and aliquoted into HPLC vials (1 ml) which were frozen in a dry ice-acetone bath right afterwards. Samples were then placed on the freeze drier for 48 h. The material was amorphous by XRPD and consistent with the proposed structure by 1 H-NMR, with 0.4 mol of t-butanol per molecule of compound Ex. 1 present. This material was heated to 150° C. and held at 150° C. for 10 min. The final sample was analyzed by XRPD and 1 H-NMR and it was determined to be amorphous and that 0.03 mol of t-butanol remained per molecule of compound Ex. 1.
  • Embodiments 1-18 in Table 2 and FIG. 2 are crystalline, and embodiment 19 in FIG. 2 is amorphous.
  • Embodiments 1s and 1a through 1h are isostructural.
  • Embodiment 1s crystallizes in a centro-symmetrical triclinic space group P-1.
  • the term “embodiment 1” collectively refers to the isostructural embodiments 1s and 1a through 1h. Any one of such 1s and 1a through 1h embodiments is sometimes referred to as an isostructural member of embodiment 1 or just as a member of embodiment 1.
  • Embodiments 3b, 3c, 3d and 3e are isostructural and crystallize in the monoclinic system, space group C 2/c.
  • invention 3 collectively refers to the isostructural embodiments 3b, 3c, 3d and 3e. Any one of such 3b, 3c, 3d and 3e embodiments is sometimes referred to as an isostructural member of embodiment 3 or just as a member of embodiment 3.
  • Isostructural embodiments are such that they possess similar crystal structure properties (same symmetries and similar unit cell parameters and crystal packing) while having different chemical compositions (i.e., different solvent and/or water molecules incorporated in the crystal lattice). Unit cell parameters in isostructural embodiments can slightly differ due to the different composition (solvent or water incorporated into the crystal structure).
  • Embodiments referred to in Table 2 were prepared and/or inter-converted as schematically shown in FIG. 2 and as described in more detail as follows.
  • Crystallization protocols used in these preparations included solvent equilibration in neat solvents, evaporative crystallization, cooling crystallization with hot filtration, crash-crystallization with anti-solvent, crystallization by thermocycling, incubation at low temperature, heat/cool maturation, incubation at elevated temperature, high temperature maturation using amorphous material (embodiment 19), and thermocycling using amorphous material (embodiment 19).
  • Solids were analyzed by HT-XRPD or XRPD. When applicable, mother liquors were evaporated completely and the remaining solids were also analyzed by HT-XRPD or XRPD.
  • the starting material embodiment 1s as a monohydrate was a predominant solid form.
  • the starting material as embodiment 1s was obtained. From several crystallization solvents, HT-XRPD patterns were found to be similar to those of the initial embodiment 1s. In most of these diffraction patterns, peak shifts and/or additional peaks were identified. Each of these patterns corresponded to an embodiment that was labeled as one of 1a through 1h, and based on the similarities in the HT-XRPD diffraction patterns for such embodiments, they are presented as embodiments that are isostructural members of embodiment 1. All isostructural members of embodiment 1 converted to embodiment 1a after exposure to 40° C. and 75% RH for two days.
  • Embodiment 1s converted to hydrated embodiment 10 when it was exposed to 100% RH at 25° C. Nevertheless, embodiment 10 was physically not stable at ambient conditions. Whereas embodiment 1s crystallized in the triclinic system, space group P-1, embodiment 10 was found to crystallize in the monoclinic system, space group C 2/c. Embodiment 10 had limited physical stability under ambient conditions and it converted to another embodiment such as 1s or 1a. This behavior is attributable to an unequally strong binding of all the hydration/solvation molecules. In this case, embodiment 10 would have a less strongly bound second water molecule that would be lost under ambient conditions. More precisely, the physical stability of embodiment 1s was investigated in climate chambers by exposing a 20 mg sample of such embodiment to 40° C.
  • the third diffractogram corresponds to embodiment 1s after a 4-day exposure to 25° C. and 100% relative humidity, noted as “10” in the same figure. These conditions lead to the conversion of embodiment 1s into embodiment 10, with a small contamination of initial embodiment 1s, and solvation as characterized in Table 2. Upon dehydration, both embodiments 1s and 10 re-crystallized to the anhydrous form with a melting point of 148° C.
  • Embodiment 10 was also prepared by slurry conversion of embodiment 1s or embodiment 19 in water, with temperature cycling of 25-5° C.
  • the slurry was prepared by suspending 50 mg of material in 1 mL water. Temperature cycling of 25-5° C. is: the mixture was heated at 25° C. for 1 hour and then the temperature was decreased to 5° C. over a 2 hour period. The mixture was then held at a temperature of 5° C. for 1 hour. The temperature was then increased to 25° C. over a 2 hour period. This temperature cycling regime was repeated for a total of about 24 hours.
  • the solids were isolated by vacuum filtration and then dried on a filter for 10 minutes.
  • Embodiment 10 converted to embodiment 1s during drying under ambient conditions and under vacuum.
  • Solvent equilibration at room temperature yielded embodiment 1b out of toluene as the crystallization solvent, and embodiment 1f out of p-xylene as the crystallization solvent.
  • Embodiment 2 whose TGA and DSC are shown in FIGS. 21A and 21B , respectively, was identified from the solvent equilibration experiment performed at room temperature in 1,4-dioxane while embodiment 7 was found as a mixture with embodiment 1s in the single solvent equilibration experiment at 50° C. from heptane.
  • Several similar but not identical diffractograms were identified which were grouped as embodiments 3b, 3c, 3d and 3e that are isostructural members of embodiment 3. Isostructural members of embodiment 3 were found mixed with members of embodiment 1.
  • the mixtures containing members of embodiment 3 transformed in some cases to embodiment 1a or to mixtures of embodiments 1a and 3e.
  • Embodiment 7 appeared to be physically stable, but embodiment 2 converted to embodiment 3e after exposure to AAC for two days.
  • the mother liquors saved from the solvent equilibration experiments performed at RT were used for slow evaporative crystallization experiments.
  • the mother liquors were filtered to remove any particulate matter and allowed to slowly evaporate under ambient conditions.
  • the obtained solids were analyzed by HT-XRPD and again after exposure to AAC for two days.
  • the mother liquors of the solvent equilibration experiments performed at 50° C. were filtered at 50° C. to remove any particulate matter.
  • the suspensions at 50° C. were filtered using 0.2 m PTFE filters, and the solutions were placed at 5° C. and aged for 72 hours.
  • HT-XRPD 0.2 m PTFE filters
  • the remaining mother liquors were allowed to slowly evaporate and the remaining solids were analyzed by HT-XRPD.
  • the samples in which no precipitation occurred were placed under vacuum and the dried solids were analyzed by HT-XRPD. All the solids were then exposed to AAC (2 days at 40° C./70% RH) and re-analyzed by HT-XRPD.
  • Embodiment 6 was identified after evaporation of a single cooling crystallization experiment at mL scale in 800 ⁇ L acetonitrile, concentration of 25 mg/mL. Embodiment 6 seemed to be a stable solid form after 2 days AAC, and it appeared as a non-solvated embodiment.
  • the cooling/evaporative crystallization experiments at ⁇ L scale were performed in a 96-well plate, using 12 neat solvents and 12 solvent mixtures and applying four temperature profiles. In each well approximately 4 mg of embodiment 1s was solid dosed. Subsequently, the crystallization solvents (80 ⁇ L) and solvent mixtures were added to reach a concentration of 50 mg/mL, and the plate, with each well individually sealed, to subsequently undergo one of the four temperature profiles. Upon completion of the temperature profile the solvents were allowed to evaporate at low ambient pressure (24 hours) and the remaining solids were analyzed by HT-XRPD before and after exposure to AAC for 2 days (40° C./70% RH).
  • Embodiment 1b was mainly identified from the short temperature profiles (3 hours aging). Nevertheless, the same solvent systems with long aging times led to the identification of embodiment 1f, members of embodiment 3 or mixtures of members of embodiments 1 and 3.
  • Embodiment 3c was obtained with 1,4-dioxane as crystallization solvent and a temperature profile of 50° C.
  • embodiment 3d was obtained with tetrahydrofuran as crystallization solvent and the same temperature profile as for embodiment 3c.
  • Embodiment 4 was identified in experiments performed in methanol/water (50/50, v/v), THF and DCM/IPA (50/50, v/v) when short aging conditions were applied.
  • Embodiment 4 was obtained by treating embodiment 1s with a mixture (50/50) of water and methanol and a temperature profile of 50° C. as initial temperature, held for 60 min, followed by cooling at a rate of 20° C./h to a final temperature of 5° C., held for 3 h, which yielded embodiment 4 together with embodiment 1b.
  • Embodiment 4 together with embodiment 1b was also obtained by treating 1s with a mixture (50/50) of water and methanol and a temperature profile of 50° C.
  • Embodiment 4 did not appear to be physically stable under ambient conditions. Cooling crystallization experiments yielded embodiment 1c out of ethyl acetate/1,4-dioxane (50/50, v/v) as the crystallization solvent and a temperature profile of 50° C.
  • Embodiment 5 was identified in experiments performed in chloroform as the crystallization solvent and a temperature profile of 50° C. as initial temperature, held for 60 min, followed by cooling at a rate of 1° C./h to a final temperature of 20° C., held for 48 h.
  • the cooling crystallization experiments with hot filtration were performed from supersaturated solutions of compound Ex. 1 prepared at 50° C. in different solvent mixtures.
  • the hot filtrated solutions underwent a 48-hour cooling profile.
  • the vials in which solids had precipitated after the temperature profile were centrifuged and the solids were separated from the liquid and analyzed by HT-XRPD (after drying under vacuum). If no solids had precipitated the solutions were evaporated under vacuum and the solids analyzed by HT-XRPD. All the solids were exposed to AAC (2 days at 40° C./70% RH) and re-analyzed by HT-XRPD.
  • Suspensions of about 6 mg of embodiment 1s were prepared in 10 solvents at room temperature. The suspensions were cycled between 5° C. and 50° C. Upon completion of the thermo-cycling, the solids were separated by centrifugation and dried under ambient conditions and under vacuum (5 mbar) before being analyzed by HT-XRPD. Subsequently, all solids were exposed to AAC for two days and again analyzed by HT-XRPD. Thermo-cycling experiments usually promote the formation of the more stable polymorphic form. With the exception of the experiment performed in cyclohexanone all vials contained solids after the thermo profile. The cyclohexanone solution was slowly evaporated under mild vacuum.
  • Embodiments 1, 3 or mixtures of them were identified mainly in the wet solids. Upon drying these solids, conversion to embodiment 1s was observed.
  • Embodiments 3b and 3e were obtained from thermo-cycling in 300 ⁇ L of cyclohexanone at a concentration of 51 mg/mL (3b), and in 400 ⁇ L of isobutanol at a concentration of 37.3 mg/mL (3e).
  • Embodiment 5 was obtained from thermo-cycling in 800 ⁇ L of chloroform at a concentration of 18.6 mg/mL.
  • FIGS. 3, 4 and 5 show an overlay of HT-XRPD patterns for some of the embodiments listed in Table 2 and also referred to in the screenings described above.
  • Embodiment 1s was recovered from most of the crystallization experiments. It is a channel hydrate having a variable number of water molecules and/or other solvents incorporated depending on ambient conditions. Conversion to embodiment 1a was observed. This form contained slightly more water (1.3 molecules of water). All isostructural members of embodiment 1 converted to embodiment 1a after exposure to 40° C. and 75% RH for two days. The shifts of some diffraction peaks in XRPD patterns for members of embodiment 1 might be attributed to the different solvent or water molecules that were incorporated into the crystal lattice.
  • FIG. 4 shows an overlay of HT-XRPD patterns for members of embodiment 1. Diffractogram 1s corresponds to compound Ex. 1 as starting material in the form of embodiment 1s.
  • Diffractogram 1a corresponds to embodiment 1a that was obtained after exposure to AAC of several embodiment 1s samples.
  • Diffractogram 1b corresponds to embodiment 1b that was obtained from the solvent equilibration experiment at RT in toluene.
  • Diffractogram 1c corresponds to embodiment 1c that was obtained from the cooling crystallization experiment at ⁇ L scale in ethyl acetate/1,4-dioxane (50/50, v/v).
  • Diffractogram 1c corresponds to embodiment 1d that was obtained from the cooling crystallization experiment at ⁇ L scale in acetonitrile/chloroform (50/50, v/v).
  • Diffractogram 1e corresponds to embodiment 1e that was obtained from the cooling crystallization experiment at ⁇ L scale in ethyl acetate/1,4-dioxane (50/50, v/v).
  • Diffractogram if corresponds to embodiment 1f that was obtained from the solvent equilibration experiment at RT in p-xylene.
  • Diffractogram 1g corresponds to embodiment 1g that was obtained from the solvent equilibration experiment at 50° C. in anisole.
  • Diffractogram 1h corresponds to embodiment 1h obtained from the cooling crystallization experiment at ⁇ L scale in p-xylene.
  • Embodiment 3 Diffractograms for members of embodiment 3 are shown in FIG. 5 .
  • the shifts observed in the different HT-XRPD patterns are most likely attributed to the different solvent molecules that were incorporated into the crystal lattice.
  • Embodiment 3 was obtained by heating embodiment 2 to 40° C. at 70% RH for 4 days.
  • Embodiments 3b through 3e were solvated forms containing a non-stoichiometric amount of solvent which varied depending on the solvent incorporated in the crystal structure (0.3-0.7 molecules).
  • the mixtures containing members of embodiment 3 were unstable upon exposure to AAC and they transformed in some cases to embodiment 1a or to mixtures of embodiments 1a and 3e. Conversion to embodiment 1a is attributed to the exchange of solvent molecules by water molecules upon exposure to high relative humidity, and re-crystallization to the 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 cyclic DSC 25-200-25-300° C. Embodiment 9 was also obtained by additional procedures.
  • One of such procedures was a two-step procedure: Embodiment 1s (1.5 g) was treated with 1,4-dioxane (10 vol) at RT. Seeds of embodiment 2 (5 mg) were added and the sample was stirred at RT for 24 hours. The resulting suspension was filtered and the sample was air-dried for 1.5 hours. This sample was determined to be embodiment 2 by XRPD.
  • embodiment 2 was heated to 210° C. at 10° C./min and held at 210° C.
  • embodiment 9 was determined to be embodiment 9 by XRPD analysis.
  • Another of such procedures was also a two-step procedure for obtaining embodiment 9.
  • embodiment 1s 1.5 g was treated with 1,4-dioaxne (10 vol). Seeds of embodiment 2 (5 mg) were added and the sample was stirred at RT for 24 hours. The resulting suspension was filtered and the sample was air-dried for 1.5 hours.
  • This sample was determined to be embodiment 2 by XRPD.
  • embodiment 2 was heated to 150° C. at 10° C./min followed by further heating to 170° C. at 2° C./min. The sample was then allowed to cool to RT.
  • the resulting solid was determined to be embodiment 9 by XRPD analysis.
  • the TGA and DSC of embodiment 9 is shown in FIGS. 22A and 22B , respectively.
  • Embodiment 1s was obtained by slurring embodiment 9 in the following solvents for 6 days at 50° C.: 2-butanone, acetone/water (90/10, v/v) and acetonitrile/water (90/10, v/v). Embodiment 1s was also obtained when the same experiment was performed at room temperature.
  • Embodiment 8 was obtained by heating embodiment 5 to a temperature of about 175° C.
  • Embodiment 8 was also obtained by additional procedures.
  • One of such procedures was a two-step procedure: Embodiment 1s (1.5 g) was treated with 1,4-dioxane 10 (vol) and stirred at RT for 72 hours. The resulting suspension was filtered and the solid that was obtained was dried in a vacuum oven at RT for 16 hours.
  • the solid obtained from this first step was determined by XRPD to be embodiment 3c.
  • embodiment 3c 100 mg was heated to 150° C. at 10° C./min, then heated at the slower rate of 2° C./min up to 180° C. The sample was then allowed to cool back to RT.
  • the resulting solid was determined by XRPD to be embodiment 8.
  • Another of such procedures was also a two step procedure for obtaining embodiment 8.
  • embodiment 19 300 mg was treated with 1,4-dioxane (3 vol) and shaken at 60° C. for 24 hours.
  • the resulting suspension was filtered and the solid obtained from this first step was determined by XRPD to be embodiment 3c.
  • embodiment 3c 300 mg was heated to 180° C. at 10° C./min.
  • the sample was then allowed to cool back to RT.
  • the resulting solid was determined by XRPD to be embodiment 8.
  • the TGA and DSC for embodiment 8 is shown in FIGS. 20A and 20B , respectively.
  • Embodiment 6 was also obtained from embodiment 11 by subjecting it to a slurry experiment. The slurry experiment was run as follows: the solvent was added to embodiment 11 (50 mg) and the mixture was stirred at the designated temperature for 0.5 hours. Seed crystals of form 9 (5 mg) were added and the mixture was stirred overnight at the designated temperature. The solids were isolated by centrifugation and analyzed by XRPD. using isopropyl acetate (0.5 mL) at both 30° C. and 50° C. The generation of embodiment 6 was confirmed by XRPD. The TGA and DSC for embodiment 6 is shown in FIGS. 19A and 19B , respectively.
  • Embodiment 5 was converted to embodiment 9 by subjecting it to slurry experiments Slurry experiments were conducted as follows using various solvents at the temperatures identified: The solvent was added to embodiment 5 (50 mg) and the mixture was stirred at the designated temperature for 0.5 hours. Seed crystals of form 9 (5 mg) were added and the mixture was stirred overnight at the designated temperature. The solids were isolated by centrifugation and analyzed by XRPD. Slurry experiments run at 50° C. were conducted using the following solvents: TBME (0.75 mL) and a 33:67 mixture of isopropyl acetate: heptane (0.5 mL). Slurry experiments run at 75° C. were conducted using the following solvents: isopropyl acetate (0.5 mL) and methyl ethyl ketone (0.5 mL).
  • Embodiment 11 was obtained as follows: A suspension of embodiment 1s (45 g) in ethanol (absolute, water content ⁇ 0.1%, 300 mL) at 50° C. was stirred for 16.5 hours. The suspension was then cooled to 5° C. at 0.25° C./minute. Subsequently, the suspension was stirred at 5° C. for 3 hours. The solids were then filtered off and washed with cold (5° C.) ethanol (absolute, water content ⁇ 0.1%, 90 mL), and dried under vacuum at 40° C. for 17 hours to yield approximately 39 g of embodiment 11. The TGA and DSC of embodiment 11 is shown in FIGS. 17A and 17B , respectively.
  • Embodiment 11 was also obtained as follows: Absolute ethanol (170 mL) was added to embodiment 1s (19 g) and heated to about the boiling point of the solvent. A small amount of the solids (5%) did not dissolve and were removed by hot filtration. It was determined that the solids that were filtered off, were embodiment 1s. So the solids were added back into the filtrate and this mixture was heated until all the solids dissolved. To this hot solution was added, heptane (535 mL), drop-wise via a separatory funnel. During this drop-wise addition of heptane, the hot solution was stirred vigorously.
  • Embodiment 11 was also subjected to static storage analysis at 40° C./75% RH for up to 48 days.
  • the samples were analyzed by XRPD and Karl Fisher (KF) after 2 days, 5 days and 48 days.
  • Embodiment 11 remained unchanged as shown by XRPD analysis with a total water uptake of 1.2% after 48 days.
  • 1 H-NMR of the material post 48 days static storage showed the material retained 0.36 mol eq of ethanol.
  • Embodiment 11 stored under ambient conditions for the period of the study was shown to contain 0.46 mol eq of ethanol by 1 H-NMR.
  • Embodiment 11b was obtained from embodiment 1s as follows: 10 mL of dried methanol was added to 3.3 g of embodiment 1s. This mixture was subjected to the following temperature cycling: The mixture was heated at 40° C. for 1 hour and then the temperature was increased to 60° C. over a 2 hour period. The mixture was then heated at 60° C. for 1 hour. The temperature was then decreased to 40° C. over a 2 hour period. This temperature cycling regime was repeated for a total of about 20 hours. At that time the mixture was cooled to 5° C. over a 2 hour period. The solids were isolated at 5° C. by vacuum filtration and then dried at ambient temperature under vacuum for about 66 hours.
  • embodiment 11b was obtained from embodiment 1s using the following procedure: 1 mL of dried methanol was added to 330 mg of embodiment 1s. This mixture was subjected to the following temperature cycling: The mixture was heated at 40° C. for 1 hour and then the temperature was increased to 60° C. over a 2 hour period. The mixture was then heated at 60° C. for 1 hour. The temperature was then decreased to 40° C. over a 2 hour period. This temperature cycling regime was repeated for a total of about 18 hours. At that time the solids were isolated by centrifugation and then dried at ambient temperature under vacuum for about 33 hours. The methanol for the above experiments was dried using molecular sieves (3 ⁇ , activated at 100° C. under vacuum for at least 24 h). The diffractogram for embodiment 11b is shown in FIG. 7E .
  • Embodiment 12 was obtained from embodiment 1s, which was exposed to humidity conditions below 10% RH at 25° C. to provide embodiment 12.
  • the diffractogram for embodiment 12 is shown in FIG. 7B .
  • Embodiment 13 was obtained as follows: To a 250 mL 4-necked flask at 25 ⁇ 5° C. was added a sample of embodiment 1s. The flask was then charged with MeOH (4.0 V, 40 mL) and purified water (10 mL, 1.0 V) and stirred until all the solid dissolved. N 2 was bubbled into the mixture for 1 hour and the mixture was then cooled to 0 to 5° C.
  • a 0.225 mL volume of a cooled solution (0 to 5° C.) of NaBH 4 /water (0.006 eq., 2.5% w/w) was prepared with purified water (40 mL) charged into a 100 mL of a 4-necked flask under N 2 at 0° C., followed by the addition of NaBH 4 (1.0 g); the mixture was stirred at 0° C. until all the NaBH 4 dissolved.
  • Such NaBH 4 solution was added into the 250-mL flask that was cooled (0 to 5° C.) and stirred at 0 to 5° C. The color of the reaction mixture changed to yellow.
  • Embodiment 14 was prepared as follows: 2-(1-((1r,4r)-4-(cyanomethyl)cyclohexyl)-6-(phenylsulfonyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)-N-(2-hydroxy-2-methylpropyl)acetamide (48.15 kg, prepared in Ex. 2, Step B), EtOH (technical grade, 481 L) and KOH (6.613 kg) were stirred at 10-20° C. for 9 hours. The reaction was then quenched with acetic acid (6.74 L) maintaining the temperature at 10-20° C.
  • Acetonitrile (240 L) was added and the solvents were evaporated under reduced pressure to a volume of about 240 L. This addition and evaporation of acetonitrile was repeated two more times. The resulting mixture was heated to 60-70° C. for 5 hours after which it was cooled to 10-15° C. and stirred for 2 h. The solids in this mixture were then filtered off and washed with acetonitrile (48 L) twice. The solids were then added to water (240 L) and the reaction mixture heated to 45-50° C. for 3-5 hours followed by cooling to 15-20° C. for 4 hours. The solids remaining were filtered off and the filter cake was washed with water (96 L, two times). This filter cake was dried at 45° C. to provide embodiment 14 (26.28 kg). The diffractogram for embodiment 14 is shown in FIG. 7D .
  • Embodiment 1s (15 mg) was treated with increasing volume of solvent until the material fully dissolved or until a maximum of 100 mL of solvent had been added.
  • the solvent was added in the following increments: 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 70 mL and 100 mL.
  • the system was held at 50° C. for 5 min with gentle stirring and visually assessed for presence of solid. This process continued until a total of 100 mL of solvent had been added. If no solid remained, then no additional solvent was added. After the assessment was completed, the solution was held at 50° C. for 1 h and then cooled from 50° C. to 5° C. at 0.1° C./min with stirring.
  • a suspension of embodiment 1s (30 mg) in each solvent was placed in a platform shaker incubator and subjected to a series of heat-cool cycles from ambient to approximately 50° C. for 24 h. This was achieved by switching the heating on and off every 4 hours. Shaking was maintained throughout. An aliquot from each sample was taken and allowed to air-dry for 2 h. The air-dried solids were analyzed by XRPD, then vacuum dried using a vacuum oven (RT, 24 h) and were re-analyzed by XRPD. Each sample obtained in this experiment was vacuum dried and after vacuum drying each sample was analyzed by XRPD incubation at elevated temperature.
  • Embodiment 1s Incubation of Embodiment 1s at 60° C.
  • Embodiment 1s (30 mg) was treated with solvent and shaken at 60° C. for 24 h. An aliquot was taken out and allowed to air-dry for 16 h. The dried samples were then analyzed by XRPD. The following solvents, where total solvent amount added is noted in parenthesis immediately after the solvent, were used according to this procedure which yielded the noted embodiment: Water (10 mL) yielded embodiment 1s whose diffractogram is shown in FIG. 5 ; ethanol (10 mL) yielded embodiment 32 whose diffractogram is shown in FIG. 14 ; 2-propanol (10 mL) yielded embodiment 33 whose diffractogram is shown in FIG.
  • tetralin (10 mL) yielded a mixture (diffractogram of the mixture not shown) of embodiment 1s whose diffractogram is shown in FIG. 5 and embodiment 19 whose modulated DSC profile is shown in FIG. 9 ; 3-methyl-1-butanol (10 mL) yielded embodiment 33 whose diffractogram is shown in FIG. 14 ; anisole (10 mL) yielded embodiment 36 whose diffractogram is shown in FIG. 14 ; 1,2-dimethoxyethane (10 mL) yielded embodiment 34 whose diffractogram is shown in FIG. 14 ; cumene (10 mL) yielded embodiment 1s whose diffractogram is shown in FIG.
  • Each of a plurality of embodiment 19 (25 mg) samples was treated with an amount of a solvent as indicated below yielding in turn a plurality of samples, each agitated at 60° C. for 24 h.
  • toluene (75 ⁇ L, suspension) yielded embodiment 1s whose diffractogram is shown in FIG. 5 ; isopropyl acetate (75 ⁇ L, suspension) yielded embodiment 37 whose diffractogram is shown in FIG. 15 ; methyl t-butyl ether (75 ⁇ L, suspension), yielded embodiment 33 whose diffractogram is shown in FIG. 14 ; 2-butanone (75 ⁇ L, suspension) yielded embodiment 1s whose diffractogram is shown in FIG. 5 ; THF (75 ⁇ L, suspension) yielded embodiment 37 whose diffractogram is shown in FIG.
  • nitromethane (75 ⁇ L, suspension) yielded embodiment 42 whose diffractogram is shown in FIG. 15 ; propylene glycol (75 ⁇ L, suspension) yielded embodiment 43 whose diffractogram is shown in FIG. 15 ; 2-methyl-tetrahydrofuran (150 ⁇ L, suspension) yielded embodiment 33 whose diffractogram is shown in FIG. 14 ; tetralin (150 ⁇ L, suspension), yielded embodiment 33 whose diffractogram is shown in FIG. 14 ; 3-methyl-1-butanol (75 ⁇ L, suspension) yielded embodiment 33 whose diffractogram is shown in FIG.
  • anisole 150 ⁇ L, suspension
  • 1,2-dimethoxyethane 75 ⁇ L, suspension
  • cumene 150 ⁇ L, suspension
  • diisopropyl ether 150 ⁇ L, suspension
  • ethanol:water 95:5, 75 ⁇ L, suspension
  • each of a plurality of embodiment 19 (25 mg) samples was treated with an amount of a solvent as indicated below yielding in turn a plurality of samples, each sample was matured by thermocycling (40° C.-60° C., 4 h cycles) for 24 h. Solids were isolated, air-dried for 16 h and analyzed by XRPD. The following solvents, where total solvent amount added is noted in parenthesis immediately after the solvent followed by the observed appearance at 24 hours, were used according to this procedure which yielded the noted embodiment: Water (125 ⁇ L, green tinge solid) yielded embodiment 1s whose diffractogram is shown in FIG.
  • any one of embodiments 11, 11b, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 and 53 of compound Ex. 1 and any combination thereof is an embodiment of compounds according to this invention.
  • Still other embodiments of compounds according to this invention include compound Ex. 1 as a non-hygroscopic solvate, such as embodiment 11 of compound Ex. 1.
  • Still other embodiments of compounds according to this invention include compound Ex. 1 in amorphous form, such as embodiment 19 of compound Ex. 1.
  • Any one of embodiments 11, 16, 17, and 18 of compound Ex. 1 and any combination thereof is an embodiment of compounds according to this invention.
  • Further embodiments of this invention include compounds according to this invention in the form of pharmaceutically acceptable co-crystals. Additional embodiments of this invention include compounds according to this invention in the form of pharmaceutically acceptable salts.
  • Examples 1-12 are JAK inhibitors and were tested in enzymatic and cellular assays. The results of the enzymatic assay are presented in Table 4 which is entitled Results of Enzymatic Inhibition Assays. Examples 1-12 were also tested in three cellular assays: IL-2 pSTAT5 (JAK1/JAK3), IFN ⁇ pSTAT4 (JAK1/TYK2) and GM-CSF pSTAT5 (JAK2/JAK2) with the results presented in Table 5 entitled Cell-Based Assay Data.
  • Substrate (NH2-KGGEEEEYFELVKK-CO2), internal standard peptide (NH2-SWGAIETDKEYYTVKD-CO2) and product peptide (for standard curve only) (NH2-KGGEEEEY-Pi-FELVKK-CO2), were purchased from AnaSpec (Fremont, Calif., USA).
  • JAK1-JH1JH2 (574-1154 with a His-GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), JAK3-JH1JH2 (512-1124 with a GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), and Tyk2-JH1JH2 (8H_tev_580-1182-C936A-C1142A with a C-terminal tev (ENLYFQ-G) cleavage site) were purified internally.
  • JAK2-JH1JH2 (532-1132 with a GST tag and C-terminal tev (ENLYFQ-G) cleavage site), was purchased from Invitrogen.
  • Dimethylsulfoxide 99.8% (DMSO) and trifluoroacetic acid 99.5% (TFA) were purchased from EMD Chemical (Gibbstown, N.J., USA).
  • Adenosine triphosphate (ATP), 4-morpholinepropanesulfonic acid (MOPS), magnesium chloride (MgCl 2 ), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), formic acid >95% (FA) and Tween-20 were purchased from Sigma (St Louis, Mo., USA).
  • 384-well polypropylene plates, Cat #781280 were purchased from Greiner (Monroe, N.C.), RapidFireTM cartridge A C4 Column (Agilent Technologies, Santa Clara, Calif.).
  • the HTMS experiments were performed in positive ionization mode on a RapidFire 300 instrument (Agilent Technologies, Santa Clara, Calif.), coupled with an ABSiex QTrap 4000 system with an Electrospray Ionization source (RF-MS) (Concord, ON, Canada).
  • the RapidFire system was run with 3 Agilent 1200 series isocratic pumps Agilent Technologies (Santa Clara, Calif.) and one peristaltic pump model ISM832C from Ismatec (Wertheim, Germany). The entire system was operated using the RapidFire software interfaced with Analyst software for the mass spectrometer.
  • 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ⁇ 0.25% in a 20 ⁇ L reaction.
  • Assay conditions for each of the proteins are summarized in Table 3.
  • the enzyme reaction was initiated by the addition of 10 ⁇ L of enzyme and ATP mixture to 10 ⁇ L of substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl 2 , 1 mM EDTA, 2 mM DTT, 0.002% Tween-20).
  • the Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation.
  • the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK3 and 90 minutes for JAK1, JAK2 and Tyk2.
  • the reaction was quenched by the addition of 20 ⁇ L of 0.5% TFA containing 0.15 ⁇ M of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction.
  • the assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-12 as tested in these assays are shown in Table 4.
  • the sample analysis on the RapidFire was performed using a mobile phase A1 consisting of Water/TFA/FA (100:0.01:0.1, v/v/v), a mobile phase B 1 consisting of ACN/Water/TFA/FA (80:20:0.01:0.1, v/v/v).
  • the following run parameters were used: state 1 (aspirate), 250 ms; state 2 (load/wash), 3000 ms; state 3 (elute), 4000 ms; state 4 (re-equlibrate), 1000 ms with a flow rate of 1.25 mL/min.
  • the samples were aspirated directly from the 384-well assay plate and delivered onto RF-MS microscale solid-phase C4 extraction cartridge (Type A).
  • the undesired component such as salt, cofactor, detergent and large protein were washed out and the retained analytes (substrate, product and IS) were coeluted directly onto the ABSiex Qtrap 4000 system.
  • the AlphaLISA assay (based on Alpha Technology from PerkinElmer) was performed by first plating freshly thawed PBMCs (Biological Specialty Corporation) in 384-well plates at 30,000 cells per 4 ⁇ L per well in HBSS (Hanks' Balanced Salt Solution) containing 0.1% IgG (immunoglobulin G)-free, protease-free BSA (bovine serum albumin) (Jackson ImmunoResearch Cat. No. 001-000-161). The cells were then treated with 2 ⁇ L/well of compounds diluted in DMSO at half-log titrated concentrations, with a highest test concentration of 10 M and 0.5% final DMSO concentration, for thirty minutes at 37° C.
  • the cells were stimulated with 2 ⁇ L/well of IL-2 (R&D Systems Cat. No. 202-IL-050) at 5 ng/mL for thirty minutes at 37° C.
  • the cellular reactions were terminated by the addition of 2 ⁇ L/well of lysis buffer (PerkinElmer Cat. No. ALSU-PST5S-A10K) followed by an incubation of five minutes at room temperature.
  • 5 ⁇ L/well of acceptor mix PerkinElmer Cat. No. ALSU-PST5-A10K was added to the cells and incubated in the dark for one hour at room temperature. Then, 5 ⁇ L/well of donor mix (PerkinElmer Cat. No.
  • the AlphaLISA assay (based on Alpha Technology from PerkinElmer) was performed by first plating freshly thawed PBMCs (Biological Specialty Corporation) in 384-well plates at 100,000 cells per 6 ⁇ L per well in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) and 1,000 I.U./mL penicillin and 1,000 ⁇ g/mL streptomycin. The cells were then treated with 2 ⁇ L/well of compounds diluted in DMSO at half-log titrated concentrations, with a highest test concentration of 10 ⁇ M and 0.5% final DMSO concentration, for thirty minutes at 37° C.
  • DMEM Dynamic fetal bovine serum
  • the cells were stimulated with 2 ⁇ L/well of IFN ⁇ (PBL Assay Science Cat. No. 11101-2) at 4 ng/mL for thirty minutes at 37° C.
  • the cellular reactions were terminated by the addition of 2 ⁇ L/well of lysis buffer (PerkinElmer Cat. No. ALSU-PST4-A1 (K) followed by an incubation of five minutes at room temperature.
  • 4 ⁇ L/well of acceptor mix PerkinElmer Cat. No. ALSU-PST4-A10K was added to the cells and incubated in the dark for one hour at room temperature. Then, 4 ⁇ L/well of donor mix (PerkinElmer Cat. No.
  • the AlphaLISA assay (based on Alpha Technology from PerkinElmer) was performed by first plating freshly thawed PBMCs (Biological Specialty Corporation) in 384-well plates at 30,000 cells per 4 ⁇ L per well in HBSS containing 0.1% IgG-free, protease-free BSA (Jackson ImmunoResearch Cat. No. 001-000-161). The cells were then treated with 2 ⁇ L/well of compounds diluted in DMSO at half-log titrated concentrations, with a highest test concentration of 10 ⁇ M and 0.5% final DMSO concentration, for thirty minutes at 37° C. Next, the cells were stimulated with 2 ⁇ L/well of GM-CSF (R&D Systems Cat. No.
  • the plates were read on a PerkinElmer EnVision for detection of the time-resolved fluorescence signal.
  • the percentage of GM-CSF-dependent pSTAT5 inhibition was determined at the compound test concentrations; and for each compound, a dose curve was generated and the IC 50 was calculated.
  • Compound IC 50 was calculated by nonlinear regression, sigmoidal dose response analysis of the half-log dilution titration curve of the compound concentration vs. Alpha signal.
  • Alpha is defined in the IL-2 pSTAT5 (JAK1/JAK3) cellular assay description.
  • Examples 1-12 were tested in solubility and permeability assays.
  • the results of the solubility assay are presented in Table 6 which is entitled Solubility Assay Data and the results of the permeability assay are presented in Table 7 entitled MDCK-MDR1 Permeability Data.
  • Solubility Assays were tested in solubility and permeability assays.
  • the results of the solubility assay are presented in Table 6 which is entitled Solubility Assay Data and the results of the permeability assay are presented in Table 7 entitled MDCK-MDR1 Permeability Data.
  • Solubility measurements were conducted in the following solubility media: Simulated gastric (34.2 mM of sodium chloride and 100 mM of hydrochloric acid) or simulated intestinal fluids (fasted state [pH 6.5]: 3 mM of sodium taurocholate, 0.75 mM of lecithin, 28.4 mM of monobasic sodium phosphate, 8.7 mM of sodium hydroxide, and 105.9 mM of sodium chloride). Test compounds were dissolved in DMSO at a concentration of 10 mM.
  • test compounds were dispensed (20 ⁇ L) into Nunc 1-mL-96-Deep-Well-PP plates, and the DMSO was evaporated via nitrogen blow down from a TurboVap 96 for 6 hours or until a dry residue was produced. Then, 400 ⁇ L of solubility media was added to the well containing the dry solid. A Pre-Slit Well Cap was securely placed over the well plate block, and the samples were vigorously stirred for 2-5 days at ambient temperature.
  • Solubility Sample ⁇ ⁇ Peak ⁇ ⁇ Area Average ⁇ ⁇ Response ⁇ ⁇ Factor ⁇ ⁇ from ⁇ ⁇ 3 ⁇ ⁇ Standards .
  • solubility values were in the range of 4-400 ⁇ M. Values outside of this range were reported as either ⁇ 4 ⁇ M or >400 M. Solubilities are reported as long as the compound under study was sufficiently stable to complete the corresponding solubility determination.
  • Permeability measurements were conducted according to the Cyprotex protocol using the MDCK-MDR1 cell line obtained from the NIH (Rockville, Md., USA). Cells between passage numbers 6-30 were seeded onto a Multiscreen PlateTM (Millipore) at a cell density of 3.4 ⁇ 10 5 cells/cm 2 and cultured for three days before permeability studies were conducted. The cells in this assay form a cohesive sheet of a single cell layer filing the surface area of the culture dish, also known as a confluent monolayer, and on day four the test compound was added to the apical side of the membrane and the transport of the compound across the monolayer was monitored over a time period of 60 min.
  • the apical (“A”) side or compartment of an entity is the side of such entity that is exposed to the lumen or exterior environment
  • the basolateral (“B”) side or compartment is the side or compartment of such entity that is exposed to the typically internal environment, encompassing the opposite side.
  • the apical side of such intestinal cell would be the side of the cell exposed to the intestinal lumen
  • the basolateral side would be the side that is exposed to the blood.
  • Test compounds were dissolved in DMSO at a concentration of 10 mM.
  • the dosing solutions were prepared by diluting test compound with assay buffer (Hanks Balanced Salt Solution), pH 7.4, at a final concentration of 5 ⁇ M.
  • assay buffer Hanks Balanced Salt Solution
  • Assay buffer Hanks Balanced Salt Solution
  • buffer was removed from the apical compartment and replaced with test compound dosing solution with or without the permeability glycoprotein (“PgP”, “P-gP”, “Pgp” or “P-gp”) inhibitor elacridar (2 ⁇ M).
  • B-A basolateral to apical
  • Incubations were carried out in duplicate at 37° C.
  • the second and third columns in Table 7 show the values of P app(A ⁇ B) for the apical-to-basolateral compound transport without (second column) and with a P-gp inhibitor (third column, noted as P e app(A ⁇ B) ) that was elacridar.
  • P app(A ⁇ B) gives an indication of permeation extent across the cells in this assay, which is envisaged to model the transcellular transport across Pgp-expressing cells, such as Pgp-expressing gastrointestinal tract cells.
  • P e app(A ⁇ B) values (P app(A ⁇ B) in the presence of the P-gp inhibitor) given in column 3 are determined to confirm the role of P-gp in the compound efflux.
  • the fourth column in Table 7 shows the values of P app(B ⁇ A) for the basolateral-to-apical compound transport.
  • Test compound efflux ratios are given in the fifth column of Table 7 as P app(B ⁇ A) /P app(A ⁇ B) by using the corresponding permeability coefficient values from the fourth and second columns in the same table.
  • the efflux ratios (fifth column, Table 7) are consistently greater than 2 for compounds (A)-(C) and also for compounds Ex. 1-12, which indicates that compound efflux occurs for all such compounds.
  • P app(A ⁇ B) values in column 2 are generally low and comparable for reference compounds (A)-(C) and also for compounds Ex. 1-12. These low values indicate low permeability for all such compounds, which is due to the P-gp effects since all such compounds are P-gp substrates as indicated by the values given in column 5 being all greater than 2.
  • the values given in the third and fourth columns for P e app(A ⁇ B) and P app(B ⁇ A) , respectively should be low. However, these data show that the P app(B ⁇ A) values for compounds (A)-(C) are greater than the corresponding values compounds Ex. 1-12.
  • mice Three non-fasted female C57BL/6 mice were orally administered test compound at a dose of 25 mg/kg p.o. as a solution in 20% hydroxypropyl-beta-cyclodextrin (HP ⁇ CD) at a dose volume of 5 mL/kg.
  • Blood samples were collected at 0.5, 2, and 4 h post dose via retro-orbital bleed or venipuncture of the dorsal metatarsal vein. Blood samples were collected into tubes containing anticoagulant (Heparin-Na) and placed on wet ice. The plasma fraction was separated by centrifugation and frozen at ⁇ 20° C. for up to 4 h and ⁇ 80° C. after 4 h unless analyzed shortly after sample collection.
  • Heparin-Na anticoagulant
  • Colon samples were collected at 4 h post dose. From the beginning of the cecum, a 4-6 cm sample of the colon was dissected, cut open on the longitudinal axis, and the solid contents removed by flushing with 2 mL of saline. The colon was further washed by putting it in 5 mL of saline and shaken for 5 seconds. The colon sample was then patted dry, weighed, and homogenized as 1 part tissue (g) to 4 parts HPLC grade water (mL). Concentrations of the compound in plasma and colon homogenate were determined using a qualified liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method. This protocol was used to evaluate the following test compounds: Compounds (B) and (C) and Examples 6 and 11.
  • LC-MS/MS liquid chromatography-triple quadrupole mass spectrometry
  • Intracolonic (IC) dose group Following anesthesia with isoflurane by inhalation, three non-fasted female C57BL/6 mice were administered the compound intracolonically through a small incision in the abdominal wall using a syringe and needle at a dose of 5 mg/kg as a solution in 20% HP ⁇ CD at a dose volume of 1 mL/kg. Blood samples were collected at 0.5, 2, and 4 h post dose via retro-orbital bleed. Blood samples were collected into tubes containing anticoagulant (Heparin-Na) and placed on wet ice. The plasma fraction was separated by centrifugation and frozen at ⁇ 20° C. for up to 4 h and ⁇ 80° C. after 4 h unless analyzed shortly after sample collection.
  • Heparin-Na anticoagulant
  • Colon samples were collected at 4 h post dose. From 2 cm below the cecum, a 4-cm sample of the colon was dissected, cut open on the longitudinal axis, and the solid contents removed by flushing with 2 mL of saline. The colon was further washed by putting it in 5 mL of saline and shaken for 5 seconds. The colon sample was then patted dry, weighed, and homogenized as 1 part tissue (g) to 4 parts HPLC grade water (mL). Concentrations of the compound in plasma and colon homogenate were determined using a qualified liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method. This protocol was used to evaluate IC dosing of the following test compounds: Examples 1, 3, and 4.
  • LC-MS/MS liquid chromatography-triple quadrupole mass spectrometry

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US10981911B2 (en) * 2016-12-16 2021-04-20 Janssen Pharmaceutica Nv Imidazopyrrolopyridine as inhibitors of the JAK family of kinases
US11827638B2 (en) 2016-12-16 2023-11-28 Janssen Pharmaceutica Nv Imidazopyrrolopyridine as inhibitors of the JAK family of kinases
US12202831B2 (en) 2016-12-16 2025-01-21 Janssen Pharmaceutica Nv Small molecule inhibitors of the JAK family of kinases
US11066406B2 (en) 2018-06-15 2021-07-20 Janssen Pharmaceutica Nv Small molecule inhibitors of the JAK family of kinases
US11787807B2 (en) 2018-06-15 2023-10-17 Janssen Pharmaceutica Nv Small molecule inhibitors of the JAK family of kinases
US12390451B2 (en) 2021-03-11 2025-08-19 Janssen Pharmaceutica Nv Small molecule inhibitor of the JAK family of kinases

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PH12020551984A1 (en) 2021-08-02
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US20200165250A1 (en) 2020-05-28
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