RU2819004C1 - Pyrrolo[2,3-d]pyrimidine derivatives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to pyrrolo{2,3-d}pyrimidine derivatives

or pharmaceutically acceptable salts thereof, as well as to pharmaceutical compositions containing said compounds.

EFFECT: novel pyrrolo{2,3-d}pyrimidine derivatives which can be used to inhibit Janus kinase (JAK) and are applicable in methods of treating conditions mediated by JAK1 are obtained.

6 cl, 6 tbl, 6 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to pyrrolo[2,3-d]pyrimidine compounds and isolated forms of these compounds. The invention also relates to the preparation of said compounds and intermediates used in said preparation, compositions containing said compounds, uses of said compounds to inhibit Janus Kinase (JAK) and methods of treating and preventing conditions mediated by JAK1.

BACKGROUND OF THE ART

Protein kinases are families of enzymes that catalyze the phosphorylation of specific residues in proteins and are broadly divided into tyrosine kinases and series/threonine kinases. Abnormal kinase activity, resulting from mutation, overexpression or inappropriate regulation, dysregulation or lack of regulation, and overproduction or underproduction of growth factors or cytokines, is the cause of many diseases, including, but not limited to, cancer, cardiovascular diseases, allergies, asthma and other respiratory diseases, autoimmune diseases, inflammatory diseases, bone diseases, metabolic disorders, and neurological and neurodegenerative disorders such as Alzheimer's disease. Inappropriate kinase activity triggers various biological cellular responses related to cell growth, cell differentiation, survival, apoptosis, mitogenesis, cell cycle control and cell motility, leading to the above and related diseases.

Thus, protein kinases have become an important class of enzymes considered as targets for therapeutic intervention. In particular, the JAK family of cellular protein tyrosine kinases (JAK1, JAK2, JAK3 and Tyk2) play a key role in cytokine signaling (Kisseleva et al., Gene, 2002, 285, 1; Yamaoka et al. Genome Biology 2004, 5, 253) . Upon binding to their receptors, cytokines activate JAKs, which then phosphorylate cytokine receptors, thereby creating docking sites for signaling molecules, in particular members of the signal transducer and activator of transcription (STAT) family, which ultimately leads to gene expression. The JAK family is known to be activated by various cytokines. These cytokines include cytokines of the IFN family (IFN-alpha, IFN-beta, IFN-omega, Limitin, IFN-gamma, IL-10, IL-19, IL-20, IL-22), gp130 family (IL-6, IL-11, OSM, LIF, CNTF, NNT-1/BSF-3, G-CSF, CT-1, leptin, IL-12, IL-23), gamma C family (IL-2, IL-7, TSLP , IL-9, IL-15, IL-21, IL-4, IL-13), IL-3 family (IL-3, IL-5, GM-CSF), single chain family (EPO, GH, PRL, TPO ), receptor tyrosine kinases (EGF, PDGF, CSF-1, HGF) and G protein-coupled receptors (ATI).

There remains a need for new compounds that effectively and selectively inhibit specific JAK enzymes, in particular JAK1 as opposed to JAK2. JAK1 is a member of the Janus family of protein kinases, consisting of JAK1, JAK2, JAK3 and TYK2. JAK1 is expressed at varying levels in all tissues. Many cytokine receptors signal through pairs of JAK kinases in the following combinations: JAK1/JAK2, JAK1/JAK3, JAK1/TYK2, JAK2/TYK2, or JAK2/JAK2. In this context, JAK1 is the most abundant paired JAK kinase and is required for signaling by the common cytokine receptor γ chain (IL-2Rγ), the IL-6 receptor family, the type I, II and III receptor families, and the IL-10 receptor family. Animal studies have shown that JAK1 is essential for the development, function and homeostasis of the immune system. Modulation of immune activity through inhibition of JAK1 kinase activity may be useful in the treatment of various immune disorders (Murray, P. J. J. 1 mmipo1., 178, 2623-2629 (2007); Kisseleva, T. et al., Gene, 285, 1-24 (2002 ); O'Shea, J. J. et al., Cell, 109, (suppl.) S121-S131 (2002)), while preventing JAK2-dependent erythropoietin (EPO) and thrombopoietin (TPO) signaling (Neubauer N., et al., Cell, 93(3), 397-409 (1998); Parganas E., et al., Cell, 93(3), 385-95 (1998)).

Safe and effective agents are needed to control JAK-related disorders such as atopic dermatitis in humans and animals. The market for the treatment of atopic dermatitis is currently dominated by corticosteroids, which cause unpleasant and unwanted side effects. Antihistamines are also used, but they are ineffective. New compounds are needed that have selective inhibitory activity against JAK1, providing an alternative to the use of steroids and helping to address the problem of chronic itching and inflammation that either persists for a long time in atopic dermatitis or slowly regresses after removal of the allergen or pathogen.

Newly identified N-((1S,3S)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide, commonly known as abrocitinib, is described in Patent US No. 9035074 of the copyright holder of the present invention, the contents of which are fully incorporated into this description by reference; The chemical formula of abrocitinib is C ₁₄ H ₂₁ N ₅ O ₂ S, the structural formula is presented below:

Abrocitinib is known to be a promising JRK inhibitor and can be used as a therapeutic agent for the treatment of various diseases, including atopic dermatitis. We have discovered novel metabolites of abrocitinib that are therapeutically active as JAK inhibitors, which have an unexpectedly high therapeutic index and therefore have the potential to be excellent pharmaceuticals for the treatment of JAK-mediated disorders.

SUMMARY OF THE INVENTION

The present invention relates to pyrrolo[2,3-d]pyrimidine compounds and isolated forms of such compounds. The isolated compounds of the present invention have been found to have unexpectedly beneficial activities, including increased efficacy and a reduced incidence of side effects. The isolated compounds can be used in pharmaceutical compositions in combination with pharmaceutically acceptable excipients and in methods of treating JAK1-mediated conditions.

The purpose of the present invention is compounds of formula I with the following structure:

or pharmaceutically acceptable salts thereof, wherein R ¹ and R ² independently represent hydrogen or a hydroxyl group; where if R ¹ represents hydrogen, then R ² is a hydroxyl group; and if R ² is hydrogen, then R ¹ is a hydroxyl group.

In other aspects, the present invention also provides compounds of Formula I or pharmaceutically acceptable salts thereof in isolated form.

In other aspects, the present invention also provides pharmaceutical compositions that include a pharmaceutically acceptable excipient and a compound of Formula I or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention also provides methods for treating conditions or disorders, including myositis, vasculitis, pemphigus, Crohn's disease, lupus, nephritis, psoriasis, multiple sclerosis, major depressive disorder, allergies, asthma, Sjögren's disease, dry eye syndrome, rejection transplant, cancer, inflammatory bowel disease, septic shock, cardiopulmonary dysfunction, acute respiratory disease or cachexia, administering to a subject in need thereof a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention also provides methods for treating conditions or disorders, including atopic dermatitis, eczema, psoriasis, scleroderma, lupus, pruritus, other pruritic conditions, allergic reactions, including mammalian allergic dermatitis, inflammatory airway disease, recurrent airway obstruction tract, airway hypersensitivity and chronic obstructive pulmonary disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention also provides methods for preparing the compounds of the present invention.

The present invention will become more clear from the description given below by way of example only. Although the present invention is not limited to the foregoing description, various aspects of the present invention will be appreciated through the following discussion and examples.

The term "subject" refers to a human, livestock or companion animal.

The term “treatment” or “cure” means alleviation of symptoms associated with a disease, disorder or condition, or stopping the further progression or development of these symptoms. Depending on the disease and condition of the subject, the term "treatment" as used herein may include one or more curative, palliative and prophylactic methods. Treatment may also include administration of a pharmaceutical composition of the present invention in combination with other therapeutic agents.

The term “therapeutically effective” refers to the ability of an agent to prevent or alleviate the severity of a disorder without causing the adverse side effects typically associated with other treatments. The phrase "therapeutically effective" should be understood as equivalent to the phrase "effective for treating, preventing or ameliorating a condition", both of which are intended to define the amount of each agent to be used in combination therapy that will achieve the goal of improving the severity of cancer, cardiovascular disease, pain and inflammation, and their incidence, compared to treatment with each agent alone, without causing the adverse side effects typically associated with other treatments.

The term "pharmaceutically acceptable" means suitable for use by a subject.

If the substituents are described as "independently selected" from a group, each substituent is selected independently of the other. Thus, each substituent may be identical to or different from the other substituent(s). In some embodiments, the metabolites of the compounds or salts thereof are substantially isolated.

The terms “isolated,” “purified,” “in purified form,” “in isolated form,” or “purified substance,” as they apply to a compound, refer to the physical state of the compound after it has been isolated from a synthetic process, such as a reaction mixture. Thus, the terms “isolated,” “purified,” “in purified form,” “in isolated form,” or “purified substance” when referring to a compound refer to the physical state of said compound after being obtained by purification or purification methods described in herein or well known to one skilled in the art (e.g., chromatography, recrystallization, etc.), in a purity sufficient to permit its characterization by standard analytical methods described herein or well known to one skilled in the art. For example, purification methods disclosed herein (such as LC-MS and LC-MS/MS methods) result in isolated forms of the subject compounds. Such isolation and purification methods are expected to result in a product containing at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of said compound or salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds that are selective JAK1 modulators useful for the treatment of diseases and conditions associated with dysregulation of JAK1. The present invention also relates to the isolation of compounds that are selective JAK1 modulators useful for the treatment of diseases and conditions associated with dysregulation of JAK1. The present invention also relates to prodrugs and pharmaceutical compositions containing these JAK1 modulators, as well as methods for treating and/or preventing such diseases and conditions.

In a first aspect, the invention relates to a compound of formula I having the structure:

or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² independently represent hydrogen or a hydroxyl group; where if R ¹ represents hydrogen, then R ² is a hydroxyl group; and where if R ² is hydrogen, then R ¹ is a hydroxyl group.

Embodiments (E) of this first aspect of the invention are described below, where for convenience E1 is identical to this aspect.

E1. A compound of formula I having the structure:

or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² independently represent hydrogen or a hydroxyl group; where if R ¹ represents hydrogen, then R ² is a hydroxyl group; and if R ² is hydrogen, then R ¹ is a hydroxyl group.

E2. The compound according to E1 or a pharmaceutically acceptable salt thereof, wherein R ¹ represents a hydroxyl group and R ² represents hydrogen.

E3. A compound according to E1 or a pharmaceutically acceptable salt thereof, wherein R ¹ represents hydrogen and R ² represents a hydroxyl group.

E4. A compound of formula IA having the structure:

or a pharmaceutically acceptable salt thereof.

E5. A compound of formula IB having the structure:

or a pharmaceutically acceptable salt thereof.

E6. A connection selected from the group consisting of the following connections:

(S) -2-hydroxy-N-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide;

3-hydroxy-N-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide;

or pharmaceutically acceptable salts thereof.

E7. (S)-2-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide or a pharmaceutically acceptable salt thereof.

E8. 3-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide or a pharmaceutically acceptable salt thereof.

E9. (S)-2-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide.

E10. 3-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide.

E11. The connection of any one of embodiments E1 to E10 in isolated form.

E12. The compound of any one of embodiments E1 to E11 in crystalline form.

E13. A method of treating or preventing a disease or condition for which a JAK1 inhibitor is indicated in a subject in need of such treatment, which comprises administering to the subject a therapeutically effective amount of a compound of any one of embodiments E1 to E12, or a pharmaceutically acceptable salt thereof.

E14. A method of treating or preventing an inflammatory or autoimmune condition, comprising administering to a subject suffering from such a condition a therapeutically effective amount of a compound according to any of embodiments E1 to E12, or a pharmaceutically acceptable salt thereof.

E15. A pharmaceutical composition comprising a compound according to any of embodiments E1 to E12 and a pharmaceutically acceptable excipient.

E16. A method of treating or preventing a disease or condition selected from inflammation, autoimmune disease, neuroinflammation, arthritis, rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, lupus nephritis, osteoarthritis, gouty arthritis, pain, fever, pulmonary sarcoidosis, silicosis, cardiovascular disease, atherosclerosis, myocardial infarction, thrombosis, congestive heart failure and cardiac reperfusion injury, cardiomyopathy, stroke, ischemia, reperfusion injury, cerebral edema, brain injury, neurodegeneration, liver disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, nephritis, retinitis , retinopathy, macular degeneration, glaucoma, diabetes (type 1 and 2), diabetic neuropathy, viral and bacterial infections, myalgia, endotoxic shock, toxic shock syndrome, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, vitiligo, Alzheimer's disease, skin hyperemia, eczema, psoriasis, atopic dermatitis, sunburn, keloid hypertrophic scars, rheumatic diseases, urticaria, discoid lupus, cutaneous lupus, lupus of the central nervous system, psoriatic arthritis, asthma, allergic asthma, type I interferonopathy, including Aicardi-Gutierrez syndrome and other Mendelian diseases associated with overexpression of type I interferon, primary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, scleroderma, alopecia areata, cicatricial alopecia, prurigo, prurigo nodosa, CPUO, lichen diseases, lichen planus, Steven-Johnson syndrome, spondylopathy, myositis, vasculitis, pemphigus, lupus, major depressive disorder , allergies, dry eye syndrome, graft rejection, cancer, septic shock, cardiopulmonary dysfunction, acute respiratory disease, ankylosing spondylitis, cachexia, chronic graft-versus-host disease, acute graft-versus-host disease, celiac sprue, idiopathic thrombocytopenic thrombotic purpura, thrombotic thrombocytopenic purpura, myasthenia gravis, Sjogren's syndrome, epidermal hyperplasia, cartilage inflammation, bone degeneration, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, rheumatoid arthritis with systemic onset, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile Rether syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, rheumatoid arthritis and with systemic onset, enteropathic arthritis, reactive arthritis , Rether's syndrome, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, dermatitis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barré disease, Graves' disease, Addison's disease, Raynaud's phenomenon, psoriatic epidermal hyperplasia, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, immune disorder associated with or resulting from the activity of pathogenic lymphocytes, non-infectious uveitis, Behçet's disease and Vogt-Koyanagi syndrome -Harady, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E12.

E17. A method of treating or preventing psoriasis, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a composition comprising a compound or a pharmaceutically acceptable salt thereof of any one of Embodiments E1 to E12.

E18. A method of treating or preventing atopic dermatitis, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of Embodiments E1 to E12.

E19. A method of treating or preventing hand eczema, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of Embodiments E1 to E12.

E20. A method of treating or preventing itching, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E12.

E21. A method of treating or preventing cutaneous lupus, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E12.

E22. A method of treating a disorder or condition associated with dysregulation of JAKs, particularly JAK1, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof in any one of embodiments E1 to E12.

E23. A method according to E22, wherein the disorder or condition being treated or prevented is selected from the group consisting of inflammation, autoimmune disease, neuroinflammation, arthritis, rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, lupus nephritis, osteoarthritis, gouty arthritis, pain, fever , pulmonary sarcoidosis, silicosis, cardiovascular disease, atherosclerosis, myocardial infarction, thrombosis, congestive heart failure and cardiac reperfusion injury, cardiomyopathy, stroke, ischemia, reperfusion injury, cerebral edema, brain injury, neurodegeneration, liver disease, inflammatory disease intestines, Crohn's disease, ulcerative colitis, nephritis, retinitis, retinopathy, macular degeneration, glaucoma, diabetes (type 1 and 2), diabetic neuropathy, viral and bacterial infections, myalgia, endotoxic shock, toxic shock syndrome, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, vitiligo, Alzheimer's disease, skin congestion, eczema, psoriasis, atopic dermatitis, sunburn , keloid hypertrophic scars, rheumatic diseases, urticaria, discoid lupus, cutaneous lupus, lupus of the central nervous system, psoriatic arthritis, asthma, allergic asthma, type I interferonopathies, including Aicardi-Gutierrez syndrome and other Mendelian diseases associated with overexpression of type I interferon , primary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, scleroderma, alopecia areata, spondylopathy, myositis, vasculitis, pemphigus, lupus, major depressive disorder, allergy, dry eye syndrome, transplant rejection, cancer, septic shock, cardiopulmonary dysfunction, acute respiratory disease, ankylosing spondylitis, cachexia, chronic graft-versus-host disease, acute graft-versus-host disease, celiac sprue, idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis, Sjogren's syndrome, epidermal hyperplasia, cartilage inflammation, bone tissue degeneration, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, juvenile rheumatoid arthritis with systemic onset, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile Rether syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, rheumatoid arthritis with systemic onset, enteropathic arthritis, reactive arthritis, Rether syndrome, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa granules, Wegener's hematosis, arteritis , polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, dermatitis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barre disease, Graves' disease, Addison's disease, Raynaud's phenomenon, psoriatic epidermal hyperplasia, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, an immune disorder associated with or resulting from the activity of pathogenic lymphocytes, non-infectious uveitis, Behcet's disease and Vogt-Koyanagi-Harada syndrome.

E24. The method according to embodiments E13 to E23, wherein the therapeutically effective amount is from 0.01 mg/kg body weight per day to 100 mg/kg body weight per day.

E25. Method according to E24, in which the therapeutically effective amount is from 0.1 mg/kg body weight per day to 10 mg/kg body weight per day.

E26. Use of a compound of any one of embodiments E1 to E12 for the manufacture of a medicament for treating a disorder for which a JAK1 inhibitor is indicated.

E29. The compound of any one of embodiments E1 to E12 for use in treating a disorder for which a JAK1 inhibitor is indicated.

E30. Use of the compound in isolated form of any one of embodiments E1 to E12 for the manufacture of a medicament for treating a disorder for which a JAK1 inhibitor is indicated.

E31. The compound in isolated form of any one of embodiments E1 to E12 for use in the treatment of a disorder for which a JAK1 inhibitor is indicated

E32. A pharmaceutical combination comprising a compound of any one of embodiments E1 to E12 or a pharmaceutically acceptable salt thereof and one or more additional pharmacologically active compounds.

Compounds or compounds in isolated form which have the same molecular formulas but different nature or sequence of atoms joining or different arrangement of atoms in space are called “isomers”. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers". Those skilled in the art will appreciate that a compound or compound in isolated form of formula I, IA or IB can exist as cis- and trans-achiral diastereomers. An example is a compound or compound in isolated form of Formula 1C.

Included within the scope of the compounds described and compounds in isolated form are all isomers (eg, cis-, trans-, or diastereomers) of the compounds described herein only, as well as any mixtures thereof. All of these forms, including enantiomers, diastereomers, cis-, trans-, syn-, anti-, solvates (including hydrates), tautomers and mixtures thereof, are included in the described compounds or compounds in isolated form. Stereoisomeric mixtures, for example mixtures of diastereomers, can be separated into the corresponding isomers by known suitable separation methods. Diastereomeric mixtures, for example, can be separated into individual diastereomers by fractional crystallization, chromatography, solvent partition and similar methods. This separation can be carried out either at the level of one of the starting compounds or in the compound of formula I, IA or IB itself. Enantiomers can be separated through the formation of diastereomeric salts, for example by salt formation with an enantiomerically pure chiral acid, or by chromatography, for example HPLC, using chromatographic substrates with chiral ligands.

When used therapeutically to treat disorders in a subject, the compounds or compounds in isolated form of the present invention or pharmaceutical compositions thereof may be administered orally, parenterally, topically, rectally, transmucosally or interstitally. Parenteral administration includes indirect injections for systemic effect or direct injections into the affected area. Topical application includes treatment of the skin or organs accessible for topical application, such as the eyes or ears. It also includes transdermal delivery for systemic effect. Rectal administration includes administration in the form of suppositories. The preferred routes of administration are oral and parenteral.

Pharmaceutically acceptable salts of compounds or compounds in isolated form of formula I, IA or IB include their acid addition salts and base salts. Suitable acid addition salts are prepared from acids that form non-toxic salts. Examples of acid addition salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, gibensate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate.

Suitable base salts are prepared from bases that form non-toxic salts. Examples of such salts include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

Hemisalts of acids and bases, for example hemisulfate and hemicalcium salts, can also be prepared. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of compounds and compounds in isolated form of Formula I, IA or IB can be prepared, respectively, by one or more of three methods: (i) reacting the compound or compound in isolated form of Formula I, IA or IB with the desired acid or base; (ii) deprotection, which may be carried out by acid or base, from a suitable precursor compound or compound in isolated form of formula I, IA or IB or ring opening of a suitable cyclic precursor, for example a lactone or lactam, using the desired acid or base; or (iii) converting one salt of a compound or compound in isolated form of formula I, IA or IB into another by reaction with an appropriate acid or base or using a suitable ion exchange column. All three reactions are usually carried out in solution. The resulting salt may precipitate and be collected by filtration or may be isolated by evaporation of the solvent. The degree of ionization in the resulting salt can vary from fully ionized to almost non-ionized.

The pharmaceutical compositions of the present invention can be prepared by methods well known in the art, such as conventional methods of mixing, dissolving, granulating, panning, grinding, emulsifying, encapsulating, entrapping, lyophilizing or spray drying.

Pharmaceutical compositions for use in accordance with the present invention can be prepared in conventional manner using one or more pharmaceutically acceptable carriers, including excipients and excipients, which facilitate processing of the active compound or the compound into isolated forms to prepare drugs that can be used pharmaceutically. The choice of the appropriate drug determines the choice of route of administration. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present invention. Such excipients and carriers are described, for example, in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991). The preparations of the present invention may be formulated as short-acting preparations, rapid-release preparations, long-acting preparations and sustained-release preparations. Thus, pharmaceutical compositions can also be formulated for controlled release or slow release of the active substance.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount sufficient to achieve the intended purpose, i.e. to control or treat disorders or diseases. More specifically, the term “therapeutically effective amount” means an amount of a compound or compound in isolated form effective to prevent, alleviate, or ameliorate the symptoms/signs of a disease or increase the survival of a subject receiving treatment.

The amount of the active component, which is the compound or compound in isolated form of the present invention, in the pharmaceutical composition and its unit dosage form can be varied or adjusted within wide limits depending on the route of administration, the effectiveness of the particular compound or compound in isolated form and the desired concentration. One skilled in the art will be able to determine a therapeutically effective amount. Typically, the amount of active ingredient will range from 0.01% to 99% by weight of the composition.

Typically, a therapeutically effective dose amount of the active ingredient will range from about 0.01 to about 100 mg/kg body weight per day, preferably from about 0.1 to about 10 mg/kg body weight per day, more preferably from about 0 3 to 3 mg/kg body weight per day, even more preferably about 0.3 to 1.5 mg/kg body weight per day. It should be understood that dosages may vary depending on the needs of each subject and the severity of the disorder or disease being treated.

The desired dose may conveniently be presented as a single dose or divided into several doses administered at appropriate intervals, for example two, three, four or more parts of the daily dose. The portion of the daily dose itself may be further divided, for example, into a number of separate administrations at short intervals, such as multiple inhalations from an insufflator or the administration of multiple drops into the eye.

It should also be borne in mind that the initial dose administered may be increased from the above upper level to quickly achieve the desired plasma concentration. On the other hand, the initial dose may be less than optimal, and the daily dose may be gradually increased during the course of treatment, depending on the specific situation. If desired, the daily dose may also be divided into several doses for administration, for example two to four times a day.

The compounds and compounds in isolated form of the present invention can be administered in a pharmaceutically acceptable form either alone or in combination with one or more additional agents that modulate the mammalian immune system or anti-inflammatory agents. These agents may include, but are not limited to, a 5-lipoxygenase activating protein (FLAP) antagonist; a leukotriene antagonist (LTRA), such as an LTB4, LTC4, LTD4, LTE4, CysLT1 or CysLT2 antagonist, such as montelukast or zafirlukast; a histamine receptor antagonist, such as a histamine receptor type 1 antagonist or a histamine receptor type 2 antagonist, for example loratidine, fexofenadine, desloratidine, levocetirizine, metapyrylene or cetirizine; an al-adrenergic agonist or an α2-adrenergic agonist, for example phenylephrine, methoxamine, oxymetazoline or methylnorephrine; a muscarinic M3 receptor antagonist, such as tiotropium or ipratropium; dual muscarinic M3 receptor antagonist/β2 agonist; a phosphodiesterase inhibitor (PDE), such as a PDEZ inhibitor, a PDE4 inhibitor or a PDE5 inhibitor, for example theophylline, sildenafil, vardenafil, tadalafil, ibudilast, cilomilast or roflumilast; sodium cromoglycate or nedocromil sodium; a cyclooxygenase (COX) inhibitor, such as a non-selective inhibitor (eg, aspirin or ibuprofen) or a selective inhibitor (eg, celecoxib or valdecoxib); a glucocorticosteroid, such as fluticasone, mometasone, dexamethasone, prednisolone, budesonide, ciclesonide or beclamethasone; an anti-inflammatory monoclonal antibody, such as infliximab, adalimumab, tanezumab, ranibizumab, bevacizumab or mepolizumab; a β2-agonist, for example salmeterol, albuterol, salbutamol, fenoterol or formoterol, in particular a long-acting β2-agonist; an integrin antagonist, such as natalizumab; an adhesion inhibitor molecule such as a VLA-4 antagonist; kinin B1 or B2 receptor antagonist; an immunosuppressive agent such as an IgE pathway inhibitor (eg, omalizumab) or cyclosporine; a matrix metalloprotease (MMP) inhibitor such as an MMP-9 or MMP-12 inhibitor; tachykinin receptor antagonist NK1, NK2 or NK3; a protease inhibitor such as an elastase, chymase or catheopsin G inhibitor; adenosine A2a receptor agonist; adenosine A2b receptor antagonist; urokinase inhibitor; a dopamine receptor agonist (eg ropinirole), in particular a dopamine D2 receptor agonist (eg bromocriptine); an NFkB pathway modulator such as an IKK inhibitor; an additional cytokine signaling pathway modulator such as an inhibitor of JAK kinase, syk kinase, p38 kinase, SPHK-1 kinase, Rho kinase, EGF-R or MK-2; mucolytic, mucokinetic or antitussive agent; antibiotic; antiviral agent; vaccine; chemokine; epithelial sodium channel blocker (ENaC) or epithelial sodium channel inhibitor (ENaC); a nucleotide receptor agonist such as a P2Y2 agonist; thromboxane inhibitor; niacin; a 5-lipoxygenase (5-LO) inhibitor, such as zileuton; an adhesion factor such as VLAM, ICAM or ELAM; CRTH2 receptor antagonist (DP2); prostaglandin D2 receptor antagonist (DPI); hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor; interferon-β; soluble human TNF receptor, such as etanercept; HDAC inhibitor; phosphoinositotide 3-kinase gamma (PI3Kγ) inhibitor; phosphoinositotide 3-kinase delta (PI3Kδ) inhibitor; CXCR-1 or CXCR-2 receptor antagonist; IRAK-4 inhibitor; and a TLR-4 or TLR-9 inhibitor, including pharmaceutically acceptable salts of the specifically named compounds. These agents may be administered with another active agent, wherein the second active agent may be administered orally or topically.

Suitable specific drugs for use in combination therapy with a compound or compound in isolated form of formula I, IA or IB or pharmaceutically acceptable salts thereof include sulfasalazine, mesalazine, prednisone, azathioprine, infliximab, adalimumab, belimumab, becertolizumab, natalizumab, vedolizumab , hydrocortisone, budesonide, cyclosporine, tacrolimus, fexofenadine, 6-mercaptopurine, methotrexate, ursodeoxycholic acid, obeticholic acid, antihistamines, rifampicin, prednisolone, methotrexate, azathioprine, cyclophosphamide, hydroxychloroquine, mofetil, mycophenolate sodium, rolimus, leflunomide, chloroquine and quinacrine , thalidomide, Rituxan, NSAIDs, solumedrol, depomedrol and dexamethasone.

Chemical synthesis

The following diagrams and their descriptions provide general information regarding the preparation of the compounds of the present invention.

Scheme 1

The synthesis method described in Example 1 and Example 2 is presented in Scheme 1. The primary amine (J. Med. Chem. 2018, 61(3), 1130-1152) is treated with 2-oxo-1-propanesulfonyl chloride (prepared in two steps from chloroacetone) in dichloromethane containing triethylamine. The resulting sulfonamide is then treated with sodium borohydride in methanol to reduce the ketone to produce an isomeric mixture of secondary alcohols. The tosyl protecting group is then removed from the pyrrolopyrimidine ring. The secondary alcohols are then separated by supercritical fluid chromatography (SFC) to obtain the compound of Example 1 (Peak 1), corresponding to Compound IA, and the compound of Example 2 (Peak 2) under the described conditions.

An alternative method for preparing the compound of Example 1 is presented in Scheme 2.

Scheme 2

(S)-1-chloro-2-propanol is protected using tert-butyldimethylsilyl chloride in dimethylformamide as solvent and pyridine as base to produce silyl ether. The primary chloride undergoes a displacement reaction using potassium thioacetate to produce the thioester. The thioether undergoes oxidation with chlorine gas to produce sulfonyl chloride. Under the reaction conditions, the silyl protecting group can be simultaneously removed. cis-N-Methyl-N-{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutan-1,3-diamine is reacted with sulfonyl chloride to produce the sulfonamide . The pyrrolopyrimidine is then deprotected with lithium hydroxide in a mixture of water and tetrahydrofuran to obtain, after purification with SFC, compound IA (Example 1).

Scheme 3

(R)-1-Chloro-2-propanol is protected using tert-butyldimethylsilyl chloride with dimethylformamide as solvent and pyridine as base to produce silyl ether. The primary chloride undergoes a substitution reaction with potassium thioacetate to produce the thioester. The thioether undergoes oxidation with chlorine gas to produce sulfonyl chloride; Some loss of the strong protecting group is observed. cis-N-Methyl-N-{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutan-1,3-diamine is reacted with sulfonyl chloride to produce the sulfonamide . The silyl protective agent is removed with trifluoroacetic acid to produce a secondary alcohol. Next, the tosyl protecting group is removed from the pyrrolopyrimidine using lithium hydroxide in a mixture of water and tetrahydrofuran to obtain the compound of Example 2 after SFC purification.

The preparation of compound IB is presented in Scheme 4 and described in Example 5. cis-N-Methyl-N-{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutane -1,3-diamine is reacted with methyl 3-(chlorosulfonyl)propanoate to produce the sulfonamide. The sulfonamide is reduced with lithium aluminum hydride to give 3-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IB).

An alternative preparation of compound IB is presented in Scheme 5 and described in Example 6. Dihydrobromide cis-N-methyl-N-{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]-pyrimidine-4- yl}cyclobutan-1,3-diamine is treated with a base such as triethylamine and ethyl 3-(chlorosulfonyl)propanoate to produce ethyl 3-(N-(cis-3-(methyl(7-tosyl-7H-pyrrolo [2,3-d]pyrimidin-4-yl)amino)cyclobutyl)sulfamoyl)propanoate. The resulting sulfamoylpropanoate is subjected to reduction with lithium aluminum hydride and subsequent processing to obtain crude 3-hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl) propane-1-sulfonamide, which is used further without further purification. The resulting sulfonamide is treated with a base such as LiOH to give 3-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1- sulfonamide (IB).

One skilled in the art will appreciate that when synthesizing the compounds of the invention, it is necessary to sample and analyze reaction mixtures prior to processing in order to monitor the progress of the reactions and decide whether the reaction should be continued or whether the reaction mixture is ready to be processed to obtain the desired product. Standard methods for analyzing reaction mixtures include thin layer chromatography (TLC), liquid chromatography/mass spectroscopy (LCMS), and nuclear magnetic resonance (NMR).

One skilled in the art will also understand that the compounds and compounds in isolated form of the invention can be obtained as mixtures of diastereomers or geometric isomers (eg, as a result of cis and trans substitution on the cycloalkane ring). These isomers can be separated by standard chromatographic techniques such as normal-phase silica gel chromatography, preparative reverse-phase high-pressure liquid chromatography, or supercritical fluid chromatography (SFC). One skilled in the art will also understand that some of the compounds of the invention are chiral and thus can be prepared as racemic or scalemic mixtures of enantiomers. There are several methods for separating enantiomers that are well known to those skilled in the art. The preferred method for conventional enantiomer separation is supercritical liquid chromatography using a chiral stationary phase.

EXPERIMENTAL PART

Except where otherwise noted, reactions are carried out under a nitrogen atmosphere. Silica gel chromatography is performed using 250-400 mesh silica gel and pressurized nitrogen (~10-15 psi (68.95-103.43 kPa)) to force solvent through the column (“flash chromatography”). When indicated, solutions and reaction mixtures are concentrated by rotary evaporation in vacuo.

Receipt and examples

Preparation 1. Sodium salt of 2-oxopropane-1-sulfonic acid

A mixture of chloroacetone (9.25 g, 100 mmol) and sodium sulfite (14.5 g, 115 mmol) in water (100 ml) was stirred for 24 hours. The resulting mixture was evaporated to dryness and the solid residue was suspended in methanol (300 ml). The mixture is treated with ultrasound for 3 minutes, then filtered. The filter cake was washed with methanol and the filtrates were concentrated to give the title compound (15.5 g) which was used without further purification.

Preparation 2. 2-Oxopropane-1-sulfonyl chloride

Phosphorus oxychloride (40 ml) was added to a stirred suspension of 2-oxopropane-1-sulfonic acid sodium salt (15.5 g, 97 mmol) in dry toluene (40 ml). The mixture is refluxed for 3 hours, then the heat is removed and the mixture is cooled to 20°C. The mixture is concentrated under reduced pressure and the residue is treated with dichloromethane (100 ml). The mixture is filtered and the filter cake is washed with dichloromethane (20 ml). The filtrate was concentrated to give the title compound (11.8 g). ^1H NMR (400 MHz, CDCl ₃ -d) δ 4.76-4.53 (m, 2H), 2.49 (s, 3H).

Preparation 3. cis-N-(3-(Methyl-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)-2-oxopropane-1-sulfonamide

To a solution of cis-N ¹ -methyl-N ¹ -(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclobutan-1,3-diamine (2.4 g, 5.3 mmol ) in dichloromethane (45 ml) add triethylamine (2.68 g, 26.5 mmol). A solution of 2-oxopropane-1-sulfonyl chloride (1.24 g, 6.27 mmol) in dichloromethane (5 ml) was added to the mixture at 0° C., then the mixture was allowed to warm to 20° C. and the mixture was stirred for 3 hours. The resulting reaction mixture was quenched with saturated ammonium chloride solution (30 ml). The layers are separated, the aqueous layer is extracted twice with dichloromethane (2×10 ml). The combined organic layers are washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography (petroleum ether:ethyl acetate, 1:2) to give the title compound (801 mg). LC/MS [M+H]=491.9.

Preparation 4. 2-Hydroxy-N-{cis-3-(methyl-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a solution of N-(3-{methyl[7-(4-methylbenzene-1-sulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}cyclobutyl)-2-oxopropane-1-sulfonamide (0.801 g, 1.62 mmol) in methanol (10 ml) at 0°C add sodium borohydride (123 mg, 2.26 mmol) in a 10% solution of sodium hydroxide (1.6 ml) in methanol (1.6 ml ). The mixture is stirred for 30 minutes, then the cooling bath is removed and the mixture is stirred at 20°C for 30 minutes. Methanol is removed under reduced pressure. The residue was dissolved in ethyl acetate (15 ml), washed with brine (10 ml), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography (0-80% ethyl acetate in petroleum ether) to give the title compound (0.76 g). LC/MS [M+H]=494.2.

Preparation 5. 2-Hydroxy-N-(cis-3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a mixture of 2-hydroxy-]-N-(cis-3-{methyl-[7-(4-methylbenzene-1-sulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino}cyclobutyl )propane-1-sulfonamide (1.13 g, 2.3 mmol), tetrahydrofuran (10 ml) and water (10 ml) add lithium hydroxide monohydrate (273 mg, 11.4 mmol) at 20°C for 24 hours . The mixture is concentrated and purified by flash chromatography (0-10% methanol in dichloromethane). The reaction mixture is dissolved in water/acetonitrile and then lyophilized. The residue is purified by preparative thin layer chromatography (dichloromethane:methanol, 10:1). The residue is dissolved in a water/acetonitrile mixture and lyophilized. The residue is then purified by supercritical liquid chromatography (Chiralpak AD-H™ 250×30 mm ID, 5 µm, mobile phase: 45% methanol (0.1% NH ₄ OH) in CO ₂ , flow rate 50 ml/min, temp. . 35°C, RT 3.53 and 4.07 min.) to obtain the title compound (552 mg, 71%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.64 (br.s, 1H), 8.11 (s, 1H), 7.44 (d, J=9.0 Hz, 1H), 7 ,16-7.15 (m, 1H), 6.65-6.64 (m, 1H), 5.02-4.81 (m, 2H), 4.15-3.94 (m, 1H), 3 , 67-3.54 (m, 1H), 3.26 (s, 3H), 3.14-2.95 (m, 2H), 2.71-2.56 (m, 2H), 2.31 -2.19 (m, 2H), 1.22 (d, J=6.0 Hz, 3H), LC/MS [M+H]=339.9.

Example 1. (S)-2-Hydroxy-N-(cis-3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IA)

2-Hydroxy-N-{3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (532 mg, 1.57 mmol) purified with SFC ((Chiralpak AD-H™ 250×30 mm ID, 5 µl mobile phase: 45% ethanol (0.1%) NH ₄ OH) in CO ₂ , flow rate 50 ml/min., temp. 35°C, RT 3.44 min.) to obtain the title compound (223.7 mg, 42%). ^1H NMR (400 MHz, DMSO-d ₆ ) δ=11, 69-11.57 (m, 1H), 8.10 (s, 1H), 7.43 (d, J=9.0 Hz, 1H ), 7.16-7.12 (m, 1H), 6.67-6.61 (m, 1H), 4.92 (d, J=5.0 Hz, 1H), 4.90-4.84 ( m, 1H), 4.11-4.01 (m, 1H), 3.63-3.53 (m, 1H), 3.27-3.23 (m, 3H), 3.11-2, 96 (m, 2H), 2.63-2.53 (m, 2H), 2.29-2.20 (m, 2H), 1.21 (d, J=6.0 Hz, 3H), LC /MS [M+N]=340.2.

The crystalline product can be obtained from ethyl acetate.

X-ray structure of a single crystal

Data collection is carried out on a Bruker D8 Quest diffractometer at room temperature. Data collection includes omega and fiscal scans. The structure is determined by internal phasing in the triclinic space group P1 using the SHELX software package. The structure is then refined using the least squares method in the full matrix approximation. All non-hydrogen atoms are identified and their location is refined using anisotropic substitution parameters.

The positions of hydrogen atoms on nitrogen and oxygen atoms are determined from the Fourier difference map and refined with distance restrictions. The positions of the remaining hydrogen atoms are calculated and their connection with the atoms that are their carriers is assumed. The final refinement includes isotropic substitution parameters for all hydrogen atoms. Absolute structure analysis using the likelihood method (Hooft 2008) is carried out using PLATON (Spek 2 010). Provided that the submitted sample is inantiomerically pure, the results indicate that the absolute structure is correctly determined. Calculation using this method shows that the probability of correctly determining the structure is 100%. The Hooft parameter is 0.104 at Esd (15), the Parson parameter is 0.097 at Esd (16). The absolute configuration at C13 and C2 7 for two identical molecules per asymmetric cell is confirmed as (-S) (-S). The final R-index is 5.9%. The final Fourier difference shows no missing or misplaced electron density. Basic information about the crystal, the totality of the data obtained and the refinement are presented in Table 1. The coordinates of the atoms are presented in Table 2.

Software and links

SHELXTL, Version 5.1, Bruker AXS, 1997. PLATON, A.L. Speck, J. Appl. Christ. 2003, 36, 7-13. MERCURY, C.F. Macrae, P.R. Edington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor, M. Towler and J. van de Streek, J. Appl. Christ. 39, 453-457, 2006. OLEX2, Dolomanov, O.V.; Bourhis, L. J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H., (2009). J. Appl. Cryst., 42, 339-341. R.W.W. Hooft et al. J. Appl. Christ. (2008). 41.96-103. H.D. Flack, Acta Cryst. 1983, A39, 867-881.

Example 2: (R)-2-Hydroxy-N-(cis-3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IC)

2-Hydroxy-N-{3-[methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (532 mg, 1.57 mmol) was purified using SFC ((Chiralpak AD-H™ 250×30 mm ID, 5 µm, mobile phase: 45% ethanol (0.1%) NH ₄ OH) in CO ₂ , flow rate 50 mg/min., temp. 35°C , RT, 3.88 min.) to obtain the title compound (222.7 mg, 42%). ^1H NMR (400 MHz, DMSO-d ₆ ) δ=11, 69-11.56 (m, 1H), 8.10 (s, 1H), 7.43 (d, J=9.0 Hz, 1H ), 7.18-7.11 (m, 1H), 6.66-6.60 (m, 1H), 4.96-4.84 (m, 2H), 4.12-3.99 (m , 1H), 3.66-3.54 (m, 1H), 3.24 (s, 3H), 3.12-2.94 (m, 2H), 2.64-2.56 (m, 2H ), 2.30-2.18 (m, 2H), 1.20 (d, J=6.5 Hz, 3H), LC/MS [M+H]=340.2. The crystalline product can be obtained from ethyl acetate.

X-ray structure of a single crystal

Data collection is carried out on a Bruker D8 Quest diffractometer at room temperature. Data collection consists of omega and phi scans. This lot yields wafers of crystals stacked together, the crystal is separated and mounted for data collection. The structure is determined by internal phasing in the triclinic space group P1 using the SHELX software package. The structure is then refined using the least squares method in the full matrix approximation. All non-hydrogen atoms are identified and their location is refined using anisotropic substitution parameters. The positions of hydrogen atoms on nitrogen and oxygen atoms are determined from the Fourier difference map and refined by limiting the distances. The positions of the remaining hydrogen atoms are calculated and an assumption is made about their connection with the atoms that are their carriers. The final refinement includes isotropic substitution parameters for all hydrogen atoms. Absolute structure analysis using likelihood methods (Hooft 2008) is carried out using PLATON (Spek 2010). Provided that the submitted sample is inantiomerically pure, the results indicate that the absolute structure is correctly determined. Calculation using this method shows that the probability of correctly determining the structure is 100%. The Hooft parameter is 0.044 at Esd (18), the Parson parameter is 0.050 at Esd (19). The absolute configuration at C13 and C27 for two identical molecules per asymmetric cell is confirmed as (-R)_(-R). Pseudo-symmetry to P-1. The final R-index is 4.5%. The final Fourier difference shows no missing or misplaced electron density. Basic information about the crystal, the totality of the data obtained and refinement are presented in Table 3. The coordinates of the atoms are presented in Table 4.

Software and links

SHELXTL, Version 5.1, Bruker AXS, 1997. PLATON, A.L. Spek, J. Appl. Christ. 2003, 36, 7-13. MERCURY, C.F. Macrae, P.R. Edington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor, M. Towler and J. van de Streek, J. Appl. Cryst.39, 453-457, 2006.OLEX2, Dolomanov, O.V.; Bourhis, L. J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H., (2009). J. Appl. Cryst., 42, 339-341. R.W.W. Hooft et al. J. Appl. Christ. (2008). 41.96-103. H.D. Flack, Acta Cryst. 1983, A39, 867-881.

Example 3 - Alternative preparation of (S)-2-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IA )

Step 1. (S)-tert-Butyl-((1-chloropropan-2-yl)oxy)dimethylsilane

Pyridine (1.0 g, 12.7 mmol) and tert-butyldimethylsilyl chloride (1.91 g, 12.7 mmol). After stirring at 20°C, the reaction mixture is diluted with water (40 ml). The resulting mixture was extracted with methyl tert-butyl ether (2×40 ml). The organic extracts are combined, washed with brine (50 ml) and dried (Na ₂ SO ₄ ). The solvent was removed to give the title compound (2.2 g), which was used without further purification. ^1H NMR (4 00 MHz, CDCl ₃ ) δ 3.96 (m, 1H), 3.42 (dd, J=10.8, 5.9 Hz, 1H), 3.34 (dd, J=10 .7, 5.9 Hz, 1H), 1.23 (d, J=6.1 Hz, 3H), 0.89 (s, 9H), 0.08 (d, J=4.1 Hz, 6H ).

Step 2. (S)-S-(2-((tert-Butyldimethylsilyl)oxy)propyl)ethanethoate

Potassium thioacetate is added to a solution of (S)-tert-butyl((1-chloropropan-2-yl)oxy)dimethylsilane (2.2 g, 10.5 mmol) in N,N-dimethylformamide (20 ml) at 20°C (2.41 g, 21.1 mmol) and potassium iodide (17.5 mg, 0.10 mmol). The reaction mixture is heated to 80°C and stirred for 20 hours. The reaction mixture is cooled to room temperature, diluted with water (50 ml) and the resulting mixture is extracted with methyl methyl ether (40 ml x 2). The organic extracts were cooled, washed with brine (60 mL), dried (Na ₂ SO ₄ ) and the solvent was removed to give the title compound (2.40 g) which was used without further purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.91 (m, 1H), 3.00-2.91 (m, 2H), 2.33 (s, 3H), 1.18 (d, J= 6.1 Hz, 3H), 0.89 (s, 9H), 0.07 (d, J=6.8 Hz, 6H).

Step 3. (S)-2-Hydroxypropane-1-sulfonyl chloride

Cl ₂ (gas) is passed through a solution of (S)-S-(2-((tert-butyldimethylsilyl)oxy)propyl)ethanethioate (0.5 g, 2.01 mmol) in dichloromethane (20 ml) and water (10 ml) for 5 minutes at 0°C, as a result the solution becomes yellow. The Cl ₂ (gas) flow is stopped and the reaction mixture is stirred at 0°C for 5 hours. N ₂ (gas) is then bubbled through the reaction mixture to obtain a colorless solution. TLC shows the consumption of the starting material and the appearance of a new spot. Water (20 ml) was added to the reaction mixture, and the resulting mixture was extracted with dichloromethane (40 ml). The organic extract is washed with 10% NaHCO ₃ (50 ml), brine (50 ml) and dried (Na ₂ SO ₄ ). The solvent was removed to give the crude title compound (0.6 g) as a colorless oil, which was used in the next step without further purification.

Step 4. (S)-2-Hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

Add cis-N ¹ -methyl-N ¹ -(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclobutan-1,3-diamine (200 mg, 0. 37 5 mmol), Na ₂ CO ₃ (199 mg, 1.8 8 mmol), tetrahydrofuran (7 ml) and water (2.5 ml). After 2 minutes, the solution obtained in step 3 of Example 1 (292 mg) in tetrahydrofuran (2 ml) was added. The resulting suspension is heated to 50°C and stirred for 18 hours. The reaction mixture was cooled to room temperature and water (20 ml) was added to the mixture. The resulting mixture was extracted with ethyl acetate (20 ml×2). The organic extracts are cooled, dried (Na ₂ SO ₄ ), the solvent is removed, and the resulting crude product is purified by chromatography (silica, EtOAc/PetEther, 20-100%) to give the title compound (30 mg). LC/MS m/z (M+H) ⁺ =4 94, 3.

Step 5. (S)-2-Hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a solution of (S)-2-hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide ( 45 mg, 0.0 91 mmol) in a mixture of tetrahydrofuran: water (5 ml: 1 ml) at 15°C add lithium hydroxide monohydrate (23.0 mg, 0.547 mmol). After this, the reaction mixture is heated to 60°C and stirred at the specified temperature for 16 hours. Water (10 ml) was added to the reaction mixture, and the resulting mixture was extracted with ethyl acetate (15 ml x 4). The organic extracts are combined, dried (Na ₂ SO ₄ ) and the solvent is removed, the resulting crude product (30 mg) is purified by RP-HPLC (Phenomenex Gemini C-18™, 250 × 50 mm, 10 µm, H ₂ O/CH ₃ CN + 0.05% NH ₄ OH, 15-35% for 10 min) to obtain the title compound (16 mg). ^1H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 8.09 (s, 1H), 7.41 (s, 1H), 7.14 (d, J=3.6 Hz, 1H), 6.62 (d, J=3.6 Hz, 1H), 4.89 (ddd, J=17.2, 7.9, 5.9 Hz, 2H), 4.04 (m, 1H ), 3.58 (m, 1H), 3.24 (s, 3H), 3.14-2.86 (m, 2H), 2.62-2.51 (m, 2H), 2.23 ( m, 2H), 1.20 (d, J=6.2 Hz, 3H); LC/MS m/z (M+H)+=340.1; chiral SFC™ (Chiralpak AD-3™, 150 × 4.6 mm, 3μ, 40% MeOH (0.05% DEA) in CO ₂ (isocrat.) 10 min., 2.5 mg/min., T= 35°С), R _t =5.54 min., 99% e.e.

Example 4 - Alternative preparation of (R)-2-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IC )

Step 1. (R)-tert-Butyl((1-chloropropan-2-yl)oxy)dimethylsilane

To a solution of (R)-(-)-1-chloropropan-2-ol (2.0 g, 21.2 mmol, CAS: 19141-39-0) in N,N-dimethylformamide (40 ml) at 20°C add pyridine (2.0 g, 25.4 mmol) and tert-butyldimethylsilyl chloride (3.83 g, 25.4 mmol). After stirring for 20 hours, the reaction mixture was diluted with water (60 ml) and the resulting mixture was extracted with methyl tert-butyl ether (2×80 ml). The organic extracts are cooled, washed with brine (100 ml) and dried (Na ₂ SO ₄ ). The solvent was removed to give the title compound (4.60 g) which was used without further purification. ^1H NMR (400 MHz, CDCl ₃ ) δ 3.96 (m, 1H), 3.42 (dd, J=10.8, 5.9 Hz, 1H), 3.34 (dd, J=10, 7, 5.9 Hz, 1H), 1.23 (d, J=6.l Hz, 3H), 0.89 (s, 9H), 0.08 (d, J=4.l Hz, 6H) .

Step 2. (R)-S-(2-((tert-Butyldimethylsilyl)oxy)propyl)ethanethoate

Potassium thioacetate (5, 03 g, 44.1 mmol) and potassium iodide (36.6 mg, 0.22 mmol). The mixture is kept for 20 hours at 80°C, then the reaction mixture is cooled to room temperature, diluted with water (100 ml) and the resulting mixture is extracted with methyl tert-butyl ether (100 ml x 2). The organic extracts are combined, dried (Na ₂ SO ₄ ) and the solvent is removed to give the title compound (5.0 g). ^1H NMR (400 MHz, CDCl ₃ ) δ 3.90 (m, 1H), 3.00-2.91 (m, 2H), 2.33 (s, 3H), 1.18 (d, J= 6.l Hz, 3H), 0.88 (s, 9H), 0.06 (d, J=6.7 Hz, 6H).

Step 3. (R)-2-((tert-Butyldimethylsilyl)oxy)propane-1-sulfonyl chloride

Cl ₂ (gas) is passed for 5 minutes at 0°C through a solution of (R)-S-(2-((tert-butyldimethylsilyl)oxy)propyl)ethanethioate (0.5 g, 2.01 mmol) in dichloromethane (20 ml) and water (10.0 ml), as a result of which the solution turns yellow. The flow of C1 ₂ (gas) is stopped and the reaction mixture is stirred at 0°C for 0.5 hours. N ₂ gas is passed through the reaction mixture to obtain a colorless solution. TLC shows consumption of the starting material with the formation of a new spot. Water (20 ml) was added to the reaction mixture, and the reaction mixture was extracted with dichloromethane (40 ml). The organic extract is washed with 10% NaHCO ₃ (50 ml), brine (50 ml) and dried (Na ₂ SO ₄ ). The solvent was removed to give the title compound (0.4 g) as a colorless oil, which was used in the next step without further purification.

Step 4. (R)-2-((tert-Butyldimethylsilyl)oxy)-N-((1S,3S)-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidine-4 - yl)amino)cyclobutyl)propane-1-sulfonamide

Add cis-N ¹ -methyl-N ¹ -(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclobutan-1,3-diamine (150 mg, 0. 2 81 mmol), Na ₂ CO ₃ (149 mg, 1.41 mmol), tetrahydrofuran (5 ml) and water (2.0 ml). After 2 minutes, a solution of the compound obtained in step 3 of Example 2 (292 mg, 0.75 mmol) in THF (2 ml) was added. The resulting suspension is heated to 50°C and stirred for 20 hours. The reaction mixture was cooled to room temperature and water (20 ml) was added to the mixture. The resulting mixture was extracted with EtOAc (20 ml x 2). The organic extracts are cooled, dried (Na ₂ SO ₄ ) and the solvent is removed, the resulting crude product is purified by chromatography (silica, EtOAc/petroleum ether, 20-100%) to obtain the title compound (60 mg) and the product without the TBS group (20 mg). LC/MS m/z (M+H)+=608.3 (peak 1); LC/MS m/z (M+H)+=494.3 (peak 2).

Step 5. (R)-2-Hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a solution of (R)-2-((tert-butyldimethylsilyl)oxy)-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl )propane-1-sulfonamide (60 mg, 0.099 mmol) in dichloromethane (5 ml) add trifluoroacetic acid (1.5 ml) at 0°C. The mixture is kept for 16 hours at 15°C, then the solvent is removed and the residue is dissolved in ethyl acetate (15 ml). The mixture was washed with saturated NaHCO ₃ (aq), dried (Na ₂ SO ₄ ) and the solvent was removed to give the title compound (50 mg), which was used in the next step without further purification. LC/MS m/z (M+H) ⁺ =494.3.

Step 6. (R)-2-Hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a solution of (R)-2-hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide ( 50 mg, 0.10 mmol) in a mixture of tetrahydrofuran: water (5 ml: 1 ml) at 15°C add lithium hydroxide monohydrate (42.5 mg, 1.01 mmol). The mixture is kept for 16 hours at 65°C, water (10 ml) is added and the resulting mixture is extracted with ethyl acetate (4×10 ml). The organic extracts are combined, dried (Na ₂ SO ₄ ) and the solvent is removed, the resulting crude product is purified by pre-HPLC (Phenomenex Gemini C-18™, 250 × 50 mm, 10 μm, H ₂ O/CH ₃ CN + 0.05% NH ₄ OH, 15-35% for 10 minutes) to obtain the title compound (18 mg). ^1H NMR (4 00 MHz, DMSO) δ 11.63 (s, 1H), 8.10 (s, 1H), 7.43 (d, J=9.1 Hz, 1H), 7.15 (dd , J=3.6, 2.4 Hz, 1H), 6.64 (dd, J=3.6, 1.9 Hz, 1H), 4.97-4.83 (m, 2H), 4, 12-3.98 (m, 1H), 3.59 (m, 1H), 3.25 (s, 3H), 3.13-2.92 (m, 2H), 2.58 (m, 2H) , 2.24 (m, 2H), 1.21 (d, J=6.2 Hz, 3H); LC/MS m/z (M+H) ⁺ =340.0; chiral SFC (Chiralpak AD-3™, 150 × 4.6 mm, 3 µm, 40% MeOH (0.05% DEA) in CO ₂ isocratic. 10 minutes, 2.5 mg/min., T=35°C ), R _t =6.75 min., 99% ee.

Example 5-3-Hydroxy-N-(cis-3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (IB)

To a stirred mixture of cis-N-Methyl-N-7H-pyrrolo[2,3-d]pyrimidin-4-ylcyclobutan-1,3-diamine (190 mg) and potassium carbonate (240 mg) in a 1:1 mixture of tetrahydrofuran: water (5 ml each) used as a solvent, methyl 3-(chlorosulfonyl)propanoate (250 mg) is added at 10°C. The mixture is warmed to room temperature and stirred for 10 minutes. The mixture is concentrated in vacuo. The residue was purified by column chromatography (MeOH/DCM 1:20) to give methyl 3-(N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)sulfamoyl )propanoate (140 mg) as a white solid. ^1H NMR (400 MHz, methanol- _d4 ): 8.13 (s, 1H), 7.13 (d, 1H), 6.70 (d, 1H), 4.89 (m, 1H), 3 .73 (s, 3H), 3.69 (m, 1H), 3.4 (m, 5H), 2.82 (m, 4H), 2.34 (m, 2H), MS m/z for C ₁₅ H ₂₁ N ₅ O ₄ S: 390.2 (M+Na)+. HPLC: column Ultimate ХВ-С18 3pm 3.0*50 mm, retention time: 2.13 min., mobile phase: from 0% acetonitrile (0.1% TFA) in water to 60% acetonitrile (0.1% TFA ) in water, wavelength: 220 nm.

To a stirred solution of methyl 3-(N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)sulfamoyl)propanoate (120 mg) in dry tetrahydrofuran (5 ml) at 0°C in a nitrogen atmosphere add lithium aluminum hydride (50 mg). The reaction mixture is warmed to room temperature and stirred for 0.5 hours. The mixture was quenched by careful addition of methanol and purified by column chromatography (MeOH/DCM 1:10) to give 3-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino )cyclobutyl)propane-1-sulfonamide (50 mg) as a white solid. 1H NMR (400 MHz, methanol- _d4 ): 8.12 (s, 1H), 7.13 (d, 1H), 6.70 (d, 1H), 4.89 (m, 1H), 3. 73 (m, 3H), 3.36 (s, 3H), 3.15 (m, 2H), 2.81 (m, 2H), 2.35 (m, 2H), 2.03 (m, 2H ); MS m/z for C ₁₄ H ₂₁ N ₅ O ₃ S: 340.2 (M+H)+; HPLC: Ultimate ХВ-С18 3 pm column 3.0 × 50 mm, retention time: 1.85 min., mobile phase: from 0% acetonitrile (0.1% TFA) in water to 60% aacetonitrile (0.1% TFA) in water, wavelength: 22 0 nm.

Example 6 - Alternative preparation of 3-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide

To a mixture of cis-N-methyl-N-{7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]-pyrimidin-4-yl}cyclobutan-1,3-diamine dihydrobromide (988 mg ) triethylamine (562 mg) and ethyl 3-(chlorosulfonyl)propanoate (743 mg) are added to 50 ml of dichloromethane at 0°C. The reaction mixture is heated to 25°C and stirred at the specified temperature for 2 hours. The reaction mixture was evaporated in vacuo and the residue was purified by chromatography (silica gel, Biotage column, 20 g silica, petroleum ether: ethyl acetate - 1:0 to 0:1, ethyl acetate: methanol 20:1) to obtain ethyl-3-(N- (cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)sulfamoyl)propanoate. _{LCMS m/z for C23H29N5O6S2 : 535.9 ( M} +H)+.

To a solution of ethyl 3-(N-(cis-3-(methyl-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)sulfamoyl)propanoate (7 00 mg) lithium aluminum hydride (74.4 mg) is added to 50 ml of tetrahydrofuran at 0°C. The reaction mixture is allowed to warm to a temperature of 25°C for 15 hours. The reaction mixture is carefully quenched by adding 1 ml of water, then filtered. The filtrate is evaporated in vacuo to obtain 3-hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane as a crude product. 1-sulfonamide, which is used further without further purification. LCMS m/z for C ₂₁ H ₂₇ N ₅ O ₅ S ₂ : 494.0 (M+H)+.

To a solution of 3-hydroxy-N-(cis-3-(methyl(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide (400 mg) in LiOH (97 mg) is added to 20 ml ethanol and 10 ml water. The reaction mixture is heated to 90°C and stirred for 2 hours. The reaction mixture was cooled and evaporated in vacuo, the resulting crude product was purified by pre-HPLC to obtain 181 mg of 3-hydroxy-N-(cis-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino )cyclobutyl)propane-1-sulfonamide as a white solid. HPLC conditions: DuraShell™ column 150 × 25 mm × 5 µm, water (0.05% ammonium hydroxide v/v)-acetonitrile, 1-41%B, gradient 10 min, retention time 1 min. 100%B, flow rate 25 mg/min; ^1H NMR (400 MHz, DMSO- _d6 ): 11.6 (s, 1H), 8.11 (s, 1H), 7.53 (s, 1H), 7.15 (d, 1H), 6 .65 (d, 1H), 4.89 (m, 1H), 4.68 (s, 1H), 3.58 (m, 1H), 3.49 (m, 2H), 3.26 (s, 3H), 3.0 (m, 2H), 2.58 (m, 2H), 2.24 (m, 2H), 1.81 (m, 2H). Biological Activity Assessment JAK Caliper Enzymatic Assay at 1 mM ATP The test sample is dissolved in dimethyl sulfoxide (DMSO) to an initial concentration of 30 mM. Prepare an 11-point semi-log dilution series in DSMO with an upper concentration of 500 µM. The test compound plate also contains positive control wells containing a known inhibitor to determine 100% inhibition, and negative control wells containing DMSO to determine no inhibition. The plates with the compound are diluted 1 to 60, obtaining a final concentration of the test compound of 10 μM and a DMSO concentration of 2%. The experimental compound and control wells are placed in a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP, and 1 μM peptide substrate. The JAK1 and TYK2 tests contain 1 μM IRStide peptide (5FAM-KKSRGDYMTMQID), the JAK2 and JAK3 tests contain 1 μM JAKtide peptide (FITC-KGGEEEEYFELVKK). Assays are initiated by adding 20 nM JAK1, 1 nM JAK2, 1 nM JAK3, or 1 nM TYK2 enzyme and incubate at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3, or 135 minutes for TYK2. Enzyme concentrations and incubation times are optimized for each new enzyme preparation and adjusted slightly over time to achieve 20% to 30% phosphorylation. Testing is stopped with a final concentration of 10 mM EDTA, 0.1% coating reagent and 100 mM HEPES, pH=7.4. Test plates are placed on a Caliper Life Science Lab Chip 3000 (LC3000) and each well is sampled using appropriate separation conditions to quantify unphosphorylated and phosphorylated peptide. The obtained data are presented in Table 5.

Cellular Activity Assessment: Cytokine-Induced STAT Phosphorylation in Human Whole Blood, Human Keratinocytes, and IFNγ-Primed THP-1 Cells

Human whole blood is collected from healthy donors by venous puncture into vacuum collection tubes containing sodium heparin in accordance with Pfizer protocols (Protocol No. GOHW RDP-01) approved by the Shulman Institutional Review Board. The blood is heated to 37°C before use. Human whole blood is aliquoted (90 μl/well) into 96-well deep-well V-bottom plates and treated with compounds (5 μl/well) at varying concentrations up to 60 μM (final concentration 0.2% DMSO) at 37°C C for 60 minutes. Then IFNcx (5000 U/ml), IFNγ (100 ng/ml), IL-6 (50 ng/ml), IL-10 (30 ng/ml), IL-12 (30 ng/ml), IL -15 (30 ng/ml), IL-21 (50 ng/ml), IL-23 (25 ng/ml), IL-27 (1000 ng/ml), EPO (2U/ml), TSLP (50 ng /ml) or PBS and incubate for 15 minutes. Anti-cell surface antibodies are added 15 minutes before cytokine stimulation; anti-CP3-VU421 (0.5 μl/well) to IL-6-treated samples and TSLP-treated samples, and anti-CD14-BV421 (0.5 μl/well) to IFNγ-treated samples. Samples are treated with warm 1× Lyse/Fix buffer (700 µl/well) to stop activation and further incubated at 37°C for 20 minutes to lyse red blood cells. The plates are centrifuged at 300 × g for 5 minutes, the supernatant is aspirated, and the cells are washed with 800 μl/well of staining buffer (D-PBS containing 0.1% FBS and 0.01% sodium azide). The washed cell pellets were resuspended in pre-cooled 90% methanol (350 μl/well) and incubated at 4°C for 30 minutes. After 90% of the methanol has been removed, the cells are washed once with staining buffer. All samples are then suspended in solutions of the desired anti-phospho-STAT antibodies (150 μl/well) at a dilution of 1:150: anti-pSTAT1-AF647 in IFNγ-treated samples; anti-pSTAT1-AF488 and anti-pSTAT3-AF647 in samples treated with IL-6, anti-pSTAT3-AF647 in samples treated with IFNα, IL-10, IL-21, IL-23 and IL-27, anti-pSTAT4- AF647 in IL-12 treated samples, anti-pSTAT5-AF-647 in EPO, IL-15 and TSLP treated samples.

Primary human keratocytes are cultured in DermaLife™ medium with a range of additives to increase the cell population. In the experiments, cells of 2-5 passages were used. Cells are harvested at -80% confluence, suspended in warm DermaLife™ medium, aluquoted (90 μl/well) into 96-well deep-well V-bottom plates and incubated at 37°C for 30 minutes. Cells are then treated with compounds (0.0003 to 20 μM) for 60 minutes followed by stimulation with IL-4 (2 ng/ml) or IL-13 (20 ng/ml) at 37°C for 15 minutes. For the pSTAT3-induced IL-22 assay, keratocytes were seeded in 24-well plates and cultured for 18 hours. The cell medium is changed to DermaLife™ basal medium, the cells are treated with compounds (0.0003 to 20 μM) for 60 minutes and then stimulated with IL-22 (100 ng/ml) for 30 minutes. Cells are separated by treatment with a mixture of 0.25% trypsin/EDTA. Stimulated keratinocytes are fixed with 2% paraformaldehyde and permabilized with 90% methanol. Fixed and permeabilized cells are stained with AlexaFluor647™ labeled anti-pSTAT6 antibody (1 in 150 dilution) for IL-4 and IL-13 treated samples and AlexaFluor647 labeled anti-pSTAT3 antibody (1 in 150 dilution) for IL-treated samples -22.

THP-1 cells are maintained in RPMI 1640 medium containing 10% FBS, 50 μM 2 mercaptoethanol, 50 U/ml penicillin, 50 μg/ml streptomycin and 2 mM L-glutamine. THP 1 cells are treated with IFNγ (20 ng/ml) for 18 hours. IFNγ-primed THP-1 cells were resuspended in fresh RPMI 1640 medium, treated with compounds (0.0003 to 20 μM) for 60 minutes, followed by stimulation with IL-31 (1 μg/ml) for an additional 10 minutes. The cells are then fixed with 2% paraformaldehyde and permeabilized with 90% methanol.

Fixed and permeabilized cells are stained with AlexaFluor647™ labeled anti-pSTAT3 antibody (1 in 150 dilution).

After overnight incubation at 4°C, anti-pSTAT-stained samples were transferred to 96-well polypropylene U-plates and flow cytometric analysis was performed on an LSR Fortessa™ equipped with an HTS plate loader. In human whole blood samples, the lymphocyte population is selected for pSTAT histogram analysis for samples treated with IFNα, IL-10, IL-12, IL-15, IL-21, IL-23, and IL-27; CD14 ⁺ cells for IFNγ-treated samples; CD3 ⁺ cells for samples treated with IL-6 and TSLP; all events (full populations) for EPO-treated samples. Complete populations of keratinocytes and THP-1 cells were selected for analysis of IL-4, IL-13, IL-22 and IL-31. Background fluorescence is determined using unstimulated cells, and a gate is placed at the base of the peak to include -0.5% of the population of cells producing signal above the threshold. Statistical analysis of the histogram is carried out using FACSDiva version 8.0. The relative unit of fluorescence that changes the level of pSTAT is calculated by multiplying the percentage of a positive population by its average fluorescence. Inhibition curves and half concentration (IC ₅₀ ) values are determined using Prism™ software (Version 8) or Activity Base data analysis software (ID Business Solutions).

Cellular activity towards cytokine-induced STAT phosphorylation

The compounds of formulas IA and IB, prepared in accordance with Examples 1 and 3 and Examples 5 and 6, respectively, are formed in vivo by human metabolism upon administration of abrocitinib and demonstrate activity similar to abrocitinib, a potent JAK1 inhibitor, against IL-induced STAT phosphorylation -4, IL-13, IL-22, IL 31 and TSLP, in human keratocytes, interferon-γ THP-1 primed cells or human whole blood. Gooderham M.J. et al. JAMA Dermatology, 155(12), 1371-1379 (October 2019). Notably, like abrocitinib, all of these compounds are more effective against cytokines that signal through JAK1-dependent pathways compared to those that signal through JAK1-dependent pathways, and therefore likely contribute to pharmacological activity mainly through effects on JAK1. A comparison of the cellular activity of abrocitinib and compounds of formulas IA and IB along similar signaling pathways is presented in Table 6.

Claims (10)

1. A compound of formula IA having the structure or a pharmaceutically acceptable salt thereof. 2. A compound of formula IB having the structure or a pharmaceutically acceptable salt thereof. 3. (S)-2-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide or a pharmaceutically acceptable salt thereof. 4. 3-Hydroxy-N-(3-(methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutyl)propane-1-sulfonamide or a pharmaceutically acceptable salt thereof. 5. A compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-4 in isolated form. 6. Pharmaceutical composition for inhibiting Janus kinase (JAK), containing an effective amount of a compound according to claims. 1-5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.