EA041895B1 - NEW SALTS AND THEIR PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF INFLAMMATORY DISEASES - Google Patents

Description

The field to which the invention relates

The present invention relates to salt and crystalline forms of a compound according to formula I useful in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disturbance of cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons. In particular, the salt of the present invention inhibits JAK kinase, the tyrosine kinase family, and more specifically JAK1 kinase. The present invention also provides pharmaceutical compositions comprising a salt of the present invention and methods for the prevention and/or treatment of diseases, including inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons by administering the salt of the present invention in accordance with formula I.

Prerequisites for the creation of the invention

Current therapies for the treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases accompanied by impaired cartilage renewal, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons, in particular rheumatoid arthritis, are far from satisfactory. , and there remains a need to identify new therapeutic agents that can be used in their treatment. These conditions are chronic diseases that require long-term treatment and repeated administration of the drug. Long-term treatment can be a heavy burden for the patient and physician as the patient may have or become intolerant to the drug, and in addition, high doses or frequency of drug administration may cause unpleasant side effects and/or low patient compliance where the patient may intermittently , intentionally or accidentally, skip a dose. The consequences of non-compliance lead to various chronic diseases and range from minimal to very significant (Ingersoll & Cohen, 2008). Therefore, there is a need to identify new agents to enhance the practitioner's arsenal and low frequency regimen compounds to improve the lives of patients.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transduce cytokine signals from membrane receptors to the STAT transcription factors. Four members of the JAK kinase family have been described: JAK1 kinase, JAK2 kinase, JAK3 kinase, and TYK2. Upon binding of a cytokine to its receptor, members of the JAK kinase family auto- and/or transphosphorylate each other, followed by phosphorylation of STATs, which then migrate to the nucleus to modulate transcription. Intracellular signal transduction of JAK-STAT kinases serves interferons, most interleukins, and a number of cytokines and endocrine factors such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF, and PRL (Vainchenker, Dusa, & Constantinescu, 2008).

The study of combinations of genetic models and small molecule JAK inhibitors has opened up the therapeutic potential of several JAK kinases.

JAK1 kinase is a target in immunoinflammatory diseases. JAK1 kinase heterodimerizes with other JAK kinases to transduce cytokine-dependent pro-inflammatory signals. Thus, inhibition of JAK1 kinase is of interest for immunoinflammatory diseases with pathology-associated cytokines that use JAK1 signaling, such as IL-2, IL-6, IL-4, IL-5, IL-13, or IFN gamma, and also for other diseases caused by JAK-mediated signal transduction.

Regarding the roles of members of the JAK kinase family, there is some overlap, as most signaling pathways involve more than one JAK kinase, however, for some growth factors, such as erythropoietin and thrombopoietin, only JAK2 is involved.

JAK3 plays an important role in blocking immune function through signaling generated by interleukin (IL)-2.

On the other hand, TYK2 kinase could probably work in combination with JAK2 kinase to transduce cytokine signals such as IL-12 and IL-23.

The role of JAK enzymes has been investigated primarily using mice, where each member of the JAK kinase family has been excluded. JAK1 knockout mice exhibit a perinatal lethal phenotype and also have a defect in lymphoid tissue development and function due to impaired cytokine signaling through JAK1 kinase. Deficiency of JAK2-kinase leads to embryonic lethality on the 12th day as a result of the absence of definitive erythropoiesis. JAK3-deficient mice have a severe combined immunodeficiency (SCID) phenotype but no non-immune defects (Verstovsek, 2009).

As observed with pan-JAK inhibitors, non-selective inhibition may be associated with side effects such as anemia, increased infection rates, decreased neutrophil and lymphocyte levels, decreased hemoglobin, and increased cholesterol levels (Dolgin, 2011).

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Thus, the development of a selective JAK kinase inhibitor would be beneficial in order to minimize such side effects.

Cartilage degeneration is a symptom of various diseases, among which rheumatoid arthritis and osteoarthritis are the most prominent. Rheumatoid arthritis (RA) is a chronic degenerative joint disease characterized by inflammation and destruction of the joint structure. If the disease is not diagnosed, it leads to significant disability and pain due to loss of joint functionality and even premature death. The goal of therapy for rheumatoid arthritis, therefore, is not only to slow the progression of the disease, but also to achieve remission in order to halt joint destruction and improve quality of life. In addition to the severity of the outcome of the disease, the high prevalence of rheumatoid arthritis (|~0.8% of adults worldwide suffer from this disease) means serious socio-economic consequences. (Smolen & Steiner, 2003) (O'Dell, 2004). JAK1 kinase is involved in intracellular signal transduction for many cytokines and hormones. Pathologies associated with any of these cytokines and hormones can be alleviated with JAK1 kinase inhibitors. Therefore, certain allergic, inflammatory and autoimmune disorders can be more effectively treated with the compounds of this invention, including rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis, osteoarthritis, asthma, chronic obstructive pulmonary disease (COPD), tissue fibrosis, eosinophilic inflammation, inflammation of the esophagus, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis), transplantation, graft-versus-host disease, psoriasis, myositis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and multiple sclerosis. (Kopf, Bachmann, & Marsland, 2010)

Psoriasis is a disease that can affect the skin. The causes of psoriasis are not fully understood, but it is believed to be an immune-mediated disease associated with the release of cytokines, in particular TNFa, which causes inflammation and rapid reproduction of skin cells. This hypothesis is supported by the observation that immunosuppressant treatment can clear psoriasis plaques (Zenz, et al., 2005). Psoriasis can also cause joint inflammation, which is known as psoriatic arthritis. Between 10 and 30% of all people with psoriasis also have psoriatic arthritis ((CHMP), 18 November 2004). Due to its chronic relapsing nature, psoriasis is a problematic disease to treat. It has recently been demonstrated that inhibition of JAK kinase can lead to a successful improvement in psoriatic conditions (Punwani, et al., 2012).

Inflammatory bowel disease (IBD) is a group of inflammatory diseases of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis. Recently, T-cell protein tyrosine phosphatase (TCPTP) was found to be a JAK/STAT, and phosphatase growth factor receptor, which is associated with the pathogenesis of type 1 diabetes, rheumatoid arthritis, and Crohn's disease through GWAS using a genome-wide association study (GWAS) (Zikherman & Weiss, 2011). Thus, inhibition of the JAK kinase pathway may provide a method for treating IBD.

Members of the JAK kinase family have been implicated in other conditions, including myeloproliferative disorders (O'Sullivan, Liongue, Lewis, Stephenson, & Ward, 2007), where JAK2 kinase mutations have been identified. This indicates that inhibitors of JAK kinases, in particular JAK2 kinase, can also be used in the treatment of myeloproliferative disorders. In addition, the JAK family of kinases, particularly JAK1 kinase, JAK2 kinase, and JAK3 kinase, have been associated with cancers, particularly leukemias (e.g., acute myelocytic leukemia (O'Sullivan, Liongue, Lewis, Stephenson, & Ward, 2007) (Xiang, et al., 2008) and acute lymphoblastic leukemia (Mullighan, 2009)), cutaneous T-cell lymphoma (Zhang, 1996) or solid tumors such as uterine leiomyosarcoma (Constantinescu, Girardot, & Pecquet, 2007), prostate cancer gland (Tam, McGlynn, Traynor, Mukherjee, Bartlett, & Edwards, 2007) and breast cancer (Berishaj, et al., 2007). These results indicate that inhibitors of JAK kinase, in particular JAK1 kinase, may also be useful in the treatment of cancers (leukemias and solid tumors, eg uterine leiomyosarcoma, prostate cancer, pancreatic cancer).

In addition, Castleman's disease, multiple myeloma, mesangial proliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma are likely due to hypersecretion of the cytokine IL6, whose biological effects are mediated by intracellular JAK-STAT signaling (Naka, Nishimoto, & Kishimoto, 2002). This result shows that the JAK inhibitor can also find use in the treatment of the above diseases.

Thus, compounds that are potent inhibitors of JAK kinase may offer potential for the treatment of a wide variety of diseases and conditions described above.

The compound {5-[4-(1,1-dioxothiomorpholin-4-ylmethyl)phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2yl}cyclopropanecarboxylic acid amide (compound 1), which has chemical structure:

- 2 041895

Disclosed in our earlier application WO 2010/149769 (Menet C. J., 2010) as an inhibitor of JAK kinase and as useful in the treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases accompanied by impaired cartilage renewal, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons. Hereinafter in this application, this compound is referred to as compound 1. The data presented in WO 2010/149769 demonstrate that despite similar in vitro activities, compound 1 has surprisingly high in vivo activity compared to structurally similar compounds.

An important characteristic of various biologically active substances (for example, but not limited to, pharmaceuticals, drugs and biocides, commonly referred to as drugs) is their bioavailability or active concentration in a form that can be absorbed and used by the target organ or organism. In many cases, bioavailability is related to the water solubility of the drug.

To be useful as a therapeutic agent, the drug must be soluble in a suitable concentration range for the required period of time. Various options are available to achieve these properties, including formulating the drug into tablets, capsules, solutions, or other similar forms. Of particular interest are uniform release drugs in which the release rate of the drug is constant. However, the development of such systems can be complex and expensive.

Often, drugs in their free base form are poorly soluble in water, but the presence of acid sites (eg carboxylic acids, phenols, sulfonic acids) or basic sites (eg amino groups, basic nitrogen sites) can advantageously be used to form drug salts. The resulting ionic compounds become much more water soluble due to their ionic nature and the dissolution energy is lowered and thus the bioavailability is improved. The 50 µg/mL standard for water solubility is provided by Lipinsky et al. (Lipinski, Lombardo, Dominy, & Feeney, 2001).

Salting agents are available in large quantities and the choice of salt must be accurately calculated. The purpose of salt selection is to identify the best form of salt suitable for development, and is mainly based on four main criteria: water solubility at various pH levels, high crystallinity, low hygroscopicity, and optimal chemical stability (Handbook of Pharmaceutical Salts: Properties, Selection and Use, Stahl, P.H. and Wermuth, C.G. Eds. Wiley-VCH, Weinheim, Germany, 2002).

If a suitable drug salt can be identified, then further studies are needed to determine if alternative crystal forms exist. The existence of such alternative forms is highly unpredictable and may require a combination of intuition, careful empirical calculation, perseverance, and serendipity. Along with the challenges associated with discovering at least one or more specific crystalline forms, the properties of any crystalline forms thus discovered must be carefully evaluated to understand whether or not one or more of them are actually suitable for pharmaceutical development. Indeed, in the first aspect, the crystallinity of drugs affects, among other things, physical and mechanical properties, solubility, dissolution rate, flowability, hardness, compressibility and melting point. In a second aspect, the crystalline form may have advantages over the amorphous form, for example, purification to a higher purity required by most regulatory authorities is more efficient and therefore less expensive for a crystalline form than for amorphous solids. In addition, the processing properties of the crystalline form are improved compared to the amorphous form, which tends to be oily or sticky, and in practice, the drying of a crystalline substance, which has a well-defined drying or desolvation temperature, is easier to control than for an amorphous solid, which has a greater affinity for organic solvents. Finally, subsequent processing of the crystalline drug allows for improved process control. In a third aspect, physical and chemical stability, and hence shelf life, are also improved for crystalline forms compared to amorphous forms.

Finally, the pharmacokinetic and pharmacodynamic properties of a drug can be related to a specific crystalline structural form, and this is of paramount importance for obtaining and maintaining the same form from receipt to administration to the patient. Therefore, obtaining salts and/or crystalline forms is highly desirable compared to amorphous substances (Hilfiker, Blatter, & von Raumer, 2006).

Thus, it is an object of this invention to disclose salt forms and salt polymorphs of the present invention which have desirable pharmacological properties and which show improvements in their pharmaceutical profile compared to the free base form and/or amorphous salt form of the present invention, in particular, improved exposure in vivo.

Description of drawings

Fig. 1 shows sample 1 of an X-ray powder diffraction pattern of compound 1;

fig. 2 shows sample 3 of the X-ray powder diffraction pattern of compound 1;

fig. 3 shows sample 4 of an X-ray powder diffraction pattern of compound 1;

fig. 4 shows an X-ray powder diffraction pattern of compound 1.HCl;

fig. 5 shows an X-ray powder diffraction pattern of compound 1 .HC1.3H ₂ O;

fig. 6 shows the results of the DVS analysis of compound 1.HC1.3H2O.

fig. 7 shows the results of DSC (differential scanning calorimetry) analysis of compound 1.HC1.3H2O;

fig. 8 shows an X-ray powder diffraction pattern of l.HCl.MeOH;

fig. 9 shows an X-ray powder diffraction pattern of compound 1.HCl. 1.5HCO ₂ H;

fig. 10 shows the effect of Compound 1 either as the free base or the HC1.3H2O salt when administered daily orally;

fig. 11 shows the crystal structure of compound 1.HC1.3H2O.

Brief summary of the invention

The present invention is based on the identification of new salts and crystalline forms of compound 1 useful in the treatment and/or prevention of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disturbance of cartilage homeostasis, congenital cartilage defects , and / or diseases associated with hypersecretion of IL6 or interferons. In particular, the salts of the present invention can act as inhibitors of JAK kinase and more particularly JAK1 kinase. The present invention also provides methods for preparing these salts, pharmaceutical compositions comprising these salts, and methods for the treatment and/or prevention of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis. tissue, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons by administering the salts of the present invention.

Therefore, in one aspect, the present invention provides a salt of the compound according to formula (I) below (hereinafter also referred to as compound 1) /H U> ^NH | Compound 1 o

where said salt is formed with hydrobromic acid, hydrochloric acid, sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalic acid, maleic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-2ethanedisulfonic acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, phosphoric acid, ethanesulfonic acid, malonic acid, 2-5-dihydroxybenzoic acid or L-tartaric acid.

Therefore, in one aspect, the present invention provides a salt of the compound according to formula (I) below (hereinafter also referred to as compound 1)

where the specified salt is formed with hydrochloric acid.

In another aspect of the present invention, the salt of the present invention is an addition product of [compound 1.HC1.3H2O] in solid crystalline form, wherein the crystalline form is characterized by at least a powder X-ray diffraction peak in

- 4 041895 any one or more of the following positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14.5,

16.3, 16.7, 17.6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8, 22.8, 23.4, 23.9, 24, 5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3,

28.6, 28.9, 29.2, 29.6, 7 and 32.7° 2θ±0.2° 2θ.

In a specific aspect of the present invention, the salt of the present invention exhibits improved solubility and activity compared to the free base, which can result in increased efficacy and reduced dosage of drug administered. In turn, lowering the dose level of a drug can potentially reduce the toxicity that may result from drug-drug interactions.

In a specific aspect, the salts of the present invention are provided for use in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects, and/ or diseases associated with hypersecretion of IL6 or interferons.

In a further aspect, the present invention provides pharmaceutical compositions comprising a salt of the present invention and a pharmaceutical carrier, excipient or diluent. In a specific aspect, the pharmaceutical composition may additionally also include therapeutically active ingredients suitable for use in combination with the salts of the present invention. In a more specific aspect, the additional therapeutically active ingredient is an agent for the treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects, and/or diseases, associated with hypersecretion of IL6 or interferons.

Moreover, the salts of the present invention useful in the pharmaceutical compositions and methods of treatment disclosed herein are pharmaceutically acceptable in both preparation and use.

In a further aspect of the present invention, this invention provides a method of treating a mammal, in particular a human, suffering from a condition selected from among those specified in this application, and, in particular, from inflammatory conditions, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases, associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons, where the method includes administering an effective amount of a pharmaceutical composition or salts of the present invention described in this application.

The present invention also provides pharmaceutical compositions comprising a salt of the present invention and a suitable pharmaceutical carrier, excipient or diluent for use in the medical field. In a specific aspect, the pharmaceutical composition is intended for use in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects, and/or diseases associated with hypersecretion of IL6 or interferons.

In additional aspects, this invention provides methods for the synthesis of salts of the present invention, with representative protocols and routes of synthesis, disclosed later in this application.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the following detailed description.

It should be understood that the salts of the present invention can be metabolized to form biologically active metabolites.

Detailed description of the present invention

Definitions

The following terms are meant to have the meanings given immediately below and are useful in understanding the description and intended scope of the present invention.

When describing the invention, which may include compounds, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms, if present, have the following meanings, unless otherwise indicated. It should also be understood that any of the groups defined herein below may be substituted with various substituents, and that the respective definitions are intended to include such substituted groups within their scope as set forth below. Unless otherwise stated, the term substituted shall be defined as follows. It should also be understood that the terms groups and radicals can be considered interchangeable when used in this document.

The articles 'a' and 'an' may be used in this application in relation to one or more than one (ie, at least one) of the element's grammatical entities. By way of example, 'analogue' means one analogue or more than one analogue.

'Salt(s) of the present invention' and equivalent expressions encompass salts of the compound according to formula (I) (compound 1) described herein, which expression includes pharmaceutically acceptable salts and solvates, for example, hydrates and solvates of pharmaceutically acceptable salts where the context permits.

Pharmaceutically acceptable means authorized or approved by a regulatory authority of a country or state government, or by an appropriate authority in countries other than the United States, or that which is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals and, more specifically, in humans.

A pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient or carrier with which the salt of the present invention is administered.

Solvate refers to forms of a compound that are in association with a solvent, usually by a solvolysis reaction. This physical interaction includes the formation of a hydrogen bond. Common solvents include water, ethanol, acetic acid, and the like. The salts of the present invention may be obtained, for example, in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates such as hydrates and further include both stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate may be isolated, for example, when one or more solvent molecules are embedded in the crystal lattice of a crystalline solid. 'Solvate' encompasses both solution-phase and recoverable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The subject includes people. The terms human, patient, and subject are used interchangeably in this application.

An effective amount means the amount of the salt of the present invention which, when administered to a subject for the treatment of a disease, is sufficient to effect such treatment of the disease. The effective amount may vary depending on the compound, the disease and its severity, and age, body weight, and the like. subject requiring treatment.

Prevention or prevent refers to reducing the risk of acquiring or developing a disease or disorder (i.e., preventing at least one of the clinical symptoms of the disease from developing in a subject who may have been exposed to or predisposed to the disease-causing agent, in advance before the onset of the disease).

The term prophylaxis refers to the prevention and refers to an action taken or a procedure whose purpose is to prevent rather than treat or cure a disease. Non-limiting examples of preventive measures may include the administration of vaccines; administration of low molecular weight heparin to hospitalized patients at risk of thrombosis due to, for example, immobilization; and administering an antimalarial such as chloroquine prior to a visit to a geographic area where malaria is endemic or where the risk of contracting malaria is high.

Treating or treating any disease or disorder refers, in one embodiment, to ameliorating the symptoms of the disease or disorder (ie, reversing the disease or reducing the severity or severity of at least one of its clinical symptoms). In another embodiment, treating or treating refers to an improvement in at least one physical parameter that may not be apparent to the subject. In another embodiment, treating and treating refers to modulating a disease or disorder, either physically (eg, stabilizing an overt symptom), physiologically (eg, stabilizing a physical parameter), or both. In yet another embodiment, treating and treating refers to slowing the progression of a disease.

In the context of this application, the term inflammatory diseases refers to a group of conditions including rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, airway allergies (e.g., asthma, rhinitis), chronic obstructive pulmonary disease (COPD), inflammatory bowel disease ( for example, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis), endotoxin-mediated disease states (eg, bypass complications or chronic endotoxin-mediated conditions contributing to chronic heart failure) and comorbidities involving cartilage, such as like diseases of the joints. In particular, the term refers to rheumatoid arthritis, osteoarthritis, allergic airway disease (eg, asthma), chronic obstructive pulmonary disease (COPD), and inflammatory bowel disease. More specifically, the term refers to rheumatoid arthritis and inflammatory bowel diseases (eg, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis).

In the context of this application, the term autoimmune disease(s) refers to a group of diseases including obstructive airway disease, including diseases such as COPD, asthma (for example, hereditary (endogenous) bronchial asthma, acquired (exogenous) bronchial asthma, dust asthma , childhood asthma), especially chronic or advanced asthma (eg, late asthma and airway hypersensitivity), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes mellitus and related complications, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto's thyroiditis and autoimmune thyroiditis), contact dermatitis and additional eczematous dermatitis, inflammatory bowel disease (eg. , Crohn's disease, Whipple's disease, chronic ulcerative coli t or colitis), atherosclerosis and amyotrophic lateral sclerosis. Especially the term refers to COPD (chronic obstructive pulmonary disease), asthma, systemic lupus erythematosus, type I diabetes mellitus and inflammatory bowel disease.

In the context of this application, the term proliferative disease(s) refers to conditions such as cancer (eg uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (eg polycythemia vera, essential thrombocythemia and myelofibrosis), leukemia (eg , acute myelocytic leukemia, acute and chronic lymphoblastic leukemia), multiple myeloma, psoriasis, restenosis, sclerodermatitis, or fibrosis. In particular, the term refers to cancer, leukemia, multiple myeloma, and psoriasis.

In the context of this application, the term cancer refers to a malignant or benign growth of cells on the skin or in organs of the body, for example, and without limitation, in the breast (breast), prostate, lung, kidney, pancreas, stomach or intestines. Cancer tends to infiltrate adjacent tissue and spread (metastasize) to distant organs, such as the bone, liver, lung, or brain. As used herein, the term cancer includes both types of metastatic tumor cells (such as, but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and tissue carcinoma types (such as, but not limited to, cancer of the colon and rectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). Specifically, the term cancer includes acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer (osteosarcoma). and malignant fibrous histiocytoma), brainstem glioma, brain tumors, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt's lymphomas, cervical cancer, chronic lymphocytic leukemia, chronic myeloblastic leukemia, colon cancer, colon cancer and rectum, craniopharyngioma, cutaneous T-cell lymphoma, fetal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing's sarcoma family tumors, eye cancer, retinoblastoma, gallbladder cancer, gastric cancer (stomach cancer), gastric squamous cell carcinoma intestinal tract, gastrointestinal stromal tumor (GIST), gastro testinal stromal cell tumor, germ cell tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma (liver cancer), hypopharyngeal cancer, intraocular melanoma, pancreatic islet tumor (pancreatic endocrine tissue), Kaposi's sarcoma, kidney cancer, langerhans cell histiocytosis, larynx cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoma, Waldenström macroglobulinemia, medulloblastoma, medulloepithelioma, melanoma, mesothelioma, oral cancer, chronic myeloid leukemia, myeloid leukemia, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, malignant fibrosis bone histiocytoma, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumors, low-grade ovarian tumor, pancreatic cancer, papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer, intermediate-differentiation pineal parenchymal tumor, pineoblastoma and supratentorial primitives neuroectodermal tumors, pituitary tumor, plasma cell neoplasms/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, family tumors Ewing's sarcoma, sarcoma, Cesari's syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric (gastrointestinal) cancer, supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, cancer of the eagle, thymoma and thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, and Wilms' tumor. More specifically, the cancer is selected from breast cancer, endometrial and cervical cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer and pancreatic cancer.

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In the context of this application, the term leukemia refers to neoplastic diseases of the blood and hematopoietic organs. Such diseases can cause bone marrow and immune system dysfunction, making the host highly susceptible to infection and bleeding. In particular, the term leukemia refers to acute myelocytic leukemia (AML) and acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL).

In the context of this application, the term allergies refers to a group of conditions characterized by hypersensitivity of the immune system, including respiratory allergies (eg, asthma, rhinitis), sinusitis, eczema, and urticaria, as well as food or insect venom allergies.

In the context of this application, the term asthma refers to any lung disorder characterized by changes in gas flow in the lungs associated with airway constriction for any reason (intrinsic, extrinsic, or both; allergic or non-allergic). The term asthma can be used with one or more adjectives to indicate the cause.

In the context of this application, the term transplant rejection refers to the acute or chronic rejection of allo- or xenografts of cells, tissues, or whole organs, e.g., pancreatic islet, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, heart-lung, kidney, liver, intestine, pancreas, trachea, or esophagus, or graft-versus-host disease.

In the context of this application, the term diseases associated with the destruction and/or disruption of cartilage homeostasis includes conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget's disease, Tietze's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydrarthrosis, periodontal disease, rheumatoid spondylitis, endemic forms of arthritis such as endemic osteoarthritis deformans, Mseleni's disease of the joints, and Handigodu's disease; degeneration due to fibromyalgia, systemic lupus erythematosus, sclerodermatitis, and ankylosing spondylitis.

In the context of this application, the term congenital cartilage malformation(s) includes conditions such as hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, in particular, but not limited to, microtia, anotia, metaphyseal chondrodysplasia and comorbid disorders.

As used herein, the term disease(s) associated with IL6 hypersecretion includes conditions such as Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

In the context of this application, the term disease(s) associated with hypersecretion of interferons includes conditions such as systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis, rheumatoid arthritis.

When ranges are specified herein, for example, but not limited to, C ₁ . ₈ alkyl, then the range specification should be considered as representing each member of the range specified.

In the context of this application, the term isotopic variant refers to a compound that contains unnatural proportions of isotopes on one or more of the atoms that make up such a compound. For example, an isotopic variant of a compound may contain one or more non-radioactive isotopes such as, for example, deuterium ( ² H or D), carbon-13 ( ¹³ C), nitrogen-15 ( ¹⁵ N), or the like. It should be understood that in a compound in which such isotone substitution occurs, the following atoms, if present, may vary such that, for example, any hydrogen may be 2H/D, any carbon may be ¹³ C, or any nitrogen may be be ¹⁵ N, and that the presence and placement of such atoms can be determined by one skilled in the art. Likewise, the present invention may include the production of isotopic variants with radioisotopes, in the case, for example, where the resulting compounds can be used for drug and/or substrate distribution studies in tissues.

The radioactive isotopes tritium, ie ³ H, and carbon-14, ie ¹⁴ C, are particularly useful for this purpose because of their ease of incorporation and ease of detection. In addition, compounds that are substituted with positron-emitting isotopes such as ^PS , ¹⁸ F, ¹⁵ O and ¹³ N can be obtained and could be useful in positron emission tomography (PET) studies to study the degree of receptor occupancy by the substrate. .

All isotopic variants of the compounds presented in this application, radioactive or non-radioactive, are intended to be included in the scope of the present invention.

Tautomers refer to compounds that have interchangeable forms of a particular compound structure, and that differ in the displacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of π-electrons and an atom (usually H). For example, enols and ketones are tautomers because they rapidly interconvert when treated with either an acid or a base. Another example of tautomerism is the aci- and nitro forms of phenylnitromethane, which are similarly formed by acid treatment.

- 8 041895 or base.

Tautomeric forms may be appropriate to achieve optimum chemical reactivity and biological activity of the compound of interest.

It should be understood that the salts of the present invention can be metabolized to form biologically active metabolites.

Invention

The present invention relates to a salt of the compound {5-[4-(1,1-dioxothiomorpholin-4-ylmethyl)phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}amide of cyclopropanecarboxylic acid (compound 1) and to methods for preparing such salts that are useful in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disturbance of cartilage homeostasis, birth defects cartilage, and/or diseases associated with hypersecretion of IL6 or interferons. In particular, the salts of the present invention inhibit the JAK family of tyrosine kinases, and more particularly the JAK1 kinase.

The present invention also provides methods for the prevention and/or treatment of diseases, including inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects and/or diseases associated with with hypersecretion of IL6 or interferons, by administering a salt of the present invention or a pharmaceutical composition containing said salt.

Therefore, in one aspect, the present invention provides a salt of a compound according to formula (I) (compound 1) below:

O

I where the specified salt is formed with a salt-forming agent selected from hydrobromic acid, hydrochloric acid, sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalic acid, maleic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-2- ethanedisulfonic acid, methanesulfonic acid, 2hydroxyethanesulfonic acid, phosphoric acid, ethanesulfonic acid, malonic acid, 2-5-dihydroxybenzoic acid and L-tartaric acid.

In one embodiment, the salt of the present invention is a salt formed with a salt forming agent selected from hydrobromic acid and hydrochloric acid, preferably hydrochloric acid.

In one embodiment, the salt of the present invention is a salt formed with a salt forming agent selected from oxalic acid, maleic acid or malonic acid, preferably maleic acid.

In one embodiment, the salt of the present invention is a salt formed with a salt-forming agent selected from toluenesulfonic acid, benzenesulfonic acid, naphthalene-2-sulfonic acid and ethanesulfonic acid; preferably toluenesulfonic acid and benzenesulfonic acid, more preferably toluenesulfonic acid, and most preferably p-toluenesulfonic acid.

In one embodiment, the salt of the present invention is the addition product [compound 1:salt forming agent] in a ratio of 3:1 to 1:3. In a preferred embodiment, the salt of the present invention is a 1:1 addition product of [compound 1:salt forming agent]. In a more preferred embodiment, the salt forming agent is selected from hydrobromic acid, hydrochloric acid, toluenesulfonic acid and maleic acid. In the most preferred embodiment, the salt-forming agent is hydrochloric acid.

In one embodiment, the salt of the present invention is a solvate. In a preferred embodiment, the salt of the present invention is a mono-, di- or trisolvate. In the most preferred embodiment, the salt of the present invention is a trisolvate. Alternatively, the salt of the present invention is not a solvate.

In one embodiment, the salt of the present invention is a hydrate. In a more preferred embodiment, the salt of the present invention is a mono-, di- or trihydrate. In the most preferred embodiment, the present salt

- 9 041895 of the invention is a trihydrate. Alternatively, the salt of the present invention is an anhydrous salt.

In one embodiment, the salt of the present invention is the addition product [compound 1:salt forming agent:solvent]. In a preferred embodiment, the salt of the present invention is the addition product [compound 1:salt forming agent:solvent] in a ratio of 1:1:0 to 1:1:4. In a more preferred embodiment, the salt of the present invention is a 1:1:0, 1:1:1, 1:1:1.5, 1:1:2 addition [compound 1:salt forming agent:solvent] or 1:1:3. In the most preferred embodiment, the salt of the present invention is the addition product [compound 1:salt forming agent:solvent] in a ratio of 1:1:3.

In another embodiment, the salt of the present invention is the addition product [compound 1:HCl:solvent]. In a preferred embodiment, the salt of the present invention is the addition product of [compound 1:HCl:solvent] in a ratio of 1:1:0 to 1:1:4. In a more preferred embodiment, the salt of the present invention is the addition product [compound 1:HCl:solvent] in a ratio of 1:1:0, 1:1:1, 1:1:1.5, 1:1:2 or 1 :1:3. In the most preferred embodiment, the salt of the present invention is the addition product [compound 1:HCl:solvent] in a ratio of 1:1:3. In an even more preferred embodiment, the solvent is selected from H ₂ O, MeOH and HCO 2 H.

In another embodiment, the salt of the present invention is the addition product [Compound 1:HCl:H ₂ O]. In a preferred embodiment, the salt of the present invention is the addition product of [compound 1:HCl:H ₂ O] in a ratio of 1:1:0 to 1:1:4. In a more preferred embodiment, the salt of the present invention is a 1:1:0, 1:1:1, 1:1:1.5, 1:1:2 addition [compound 1:H1:H ₂ O] or 1:1:3. In the most preferred embodiment, the salt of the present invention is a 1:1:3 addition product [1:HC1:H ₂ O compound].

In one embodiment, the salt of the present invention exhibits peaks in an X-ray powder diffraction spectrum.

In one embodiment, the salt of the present invention is in crystalline form.

In one embodiment, the salt of the present invention is a 1:1:0 addition product of [compound 1:H1:H ₂ O] in solid crystalline form, wherein the crystalline form is distinguished by at least a powder x-ray diffraction peak in any one or more from the following positions: 7.4, 8.9, 12.4, 14.8, 15.1, 16.9, 17.6, 19.4, 20.7, 21.1, 22.8, 24, 9, 26.0, 28.6, 29.8 and 32.6° 2θ±0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:0 addition product of [compound 1:H1:H ₂ O] in a solid crystalline form, wherein the crystalline form differs by at least a powder x-ray diffraction peak of at least 5 , 10, 15 or more of the following: 7.4, 8.9, 12.4, 14.8, 15.1, 16.9, 17.6, 19.4, 20.7, 21.1, 22.8, 24.9, 26.0, 28.6, 29.8 and 32.6° 2θ±0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:0 addition product of [compound 1:H1:H ₂ O] in solid crystalline form, wherein the crystalline form is characterized by at least a powder x-ray diffraction peak in all of the following: 7.4, 8.9, 12.4, 14.8, 15.1, 16.9, 17.6, 19.4, 20.7, 21.1, 22.8, 24.9, 26, 0, 28.6, 29.8 and 32.6° 2θ ±0.2° 2θ. In a particular embodiment, the salt of the present invention is characterized by an X-ray powder diffraction pattern expressed in terms of 2 theta angles, as shown in FIG. 4.

In one embodiment, the salt of the present invention is a 1:1:3 addition product of [compound 1:H1:H ₂ O] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder x-ray diffraction peak in any one or more from the following positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14.5, 16.3, 16.7, 17, 6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8, 22.8, 23.4, 23.9, 24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3, 28.6, 28.9, 29.2, 29.6 and 32.7° 2θ ± 0.2° 2θ.

In a preferred embodiment, the salt of the present invention is a 1:1:3 addition product of [compound 1:H1:H ₂ O] in a solid crystalline form, wherein the crystalline form differs by at least a powder X-ray diffraction peak of at least 5 , 10, 15, 20, 25, 30 or more of the following positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14, 5, 16.3, 16.7, 17.6, 19.3, 20.2, 20.6, 21.0, 21.4, 21.8, 22.8, 23.4, 23.9, 24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28.3, 28.6, 28.9, 29.2, 29.6 and 32, 7° 2θ± 0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:3 addition product of [compound 1:HCl:H ₂ O] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder X-ray diffraction peak in all of the following positions: 7.3, 8.4, 8.8, 10.7, 12.0, 12.2, 13.2, 13.7, 14.5, 16.3, 16.7, 17 .6, 19.3, 20.2, 20.6, 21.0,

21.4, 21.8, 22.8, 23.4, 23.9, 24.5, 25.2, 25.7, 25.9, 26.4, 27.2, 27.7, 28, 3, 28.6, 28.9, 29.2, 29.6 and 32.7° 2θ±0.2° 2θ. In a particular embodiment, the salt of the present invention is characterized by an X-ray powder diffraction pattern expressed in terms of 2 theta angles, as shown in FIG. 5.

In one embodiment, the addition product [compound 1:H1:H ₂ O] in a ratio of 1:1:3 in solid crystalline form has a particle size of less than 1000 microns, as measured by laser diffraction (Table II). In a preferred embodiment, the adduct [compound 1:HCl:H ₂ O] in a ratio of 1:1:3 in solid crystalline form has a particle size ranging from 50 µm to 800 µm. In a more preferred embodiment, the addition product [compound 1:HCl:H ₂ O] in a ratio of 1:1:3 in solid crystalline form has a particle size ranging from 200 µm to 600 µm.

In one embodiment, the salt of the present invention is a 1:1:1 addition product of [compound 1:HCl:MeOH] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder x-ray diffraction peak in any one or more of the following positions: 7.1, 14.4, 16.6, 17.3, 18.9, 23.4, 24.8 and 29.0° 2θ±0.2° 2θ.

In a preferred embodiment, the salt of the present invention is a 1:1:1 addition product of [compound 1:HCl:MeOH] in solid crystalline form, wherein the crystalline form differs by at least a powder x-ray diffraction peak of at least 3.5 , 7 or more of the following positions: 7.1, 14.4, 16.6, 17.3, 18.9, 23.4, 24.8 and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:1 addition product of [compound 1:HCl:MeOH] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder x-ray diffraction peak at all of the following positions: 7, 1, 14.4, 16.6, 17.3, 18.9, 23.4, 24.8 and 29.0° 2θ±0.2° 2θ. In a particular embodiment, the salt of the present invention is characterized by an X-ray powder diffraction pattern expressed in terms of 2 theta angles, as shown in FIG. 8.

In one embodiment, the salt of the present invention is a 1:1:1.5 addition product of [compound 1:HCl:HCO ₂ H] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder X-ray diffraction peak in any one or more of the following positions: 7.1, 14.4, 14.8, 16.4, 17.4, 18.6, 20.8, 23.4, 24.5, 24.9 and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:1.5 addition product of [compound 1:HCl:HCO ₂ H] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder X-ray diffraction peak of at least 3, 5, 7, 9 or more of the following: 7.1, 14.4, 14.8, 16.4, 17.4, 18.6, 20.8, 23.4, 24.5, 24.9 and 29.0° 2θ±0.2° 2θ.

In one embodiment, the salt of the present invention is a 1:1:1.5 addition product of [compound 1:HCl:HCO ₂ H] in a solid crystalline form, wherein the crystalline form is distinguished by at least a powder X-ray diffraction peak in all of the following positions: 7.1, 14.4, 14.8, 16.4, 17.4, 18.6, 20.8, 23.4, 24.5, 24.9 and 29.0° 2θ±0, 2° 2θ. In a particular embodiment, the salt of the present invention is characterized by an X-ray powder diffraction pattern expressed in terms of 2 theta angles, as shown in FIG. 9.

In one embodiment, the salt of the present invention is prepared by combining compound 1 with an acid selected from hydrobromic acid, hydrochloric acid, sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalic acid, maleic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 1-2-ethanedisulfonic acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, phosphoric acid, ethanesulfonic acid, malonic acid, 2-5-dihydroxybenzoic acid and Ltartaric acid, in an inert solvent and precipitating said salt from said solvent. In a preferred embodiment, the salt of the present invention is prepared by adding Compound 1 and a salt forming agent in a suitable solvent to achieve complete dissolution, then by controlled evaporation of the solvent to achieve supersaturation and hence crystallization of the corresponding salt.

In one embodiment, the salt of the present invention is prepared by mixing compound 1 and acid in a compound 1:acid molar ratio ranging from 5:1 to 1:5. In a preferred embodiment, the salt of the present invention is prepared by mixing compound 1 and acid in a compound 1:acid molar ratio ranging from 2:1 to 1:2. In a more preferred embodiment, the salt of the present invention is obtained by mixing compound 1 and acid in a 1:1 molar ratio.

- 11 041895

In yet another specific embodiment, the solvent for preparing the salt of the present invention is selected from ketones, alcohols, esters, C _1-10 alkyl, C _3-7 cycloalkyl, Cb-u monocyclic or fused bicyclic aryl, sulfoxide, C _1-10 alkylnitrile , C _1-10 linear, branched or cyclic ethers and C _1-10 haloalkyl. In a preferred embodiment, the solvent for preparing the salt of the present invention is selected from acetone, anisole, butanol, butyl acetate, TBME, DMSO, ethanol, ethyl acetate, heptane, isopropyl acetate, MEK, isopropyl acetate, MeCN, cyclohexane, DCM, dioxane, methanol, nitromethane, THF , methyl THF, toluene, water, 10% aqueous acetone, 10% aqueous THF and 10% methanol. In a preferred embodiment, the solvent for preparing the salt of the present invention is selected from dioxane, THF, acetone, DCM and MeOH.

In another aspect, a process for producing a salt of the present invention is provided, comprising the steps of:

i) reacting compound 1 with an acid selected from hydrobromic acid, hydrochloric acid, sulfuric acid, toluenesulfonic acid, benzenesulfonic acid, oxalic acid, maleic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1- 2-ethanedisulfonic acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, phosphoric acid, ethanesulfonic acid, malonic acid, 2-5-dihydroxybenzoic acid and L-tartaric acid, in an inert solvent; and ii) precipitating said salt from said solvent.

In a further aspect, there is provided a salt of the present invention which can be made or which has been prepared by the above process.

In one embodiment, with respect to the preparation of the salt of the present invention, the inert solvent is selected from dioxane, THF, acetone, DCM, and MeOH.

In one embodiment, with respect to the preparation of the salt of the present invention, the inert solvent is DCM.

In one embodiment, in relation to the preparation of the salt of the present invention, the inert solvent is selected from a mixture of iPrOH/water, iPrOH, iBuOH and tBuOH.

In one embodiment, a method is provided for obtaining the adduct [compound 1:HCl:3H2O] in solid crystalline form, comprising the steps of:

i) stirring compound 1 with water, ii) adding an aqueous solution of HCl, iii) further stirring the mixture obtained in step ii), iv) cooling the mixture obtained in step iii) to 15°C,

v) continuing stirring for no more than 24 hours at 15° C., vi) isolating the solid obtained in the previous step v) by filtration and vii) drying in nitrogen atmosphere for at least 4 hours of said obtained solid obtained on previous step vi).

In a preferred embodiment, a process is provided for obtaining the adduct [compound 1:HCl:3H2O] in solid crystalline form, comprising the steps of:

i) stirring compound 1 with water at 50°C, ii) adding an aqueous solution of HCl, iii) stirring the mixture obtained in step ii), then at 50°C for 15 min, iv) cooling the mixture obtained in step iii) , up to 15°С,

v) continuing stirring for a period of 12 to 24 hours at 15°C, vi) isolating the solid obtained in the previous step v) by filtration and vii) drying under nitrogen for at least 4 hours of said obtained solid obtained in the previous step vi).

In another embodiment, a method is provided for obtaining the adduct [compound 1:HCl:3H2O] in solid crystalline form, comprising the steps of:

i) mixing compound 1 suspended in DCM with MeOH, ii) adding water with stirring, iii) separating the organic layer, iv) adding HCl solution to the organic layer obtained in the previous step iii), v) isolating the one obtained in the previous step iv ) the solid by filtration, vi) drying said solid obtained in the previous step v), vii) adding the solid obtained in the previous step vi), to the formic acid/water solution, with stirring, viii) adding water to the solution obtained in the previous step vii), ix) isolation by filtration of the resulting solid obtained in the previous step viii), and

x) drying the resulting solid obtained in the previous step ix).

In another embodiment, a method is provided for obtaining the adduct [co

- 12 041895 unity 1:HC1:3H ₂ O] in solid crystalline form, comprising the steps:

i) mixing compound 1 suspended in DCM with MeOH at 35°C, ii) adding water with stirring at 35°C for at least 15 min iii) separating the organic layer, iv) adding 10% wt./wt. HCl solution in MeOH to the organic layer obtained in the previous step iii),

v) isolating the solid obtained in the previous step iv) by filtration, vi) drying said obtained solid obtained in the previous step v), vii) adding the solid obtained in the previous step vi) to a solution of 1.6/0 ,4 formic acid/water, with stirring at 55°C viii) adding water to the solution obtained in the previous step vii), ix) isolating by filtration the resulting solid obtained in the previous step viii), and

x) drying the resulting solid obtained in the previous step ix).

In another embodiment, a method is provided for obtaining the adduct [1:HC1:3H ₂ O compound] in solid crystalline form, comprising the steps of:

i) reacting compound 1 suspended in DCM with MeOH and trisodium trimercaptotriazine, ii) filtering the resulting suspension, iii) adding water with stirring, iv) separating the organic layer,

v) adding the HCl solution to the organic layer obtained in step iv), vi) isolating the obtained solid obtained in step v), by filtration, vii) drying said obtained solid obtained in step vi), viii) adding the solid substance obtained in step vii), to the formic acid/water solution with stirring, ix) adding water to the solution from step viii),

x) isolation by filtration of the solid obtained in step ix), and xi) drying the resulting solid obtained in step x).

In another aspect, a method is provided for obtaining the adduct [1:HC1:3H ₂ O compound] in solid crystalline form, comprising the steps of:

i) reacting compound 1 suspended in DCM with MeOH and trisodium trimercaptotriazine at 35°C for at least 5 hours, ii) filtering the resulting suspension, iii) adding water with stirring at 35°C for at least 15 minutes , iv) separation of the organic layer,

v) adding 10% wt./wt. HCl solution in MeOH to the organic layer obtained in step iv), vi) isolating the obtained solid obtained in step v), by filtration, vii) drying said obtained solid obtained in step vi), viii) adding the solid obtained in step vii) to a solution of 1.6/0.4 formic acid/water with stirring at 55° C., ix) adding water to the solution from step viii),

x) isolating by filtration the obtained solid obtained in step ix), and xi) drying the obtained solid obtained in step x).

Alternatively, the present invention also provides for the exclusion of one or more of these variables from a group or embodiment, or a combination thereof.

Pharmaceutical compositions

When used as a pharmaceutical preparation, the salt of the present invention is usually administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and include at least one active salt of the present invention.

Typically, the salt of the present invention is administered in a pharmaceutically effective amount. The amount of salt of the present invention actually administered will generally be determined by the physician in light of the relevant circumstances, including the condition requiring treatment, the chosen route of administration, the particular salt of the present invention administered, the age, body weight and response of the particular patient, the severity of the patient's symptoms, etc. .P.

Pharmaceutical compositions of the present invention can be administered by various routes, including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular and intranasal. Depending on the intended route of delivery, the salt of the present invention is preferably formulated as either injectable or oral compositions or as ointments, lotions or patches, all for transdermal administration.

Compositions for oral administration may be in the form of bulk liquid solutions or suspensions or bulk powders. Most often, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term unit dosage form refers to physically discrete units suitable as unit dosages for humans and other mammals, each unit containing a predetermined amount of the active substance calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, diluent or carrier. Typical unit dosage forms include ampoules or syringes pre-filled with pre-measured liquid compositions or, in the case of solid compositions, pills, tablets, capsules, or the like. In such compositions, the salt of the present invention usually represents a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with various fillers or carriers and excipients making up the remainder, useful for forming the desired dosage form.

Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous solvent with buffers, suspending and dispersing agents, coloring agents, flavoring agents, and the like. Solid forms may include, for example, any of the following ingredients, or a salt of the present invention of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a tablet disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate; an anti-caking agent such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as mint or orange flavor.

Compositions for injection are usually based on injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Also as described above, the active salt of the present invention in accordance with formula I in such compositions usually represents a minor component, most often from about 0.05 to 10% by weight, with the remainder being the injectable carrier and the like.

Transdermal compositions are usually formulated as a topical ointment or cream containing the active ingredient(s), usually in an amount in the range of from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When the composition is formulated as an ointment, the active ingredients can generally be combined with either a petroleum jelly or a water-soluble ointment base. Alternatively, the active ingredients may be formulated as a cream with, for example, an oil-in-water cream base. Such transdermal compositions are well known in the art and typically include additional ingredients to improve the stability of the active ingredients or composition upon penetration into the skin. All such known transdermal compositions and ingredients are included within the scope of the present invention.

The salt of the present invention can also be administered via a transdermal device. Accordingly, transdermal administration can be carried out using a reservoir-type patch or a porous membrane, or various solid matrices.

The components described above for orally administered, injected or topically administered compositions are representative only. Other substances, as well as processing technologies, etc. are described in Part 8 of Remington's Pharmaceutical Sciences, ^17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

The salt of the present invention can also be administered in sustained release forms or from sustained release drug delivery systems. Description of representative substances for sustained release can be found in Remington's Pharmaceutical Sciences.

The following formulation examples illustrate representative pharmaceutical compositions that can be prepared in accordance with the present invention. The present invention, however, is not limited to the following pharmaceutical compositions.

Composition 1. Tablets

The salt of the present invention can be mixed as a dry powder with a dry gelatin binder in a weight ratio of approximately 1:2. A small amount of magnesium stearate can be added as a lubricant. The mixture can be formed into 240-270 mg tablets (80-90 mg active salt of the present invention per tablet) in a tablet press.

Composition 2. Capsules

The salt of the present invention can be mixed as a dry powder with a starch diluent in a weight ratio of approximately 1:1. The mixture is filled into 250 mg capsules (125 mg active salt of the present invention per capsule).

- 14 041895

Composition 3. Liquid

The salt of the present invention (125 mg) can be mixed with sucrose (1.75 g) and xanthan gum (4 mg), and the resulting mixture can be mixed, passed through a No. 10 mesh US sieve, and then mixed with a pre-mixed solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), fragrance and color can be diluted with water and added with stirring. Sufficient water can then be added while stirring. Sufficient water may then be added to obtain a total volume of 5 ml.

Composition 4. Tablets

The salt of the present invention can be mixed as a dry powder with a dry gelatin binder in a weight ratio of approximately 1:2. A small amount of lubricant can be added as a lubricant. The mixture can be formed into 25-900 mg tablets (8-300 mg active salt of the present invention) in a tablet press.

Composition 5. Injection

The salt of the present invention may be dissolved or suspended in an aqueous injectable medium with buffered sterile saline to a concentration of approximately 5 mg/mL.

Composition 6. Topical application

Stearyl alcohol (250g) and white petrolatum (250g) can be melted at about 75°C and then a mixture of salt of the present invention (50g), methylparaben (0.25g), propylparaben (0.15g), sodium lauryl sulfate (10 g) and propylene glycol (120 g) dissolved in water (about 370 g), and stir the resulting mixture until it solidifies.

Methods of treatment

In one embodiment, the present invention provides salts of the present invention or pharmaceutical compositions comprising a salt of the present invention for use in medicine. In a preferred embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, disruption-related diseases and/ or impaired cartilage homeostasis, congenital cartilage defects, and/or diseases associated with hypersecretion of IL6 or interferons.

In one embodiment, the present invention provides the use of the salts of the present invention or pharmaceutical compositions comprising the salt of the present invention in medicine. In a preferred embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, disruption-related diseases, and / or a violation of cartilage homeostasis, congenital cartilage defects, and / or diseases associated with hypersecretion of IL6 or interferons.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and / or disruption of cartilage homeostasis, congenital cartilage defects, and / or diseases associated with hypersecretion of IL6 or interferons.

In a further method of the treatment aspects, the present invention provides methods for the prevention and/or treatment of a mammal suffering from inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects, and/ or diseases associated with hypersecretion of IL6 or interferons, wherein the methods include administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein for the treatment or prevention of said condition.

In one embodiment, the present invention provides pharmaceutical compositions comprising a salt of the present invention and another therapeutic agent. In a preferred embodiment, the other therapeutic agent is an agent for the treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of inflammatory diseases. In the preferred embodiment,

The inflammatory disease is selected from rheumatoid arthritis, osteoarthritis, respiratory allergies (eg asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis). More specifically, the inflammatory disease is selected from rheumatoid arthritis and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis).

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of inflammatory diseases. In a preferred embodiment, the inflammatory disease is selected from rheumatoid arthritis, osteoarthritis, respiratory allergies (eg asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis). More specifically, the inflammatory disease is selected from rheumatoid arthritis and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis).

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of inflammatory diseases. In a preferred embodiment, the inflammatory disease is selected from rheumatoid arthritis, osteoarthritis, respiratory allergies (eg asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis). More specifically, the inflammatory disease is selected from rheumatoid arthritis and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis).

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from inflammatory diseases, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein for the treatment or prevention of said condition. In a preferred embodiment, the inflammatory disease is selected from rheumatoid arthritis, osteoarthritis, respiratory allergies (eg asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis). More specifically, the inflammatory disease is selected from rheumatoid arthritis and inflammatory bowel diseases (eg Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis).

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of autoimmune diseases. In a preferred embodiment, the autoimmune disease is selected from COPD, asthma (e.g., hereditary (endogenous) bronchial asthma, acquired (exogenous) bronchial asthma, dust asthma, childhood asthma) in particular chronic or chronic asthma (e.g., late-onset asthma and airway hypersensitivity) , bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes mellitus and related complications, atopic eczema ( atopic dermatitis), thyroiditis (Hashimoto's thyroiditis and autoimmune thyroiditis), contact dermatitis and accessory eczematous dermatitis, inflammatory bowel disease (eg, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis), atherosclerosis, and amyotrophic lateral sclerosis. Preferably, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes mellitus, and inflammatory bowel disease.

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of autoimmune diseases. In a preferred embodiment, the autoimmune disease is selected from COPD, asthma (e.g., hereditary (endogenous) asthma, acquired (exogenous) asthma, dust asthma, childhood asthma), especially chronic or chronic asthma (e.g., late-onset asthma and airway hypersensitivity). tract), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes mellitus and related complications, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto's thyroiditis and autoimmune thyroiditis), contact dermatitis and accessory eczematous dermatitis, inflammatory bowel disease (eg, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis), atherosclerosis, and amyotrophic lateral sclerosis. Preferably the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes mellitus and inflammatory bowel disease.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of autoimmune diseases. In a preferred embodiment, the autoimmune disease is selected from COPD, asthma (e.g., hereditary (endogenous) bronchial asthma, acquired (exogenous) bronchial asthma, dust asthma, childhood asthma) in particular chronic or chronic asthma (e.g., late-onset asthma and airway hypersensitivity) , bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes mellitus and related complications, atopic eczema ( atopic dermatitis), thyroiditis (Hashimoto's thyroiditis and autoimmune thyroiditis), contact dermatitis and accessory eczematous dermatitis, inflammatory bowel disease (eg, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis), atherosclerosis, and amyotrophic lateral sclerosis. Preferably, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes mellitus, and inflammatory bowel disease.

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from autoimmune diseases, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein for the treatment or prevention of said condition. In a preferred embodiment, the autoimmune disease is selected from COPD, asthma (e.g., hereditary (endogenous) bronchial asthma, acquired (exogenous) bronchial asthma, dust asthma, childhood asthma) in particular chronic or chronic asthma (e.g., late-onset asthma and airway hypersensitivity ), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes mellitus and related complications, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto's thyroiditis and autoimmune thyroiditis), contact dermatitis and accessory eczematous dermatitis, inflammatory bowel disease (eg, Crohn's disease, Whipple's disease, chronic ulcerative colitis or colitis), atherosclerosis, and amyotrophic lateral sclerosis. Preferably, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes mellitus, and inflammatory bowel disease.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of proliferative diseases. In a preferred embodiment, the proliferative disease is selected from cancer and leukemia. In a more preferred embodiment, a proliferative disease selected from breast cancer, endometrial and cervical cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer, and pancreatic cancer.

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of proliferative diseases. In a preferred embodiment, the proliferative disease is selected from cancer and leukemia. In a more preferred embodiment, the proliferative disease is selected from breast cancer, endometrial and cervical cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer, and pancreatic cancer.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of proliferative diseases. In a preferred embodiment, the proliferative disease is selected from cancer and leukemia. In a more preferred embodiment, a proliferative disease selected from breast cancer, endometrial and cervical cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer, and pancreatic cancer.

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from proliferative diseases, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein for the treatment or prevention of said condition. In a preferred embodiment, the proliferative disease is selected from cancer and leukemia. In a more preferred embodiment, a proliferative disease selected from breast cancer, endometrial and cervical cancer, lung cancer, ovarian cancer, cancer

- 17 041895 prostate, liver cancer and pancreatic cancer.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of allergies. In a preferred embodiment, the allergy is selected from respiratory allergies (eg, asthma, rhinitis), sinusitis, eczema, and urticaria, as well as food allergies or insect venom allergies.

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of allergies. In a preferred embodiment, the allergy is selected from respiratory allergies (eg, asthma, rhinitis), sinusitis, eczema, and urticaria, as well as food allergies or insect venom allergies.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of allergies. In a preferred embodiment, the allergy is selected from respiratory allergies (eg, asthma, rhinitis), sinusitis, eczema, and urticaria, as well as food allergies or insect venom allergies.

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from allergies, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein to treat or prevent said condition. In a preferred embodiment, the allergy is selected from respiratory allergies (eg, asthma, rhinitis), sinusitis, eczema, and urticaria, as well as food allergies or insect venom allergies.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of transplant rejection.

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of transplant rejection.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of transplant rejection.

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from transplant rejection, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein for the treatment or prevention of said condition.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of diseases associated with the destruction and/or disruption of cartilage homeostasis. In a preferred embodiment, diseases associated with the destruction and/or disruption of cartilage homeostasis are selected from osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget's disease, Tietze's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydrarthrosis, periodontal disease, rheumatoid spondylitis, endemic forms of arthritis such as endemic osteoarthritis deformans, Mseleni's disease of the joints, and Handigodu's disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, sclerodermatitis and ankylosing spondylitis.

In one embodiment, the present invention provides the use of salts of the present invention or pharmaceutical compositions comprising a salt of the present invention in the prevention and/or treatment of diseases associated with the destruction and/or disruption of cartilage homeostasis. In a preferred embodiment, diseases associated with the destruction and/or disruption of cartilage homeostasis are selected from osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget's disease , Tietze's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydrarthrosis, periodontal disease, rheumatoid spondylitis, endemic forms of arthritis such as endemic osteoarthritis deformans, Mseleney's disease of the joints, and Handigod's disease; degeneration due to fibromyalgia,

- 18 041895 systemic lupus erythematosus, sclerodermatitis and ankylosing spondylitis.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the manufacture of a medicament for use in the prevention and/or treatment of diseases associated with the destruction and/or disruption of cartilage tissue homeostasis. In a preferred embodiment, diseases associated with the destruction and/or disruption of cartilage homeostasis are selected from osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget's disease, Tietze's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydrarthrosis, periodontal disease, rheumatoid spondylitis, endemic forms of arthritis such as endemic osteoarthritis deformans, Mseleni's disease of the joints, and Handigodu's disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, sclerodermatitis and ankylosing spondylitis.

In a further method of the treatment aspects, this invention provides methods for the prevention and/or treatment of a mammal suffering from diseases associated with the destruction and/or disruption of cartilage homeostasis, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more of the pharmaceutical compositions described in the present application, for the treatment or prevention of said condition. In a preferred embodiment, diseases associated with the destruction and/or disruption of cartilage homeostasis are selected from osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, achondroplasia, Paget's disease, Tietze's syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, sarcoidosis, amylosis, hydrarthrosis, periodontal disease, rheumatoid spondylitis, endemic forms of arthritis such as endemic osteoarthritis deformans, Mseleni's disease of the joints, and Handigodu's disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, sclerodermatitis and ankylosing spondylitis.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of congenital cartilage malformation(s). In a preferred embodiment, the congenital cartilage malformation(s) is selected from hereditary chondrolysis, chondrodysplasia, and pseudochondrodysplasia, in particular, but not limited to, microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

In one embodiment, the present invention provides the use of the salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, in the prevention and/or treatment of congenital cartilage malformation(s). In a preferred embodiment, the congenital cartilage malformation(s) is selected from hereditary chondrolysis, chondrodysplasia, and pseudochondrodysplasia, in particular, but not limited to, microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the preparation of a medicament for use in the prevention and/or treatment of congenital cartilage malformation(s). In a preferred embodiment, the congenital cartilage malformation(s) is selected from hereditary chondrolysis, chondrodysplasia, and pseudochondrodysplasia, in particular, but not limited to, microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

In a further method of treatment aspects, this invention provides methods for preventing and/or treating a mammal suffering from congenital cartilage malformation(s), wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein, for the treatment or prevention of said condition. In a preferred embodiment, the congenital cartilage malformation(s) is selected from hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, in particular, but not limited to, microtia, anotia, metaphyseal chondrodysplasia associated disorders.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of disease(s) associated with IL6 hypersecretion. In a preferred embodiment, the disease(s) associated with IL6 hypersecretion is selected from Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

In one embodiment, the present invention provides the use of salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, in the prevention and/or treatment of disease(s) associated with IL6 hypersecretion. In a preferred embodiment, the disease(s) associated with IL6 hypersecretion is selected from Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the manufacture of a medicament for use in the prevention and/or treatment of disease(s) associated with IL6 hypersecretion. In a preferred embodiment, the disease(s) associated with IL6 hypersecretion is selected from Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

In a further method of the treatment aspects, the present invention provides methods for preventing and/or treating a mammal suffering from disease(s) associated with IL6 hypersecretion, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more pharmaceutical compositions described herein. application, for the treatment or prevention of said condition. In a preferred embodiment, the disease(s) associated with IL6 hypersecretion is selected from Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma and/or mesangial proliferative glomerulonephritis.

In one embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the prevention and/or treatment of disease(s) associated with interferon hypersecretion. In a preferred embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis and rheumatoid arthritis.

In one embodiment, the present invention provides the use of the salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, in the prevention and/or treatment of disease(s) associated with interferon hypersecretion. In a preferred embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis and rheumatoid arthritis.

In another embodiment, the present invention provides salts of the present invention, or pharmaceutical compositions comprising a salt of the present invention, for use in the manufacture of a medicament for use in the prevention and/or treatment of disease(s) associated with interferon hypersecretion. In a preferred embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis and rheumatoid arthritis.

In a further method of the treatment aspects, the present invention provides methods for the prevention and/or treatment of a mammal suffering from disease(s) associated with interferon hypersecretion, wherein the methods comprise administering an effective amount of a salt of the present invention or one or more of the pharmaceutical compositions described herein. application, for the treatment or prevention of said condition. In a preferred embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjögren's syndrome, psoriasis and rheumatoid arthritis.

A specific mode of the method of the present invention includes administration to a subject suffering from inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with the destruction and/or disruption of cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons, an effective amount of the salt of the present invention for a period of time sufficient to reduce the severity of the above diseases in the subject, and preferably stop the processes responsible for these diseases.

Using these administration protocols, each dose provides from about 1 to about 500 mg of the salt of the present invention, with preferred doses providing each from about 10 to about 300 mg, more specifically from about 25 to about 250 mg, and especially 10 mg. 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg.

Dose levels for injections range from about 0.1 to at least 10 mg/kg/hr, all over a period of time from about 1 to about 120 hours, and especially from 24 to 96 hours. 0.1 to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 1 g/day for a human patient weighing between 40 and 80 kg.

For the prevention and/or treatment of long-term conditions such as degenerative conditions, the treatment program is extended over many months or years, so oral administration is

- 20 041895 preferred for patient comfort and patient tolerability. When administered orally, one to four (1-4) daily regular doses, especially one to three (1-3) daily regular doses, usually one to two (1-2) daily regular doses, and most preferably one (1) daily regular dose. dose are representative administration schedules. Alternatively, for drugs with long-term effect, when administered orally, representative regimens include the introduction of once every two weeks, once a week and once a day. In particular, the regimen may include administration every 1-14 days, more specifically 1-10 days, even more preferably 1-7 days, and most preferably 1-3 days.

Using these administration protocols, each dose provides from about 1 to about 500 mg of the salt of the present invention, with preferred doses providing each from about 10 to about 300 mg, more specifically from about 25 to about 250 mg, and especially 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg.

Doses for transdermal administration are generally chosen to provide similar or lower blood levels than those achieved by injection.

When used to prevent the development of a particular condition, the compounds of the present invention should be administered to a patient at risk of developing such a condition, usually on the advice or under the supervision of a physician, at the dose levels described above. Patients at risk for developing a particular condition generally include patients who have a family history of the condition, or patients who have been identified through genetic testing or screening as being particularly susceptible to developing the condition.

The salt of the present invention may be administered as the sole active agent, or may be administered in combination with other therapeutic agents, including other salts of the present invention which exhibit similar or similar therapeutic activity and which have been found to be safe and effective for such co-administration. In a particular embodiment, the co-administration of two (or more) agents provides for significantly lower doses of each agent to be administered, thereby reducing visible side effects.

In one embodiment, a salt of the present invention or a pharmaceutical composition comprising a salt of the present invention is administered as a drug. In a particular embodiment, said pharmaceutical composition further comprises an additional active ingredient.

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of a disease involving inflammation, specific agents include, but are not limited to, immunoregulatory agents, for example, azathioprine, corticosteroids (for example, prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus, mycophenolate, mofetil, muromonab-CD3 (OCTS, such as Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, and piroxicam.

In one embodiment, the salt of the present invention is co-administered with another therapeutic agent for the treatment and/or prevention of arthritis (e.g., rheumatoid arthritis), specific agents include, but are not limited to, analgesics, non-steroidal anti-inflammatory drugs (NSAIDS), steroids, synthetic disease-modifying disease-modifying antirheumatic drugs (DMARDS), such as, but not limited to, methotrexate, leflunomide, sulfasalazine, auranofin, sodium aurothiomalate, penicillamine, chloroquine, hydroxychloroquine, azathioprine, tofacitinib, baracitinib, fostamatinib, and cyclosporine, and biologic DMARDS (e.g., but not limited to, infliximab, etanercept, adalimumab, rituximab, and abatacept).

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of proliferative disorders, specific agents include, but are not limited to: methotrexate, leucovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, taxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (eg, Herceptin™), capecitabine, raloxifene hydrochloride, epidermal growth factor receptor EGFR inhibitors (eg, Iressa®, Tarceva™, Erbitux™), but not limited to VEGF inhibitors (eg Avastin™), proteasome inhibitors (eg Velcade™), Glivec® or hsp90 inhibitors (eg 17-AAG). In addition, the compound of the present invention can be administered in combination with other treatments, including, but not limited to, radiation therapy or surgery. In a specific embodiment, the proliferative disorder is selected from cancer, myeloproliferative disease, or leukemia.

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of autoimmune diseases, specific

- 21 041895 means include, but are not limited to: glucocorticoids, cytostatic agents (for example, purine analogues), alkylating agents (for example, nitrogen mustards (cyclophosphamide), nitrosoureas, platinum salts of the present invention and others), antimetabolites (for example, methotrexate, azathioprine and mercaptopurine), cytotoxic antibiotics (eg, dactinomycin, anthracyclines, mitomycin C, bleomycin, and mithramycin), antibodies (eg, monoclonal anti-CD20-, anti-CD25- or anti-CD3- (TK3) antibodies, Atgam® and Thymoglobuline ®), cyclosporine, tacrolimus, rapamycin (sirolimus), interferons (eg, IFN-β), TNF-binding proteins (eg, infliximab, etanercept, or adalimumab), mycophenolate, fingolimod, and myriocin.

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of transplant rejection, specific agents include, but are not limited to: calcineurin inhibitors (for example, cyclosporine or tacrolimus (FK506)), mTOR inhibitors (for example, sirolimus, everolimus), antiproliferatives (eg, azathioprine, mycophenolic acid), corticosteroids (eg, prednisolone, hydrocortisone), antibodies (eg, monoclonal anti-IL-2Rα receptor antibodies, basiliximab, daclizumab), polyclonal anti-T- cellular antibodies (eg, antithymocyte globulin (ATG), antilymphocyte globulin (ALG)).

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of asthma and/or rhinitis and/or COPD, specific agents include, but are not limited to: β2-adrenoceptor antagonists (for example, salbutamol, levalbuterol, terbutaline and bitolterol), epinephrine (inhaled or tablet), anticholinergics (eg, ipratropium bromide), glucocorticoids (oral or inhaled), long-acting β2-agonists (eg, salmeterol, formoterol, bambuterol, and orally administered sustained-release albuterol), combinations of inhaled steroids and long-acting bronchodilators (eg, fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonists and inhibitors (eg, montelukast, zafirlukast, and zileuton), inhibitors of mediator release (eg, cromoglycate and ketotifen) , biological regulators of IgE response (eg, omalizumab), antihis tamine preparations (eg, ceterizine, cinnarizine, fexofenadine), vasoconstrictors (eg, oxymetazoline, xylometazoline, naphazoline, and tramazolin).

In addition, the salt of the present invention may be administered in combination with asthma and/or chronic obstructive pulmonary disease rescue therapies, where such therapies include oxygen or helium-oxygen breathing, salbutamol spray, or terbutaline (optionally in combination with an anticholinergic agent ( eg, ipratropium)), systemic steroids (oral or intravenous, eg, prednisone, prednisolone, methylprednisolone, dexamethasone, or hydrocortisone), intravenous salbutamol, nonspecific beta-agonists, drugs for injection or inhalation (eg, epinephrine, isoetarine, isoproterenol, metaproterenol), anticholinergics (intravenous or nebulized, eg glycopyrrolate, atropine, ipratropium), methylxanthines (theophylline, aminophylline, bamiphylline) administered by inhalation of anesthetics that have a bronchodilator effect (eg isoflurane, halothane, enflurane), ketamine, intravenous magnesium sulfate.

In one embodiment, the salt of the present invention is co-administered with another therapeutic agent for the treatment and/or prevention of inflammatory bowel disease (IBD), specific agents include, but are not limited to: glucocorticoids (eg, prednisone, budenoside), synthetic disease-modifying immunomodulatory agents (eg, methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurine, and cyclosporine) and biological disease-modifying immunomodulating agents (infliximab, adalimumab, rituximab, and abatacept).

In one embodiment, the salt of the present invention is co-administered with another therapeutic agent for the treatment and/or prevention of SLE (systemic lupus erythematosus), specific agents include, but are not limited to, monoclonal human antibodies (belimumab (Benlysta)), generic antirheumatic drugs that modify disease course (DMARDs), such as antimalarials (eg, plaquenil, hydroxychloroquine), immunosuppressants (eg, methotrexate and azathioprine), cyclophosphamide, and mycophenolic acid;

immunosuppressants and analgesics such as non-steroidal anti-inflammatory drugs, opiates (eg dextropropoxyphene and cocodamol), opioids (eg hydrocodone, oxycodone, MC-Contin or methadone), and fentanyl transdermal patch.

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of psoriasis, specific agents include, but are not limited to: topical preparations such as washes, moisturizers, medicated creams and ointments containing tar, dithranol (anthralin), corticosteroids, eg deoxymethasone (Topicort™), fluocinonide, vitamin D3 analogs (eg, calcium

- 22 041895 potriol), retinoids based on argan oil (etretinate, acitretin, tazarotene), systemic drugs such as methotrexate, cyclosporine, retinoids, thioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, fumaric acid esters, or biologics such as Amevive™, Enbrel™, Humira™, Remicade™, Raptiva™ and ustekinumab (IL-12 and IL-23 blockers). In addition, the salt of the present invention can be administered in combination with other therapies, including, but not limited to, phototherapy or photochemotherapy (eg, a combination of phototherapy and PUFA (psoralen+UV A) therapy).

In one embodiment, the salt of the present invention is administered in conjunction with another therapeutic agent for the treatment and/or prevention of an allergic reaction, specific agents include, but are not limited to: antihistamines (for example, cetirizine, diphenhydramine, fexofenadine, levocetirizine), glucocorticoids (for example, prednisone, betamethasone, beclomethasone, dexamethasone), epinephrine, theophylline or antileukotrienes (eg montelukast or zafirlukast), anticholinergics and decongestants.

The term co-administration includes any means of delivering two or more therapeutic agents to a patient within the same treatment program, as will be apparent to those skilled in the art. Although two or more agents may be administered simultaneously in the same formulation, ie as a single pharmaceutical composition, this is not a critical factor. Means can be administered in the form of different drugs at different times.

Chemical synthesis procedures

General procedures

The compound according to formula I and the salts of the present invention can be obtained from readily available starting materials using the following general methods and techniques. It should be understood that when the usual or preferred process conditions are given (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.), other process conditions may also be used unless otherwise noted. Optimum reaction conditions may vary depending on the specific reagents or solvents used, but such conditions can be determined by one of skill in the art using conventional optimization algorithms.

In addition, as will be clear to those skilled in the art, commonly used protecting groups may be required to prevent undesired reactions of certain functional groups. The selection of an appropriate protecting group for a particular functional group, as well as suitable conditions for the introduction of protective groups and deprotection, is well known in the art. (Greene, T W; Wuts, P G M;, 1991).

Hereinafter, the following methods regarding the preparation of the salt of the present invention defined earlier in this application and comparative examples are presented in detail. The salt of the present invention can be obtained by a person skilled in the art of organic synthesis from known or commercially available starting materials and reagents.

All reagents are technical grade reagents and are used as received, without further purification, unless otherwise noted. Commercially available anhydrous solvents are used in reactions carried out under an inert atmosphere. Chemical grade solvents are used in all cases unless otherwise indicated. Column chromatography is carried out on silica gel 60 (35-70 μm). Thin layer chromatography is performed using plates pre-coated with a layer of silica gel F-254 (thickness 0.25 mm). 1H NMR spectra were recorded on a Bruker DPX 400 NMR spectrometer (400 MHz or a Bruker Advance 300 NMR spectrometer (300 MHz). Chemical shifts (5) for the 1H NMR spectrum are reported in parts per million (ppm) relative to the tetramethylsilane peak ( δ 0.00) or an appropriate residual solvent, i.e. CHCl ₃ (δ 7.27) as an internal standard.

Signal multiplicities are represented as singlet (s), doublet (d), triplet (t), quadruplet (square), multiplet (m), and wide signal (broad). Electrospray ionization mass spectrometry is performed on a Waters spectrometer with a platform for liquid chromatography and LC/MS mass spectrometry analysis or with a Waters Acquity H-Class UHPLC (high performance liquid chromatography) connected to a Waters Mass detector 3100. Columns used: Waters Acquity UPLC VEN C18 1.7 µm, 2.1 mm id. x 50 mm long, Waters Acquity UPLC VEN C18 1.7 µm, 2.1 mm id. x 30 mm long., or Waters Xterra MS 5 µm C18, 100x4.6 mm. All analyzes are performed using either MeCN/H ₂ O gradients (H ₂ O contains either 0.1% TFA or 0.1% NH3) or MeOH/H ₂ O gradients (H ₂ O contains 0.05% TFA). Microwave heating is carried out using the Biotage Initiator.

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Table I. List of abbreviations used in the experimental section

Abbreviations Definition ARMA 4-aminophenylmercury acetate app t apparent triplet ATP Adenosine 5'-triphosphate AUC Area under the curve lat. d wide doublet lat. With Wide Singlet BSA Bovine serum albumin lat. T Wide triplet Cat. Catalytic amount cDNA copy of deoxyribonucleic acid D doublet DCM dichloromethane Desc'd Described in detail DLM Data logging module DMSO Dimethyl sulfoxide DSC Differential Scanning Calorimetry DTT Dithiothreitol DVS Dynamic vapor sorption EDTA Ethylenediaminetetraacetic acid EKV. Equivalent _Et2O diethyl ether EtOAc ethyl acetate EtOH ethanol FBS Fetal bovine serum FT-IR infrared spectrophotometry with Fourier transform G gram gram Gravimetric Vapor Sorption (GSP) h hour HPLC High Performance Liquid Chromatography ΗΡ-β-CD 2-Hydroxypropyl-beta-cyclodextrin HRP horseradish peroxidase IL Interleukin int Intermediate kg kilogram l liter LC-MS Liquid Chromatography-Mass Spectrometry LPC Lysophosphatidylcholine M multiplet MeCN Acetonitrile MEK Methyl ethyl ketone Meon methanol mg milligram min minute ml milliliter mmol millimole MMR Matrix metalloproteinase MS Ms'd Mass measured by LC-MS MW Molecular mass N.A. did not define

- 24 041895

NBS N-Bromosuccinimide nBuOH n-Butanol NMR Nuclear magnetic resonance ONPG Ortho-nitrophenyl-β-galactoside Patt Sample PBF phosphate buffered formalin PBS phosphate buffered saline PCR polymerase chain reaction Pd (PPh ₃ ) 4 Tetrakis(triphenylphosphine)palladium(0) Pd/C Palladium on carbon 10% Pd ₂ (dba) 3 Tris(dibenzylideneacetone)dipalladium(0) PdC12dppf [1,1'- bis(diphenylphosphino)ferrocene]dichloropalladium(II) PEG Polyethylene glycol MLN.D. ppm XRPD powder x-ray diffraction KB. quadruplet OgbPCR Quantitative real-time PCR QTL loci of quantitative traits rel vol relative volume RH Relative Humidity PHK Ribonucleic acid c singlet sept septet TT NMR Nuclear magnetic resonance of a solid state SDTA Synchronous differential thermal analysis T triplet TBME tert-Butyl methyl ether TEA triethylamine TEA Trifluoroacetic acid TEA thermogravimetric analysis MKL microliter THF Tetrahydrofuran

- 25 041895

Table II. Salt analysis equipment

Chemical purity, determined by UHPLC Purity determination analysis was performed on a Waters Acquity system equipped with a diode array detector and a Micromass ZQ mass spectrometer using MassLynx software. TGA TGA data was obtained on a Mettler TGA/SDTA 851e equipped with a 34 position autosampler. The instrument is temperature calibrated using certified indium. Typically, 5-30 mg of each sample is loaded onto a pre-weighed aluminum crucible and heated at a rate of 10°C/min from ambient temperature to 400°C. A nitrogen purge was maintained over the sample at a rate of 50 ml/min. We used the STARe v9.10 equipment control and data analysis program. Thermodynamic solubility in water, determined by HPLC Solubility in water was determined by suspending a sufficient amount of the compound in water or buffer to obtain a maximum final concentration of >1 mg.ml ^-1 of the parent compound in free form. Quantification was carried out using HPLC with reference to a standard calibration curve. Solubility was calculated using peak areas determined by integrating the peak found at the same retention time as the main peak in the standard injection. GSP Sorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer controlled by SMS Analysis Suite. The sample temperature was maintained at 25° C. by means of equipment control. Humidity was controlled by mixing streams of dry and wet nitrogen, at a total flow rate of 200 ml/minute. Relative humidity was measured using a calibrated Rotronic probe (dynamic range 0.1-100% RH) placed near the sample. The change in mass (mass relaxation) of the sample as a function of % relative humidity was continuously monitored using a microbalance (accuracy +0.005 mg). Typically, 5-20 mg of sample was placed in a pre-weighed stainless steel wire basket at ambient conditions. The sample was loaded and unloaded at 40% relative humidity and 25°C (typical room conditions). The moisture sorption isotherm was obtained as described below (2 scans giving 1 complete cycle). A standard isotherm was obtained at 25°C at 10% RH intervals in the range of 0.5-90% RH.

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Polarized light microscopy (PLM) The samples were examined using a Leica DLM polarizing microscope with a digital video camera for imaging. A small amount of each sample was placed on a glass slide, fixed in immersion oil and covered with a glass strip, with individual particles separated as far as possible. The sample was viewed at appropriate magnification and partially polarized light, using a distorted color λ filter. DSC Perkin Elmer DSC7. Closed Au crucible, heating rate: 10 or 20°C/minute, range: -50°C to 250°C, or DSC data were collected on a Mettler DSC 823e equipped with a 34 position autosampler. The instrument has been calibrated for energy and temperature using certified indium. Typically, 0.5-3 mg of each sample, in an aluminum dish, was heated at 10°C/min from 25°C to 300°C. A nitrogen purge was maintained over the sample at a rate of 50 ml/min. We used the STARe v9.10 equipment control and data analysis program. FT-IR Data were collected on a Nicolet Avatar FT-IR spectrometer with Smart DurasamplIR accessory and monitored using the Omnic software. NMR ³ H and ¹³ C spectra were obtained using a Varian Unity Inova 400 NMR spectrometer with a 5 mm inverse triple resonant probe operating at 400.12 MHz for the proton. Samples were prepared in de-DMSO unless otherwise noted. Inverse ¹³ C NMR spectra were obtained using a Bruker DPX300 gating spectrometer using a dual ³ H/ ¹³ C probe operating at 75.46 MHz for carbon. The sample was prepared by dissolving ~50mg material in de-DMSO. D1 thirty seconds was used with 7168 scans. TT NMR ¹³ C NMR spectra of the solid were recorded using a Varian VNMRS spectrometer operating at 100.56 MHz for ¹³ C and with a 6 mm (outer diameter) rotating magic angle (MAS) probe. They were obtained using cross-polarization and MAS with a 30 sec recycle delay, 1 ms contact time and a sample rotation speed of 6.8 kHz. The spectra were correlated with an external sample of pure tetramethylsilane by tuning the high frequency line from adamantane to 38.5 ppm. The measurements were carried out in air and at ambient temperature (~25°C). Samples were analyzed as received.

- 27 041895

Particle Size Distribution (PSD) Laser Diffraction PSD was determined using Sympatec laser diffraction with a HELOS/BF particle sizer equipped with a RODOS/ASPIROS solids disperser operating at 2.5 bar, with a sliding speed of 25 mm/s, using a combination of R1 0.1/0 .18 µm 35 µm and R3 0.5/0.9 µm - 175 µm lenses for definition. Trigger conditions: 1ms, 0.2%. x-ray powder diffraction Bruker D2 Phaser Powder X-ray diffraction patterns were obtained on a Bruker AXS D2 diffractometer using CuK radiation (30 kV, 10 mA), Θ-Θ geometry, using a Lynxeye 5-42° 20 detector. The DIFFRAC program was used for data collection. SUITE and data were analyzed and presented using Diffrac Plus EVA v 13.0.0.2. Data collection: Angular range: 5 to 42° 20; step size: 0.012° 20; collection time: 0.15 sec per step. Sample preparation: Samples analyzed at ambient conditions were obtained in the form of flat plates using the powder as received, without milling. Approximately 1-2 mg of the sample was lightly pressed onto a silicon substrate to form a flat surface.

Synthesis of the salt of the present invention

Example 1 Preparation of Compound 1

1.1 Path 1

1.1.1 4-[4-(4,4,5,5-T-etramethyl-[1,3,2]dioxaborolan-2-yl)benzyl]thiomorpholine-1,1-dioxide

2-(4-Bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1 eq.) and DIPEA (2 eq.) were dissolved in a mixture of DCM/MeOH (5:1 vol:ob) under N ₂ and thiomorpholine 1,1-dioxide (2 equiv.) was added portionwise. The resulting solution was stirred at room temperature for 16 hours. After this time, the reaction was complete. The solvent was evaporated. The compound was extracted with EtOAc and water, washed with brine and dried over anhydrous MgSO ₄ . The organic layers were filtered and evaporated. The final compound was isolated without further purification.

1.1.2 Cyclopropanecarboxylic acid (5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide

1.1.2.1.

Step i): 1-(6-Bromopyridin-2-yl)-3-carboethoxythiourea

To a solution of 2-amino-6-bromopyridine (compound 1) (253.8 g, 1.467 mol) in DCM (2.5 L) cooled to 5°C was added dropwise ethoxycarbonyl isothiocyanate (173.0 ml, 1.467 mol) within 15 min. The reaction mixture was then allowed to warm to room temperature (20° C.) and stirred for 16 hours. Evaporation in vacuo gave a solid which was collected by filtration, washed thoroughly with petrol (3x600 ml) and air dried to give the desired product. The thiourea can be used directly in the next step without any purification.

1H (400 MHz, CDCl3) δ 12.03 (1H, br.s), 8.81 (1H, d), 8.15 (1H, br.s), 7.60 (1H, t), 7. 32 (1H, dd), 4.31 (2H, sq.), 1.35 (3H, t).

- 28 041895

1.1.2.2.

Step ii): 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine

To a suspension of hydroxylamine hydrochloride (101.8 g, 1.465 mol) in EtOH/MeOH (1:1, 900 ml) was added N,N-diisopropylethylamine (145.3 ml, 0.879 mol) and the mixture was stirred at room temperature (20°C ) for 1 h. Then 1-(6-bromopyridin-2-yl)-3-carboethoxythiourea (compound 2) (89.0 g, 0.293 mol) was added and the mixture was slowly heated to reflux (Note: a clarifying agent is required getter to quench the emitted H2S). After 3 hours at reflux, the mixture was allowed to cool and filtered to collect the precipitated solid. The product was then collected by evaporating the filtrate in vacuo, adding H ₂ O (250 ml) and filtering. The combined solids were washed successively with H ₂ O (250 ml), EtOH/MeOH (1:1, 250 ml) and Et ₂ O (250 ml), then dried in vacuo to give the triazolopyridine derivative (compound 3) as solid. The compound can be used directly in the next step without any purification.

1H (400 MHz, DMSO-d ₆ ) δ 7.43-7.34 (2H, m, 2x aromatic-H), 7.24 (1H, dd, J 6.8 and 1.8 Hz, aromatic-H ), 6.30 (2H, br, NH2); m/z 213/215 (1:1, M+H+, 100%).

1.1.2.3.

Step iii): (5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide cyclopropanecarboxylic acid

To a solution of 2-aminotriazolopyridine obtained in the previous step (7.10 g, 33.3 mmol) in anhydrous MeCN (150 ml) at 5°C was added Et ₃ N (11.6 ml, 83.3 mmol), with followed by the addition of cyclopropanecarbonyl chloride (83.3 mmol).

The reaction mixture was then allowed to warm to ambient temperature and stirred until all of the starting material had been consumed. If necessary, additional Et ₃ N (4.64 mL, 33.3 mmol) and cyclopropanecarbonyl chloride (33.3 mmol) were added to ensure completion of the reaction. After evaporation of the solvent in vacuo, the resulting solid was treated with 7 N ammonia in methanol (50 ml) and stirred at ambient temperature (for 1-16 h) to hydrolyse any bis-acylated product. The selection of the product was carried out by removal of volatiles in vacuum, followed by processing with Et ₂ O (50 ml). The solids were collected by filtration, washed with H ₂ O (2x50 ml), acetone (50 ml) and Et ₂ O (50 ml), then dried in vacuo to give the desired compound. 1.1.3.

Compound 1 o

O

4-[4-(4,4,5,5-T etramethyl-[ 1,3,2]dioxaborolan-2-yl)benzyl]thiomorpholin-1,1-dioxide (1.1 eq.) was added to a solution ( Cyclopropanecarboxylic acid 5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide in 1,4-dioxane/water (4:1). K2CO3 (2 equiv.) and PdCl ₂ dppf (0.03 equiv.) was added to the solution. The resulting mixture was then heated in an oil bath at 90° C. for 16 h under N ₂ . Water was added and the solution was extracted with ethyl acetate. The organic layers were dried over anhydrous MgSO ₄ and evaporated in vacuo.

The final compound was obtained after purification using flash chromatography.

Alternatively, after completion of the reaction, a palladium scavenger such as 1,2bis(diphenylphosphino)ethane was added, the reaction mixture was allowed to cool, and filtration was performed. The filter layer was resuspended in a suitable solvent (eg acetone), the solid was separated by filtration, washed with additional acetone and dried. The resulting solid was resuspended in water, an aqueous HCl solution was added, and after stirring at room temperature, the resulting solution was filtered on Celite (Celpure P300). Then an aqueous solution of NaOH was added to the filtrate and the resulting suspension was stirred at room temperature, the solid was separated by filtration, washed with water and dried with air suction. Finally, the layer was resolubilized in THF/H ₂ O, treated with a palladium scavenger (e.g. SMOPEX 234) at 50°C, the suspension was filtered, the organic solvents were removed by evaporation and the resulting suspension was washed with water and methanol, dried and sieved, with obtaining the desired compound as the free base.

1.2. Route 2

1.2.1 Step 1: Cyclopropanecarboxylic acid [5-(4-hydroxymethylphenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]amide

- 29 041895

But

4-(Hydroxymethyl)phenylboronic acid (1.1 eq.) was added to a solution of cyclopropanecarboxylic acid (5-bromo[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide in a -dioxane/water (4:1). K ₂ CO ₃ (2 eq) and PdCl ₂ dppf (0.03 eq) were added to the solution. The resulting mixture was then heated in an oil bath at 90° C. for 16 h under N ₂ . Water was added and the solution was extracted with ethyl acetate. The organic layers were dried over anhydrous MgSO ₄ and evaporated in vacuo. The resulting mixture was used without further purification.

1.2.2. Step 2: [5-(4-Bromomethylphenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylic acid amide

Phosphorus tribromide (1 0 equiv.). The reaction mixture was stirred at room temperature for 20 hours, quenched with ice and water (20 ml) and extracted with dichloromethane. The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated to dryness. The resulting white residue was triturated in dichloromethane/diethyl ether 2:1 to give the desired product.

1.2.3. Stage 3:

O

[5-(4-Bromomethylphenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxylic acid amide (1 eq) and DIPEA (2 eq) were dissolved in DCM/ MeOH (5:1 v:v) under N ₂ and thiomorpholine 1,1-dioxide (1.1 eq.) was added dropwise. The resulting solution was stirred at room temperature for 16 hours. After this time, the reaction was complete. The solvent was evaporated. The compound was dissolved in DCM, washed with water and dried over anhydrous MgSO ₄ . The organic layers were filtered and evaporated. The final compound was isolated using column chromatography using EtOAc to obtain the desired product.

Example 2 Preparation of salts of compound 1.

2.1. Protocol 1

Solvent was added to Compound 1 samples (approximately 5 mg) in relative volume parts of 10, 15 and 20 volumes and after each addition the samples were shaken at 50°C in a shaker for 20 min to promote dissolution, before being cooled to room temperature for subsequent solvent addition. . The solvent was selected from isopropanol, ethanol, methanol, isopropyl acetate, THF, TBME, acetone, methyl ethyl ketone, DCM and MeCN.

Under these conditions, almost complete dissolution of compound 1 occurred only in DCM.

After adding 20 relative volumes of solvent, HCl (1 M solution in THF) (12 μl -1 eq.) was added to each sample and observations were made. Samples were stored on a shaker, with a heat-cool cycle (50° C./room temperature, 4 hours at each temperature) for 16 hours. The resulting solids were isolated by vacuum filtration, dried under vacuum and analyzed by powder X-ray diffraction analysis.

The formation of the HCl salt was observed only in ethanol, methanol, THF, acetone, methyl ethyl ketone and MeCN.

2.2. Protocol 2

Compound 1 (-20 mg) was treated with 400 μl (20 volumes) of solvent (THF, acetone) and treated with aqueous acid solutions at room temperature (see Table III below). The selected samples were placed in a temperature cycling maturation chamber from ambient temperature to 50° C. with a 4-hour period at each temperature condition. After three days, the solids were isolated by filtration and any solutions were left to evaporate. Any

- 30 041895 further solids were isolated, or the remaining oils were treated with

EtOAc and placed back in the maturation chamber for four days. Any solids that formed further were isolated by filtration.

Solids were analyzed by powder x-ray diffraction analysis.

Using this protocol, salts were formed only in sulfuric acid, pTSA, 1,2ethane disulfonic acid, 2-hydroxyethanesulphonic acid, naphthalene 2-sulphonic acid, and maleic acid.

Table III. Acids used in protocol 2

Acid Solvent Equiv. acids 1-5-Naphthalenedisulfonic acid Water 2.1 sulfuric acid Water 2.1 1-2-Ethandisulfonic acid Water 2.1 p-Toluenesulfonic acid Water 2.1 Methansulfonic acid Water 2.1 1-5-Naphthalenedisulfonic acid Water 1.1 sulfuric acid Water 1.1 1-2-Ethandisulfonic acid Water 1.1 p-Toluenesulfonic acid Water 1.1 Methansulfonic acid Water 1.1 Naphthalene-2-sulfonic acid Water 1.1 Benzenesulfonic acid Water 1.1 Oxalic acid Water 1.1 2-Hydroxyethanesulfonic acid Water 1.1 Maleic acid Water 1.1 Phosphoric acid Water 1.1 ethanesulfonic acid Water 1.1 Malonic acid Water 1.1 2-5-Dihydroxybenzoic acid THF 1.1 L-tartaric acid Water 1.1

2.3. Protocol 3

To a suspension of compound 1 (~25 mg) and solvent (MeOH, THF, acetone or DCM) (20 relative volumes) at approximately 40° C. was added 1.05 or 2.1 eq. acids (HCl, HBr, H ₂ SO ₄ , Ph-SO ₃ H, oxalic acid, maleic acid or pTSA.H ₂ O) and visual observations were made. Samples were stored on a shaker at 26° C. for 14 hours. An aliquot was taken and the solid was isolated by vacuum filtration, dried under vacuum and analyzed by powder x-ray diffraction analysis. The mixtures were then stored on a shaker at 26°C for a further 31 hours. The remaining solid from these samples, showing powder X-ray diffraction patterns of a crystalline substance other than the free base, was isolated by vacuum filtration and further analyzed (DSC, NMR, Stability) .

The remainder of each sample was stored in a shaker with a heat/cool cycle (50° C./room temperature, 4 h at each temperature) for 136 h. Then an aliquot of each sample was taken again and the solid was isolated by vacuum filtration, dried under vacuum and analyzed by powder x-ray diffraction analysis. The remaining solid from these samples, showing powder X-ray diffraction patterns of a crystalline substance other than the free base, was isolated by vacuum filtration and further analyzed (DSC, NMR, Stability).

The samples that remained in solution were allowed to evaporate slowly at ambient temperature until the solvent evaporated, and any solids formed were isolated and analyzed by powder X-ray diffraction analysis.

Using this protocol, salts were formed only in HBr, HCl, sulfuric acid, pTSA, and maleic acid.

2.4. Protocol 4

2.4.1. HCl

HCl (1 M solution in THF) (655 µl, 0.65 mmol, 1.1 eq.) was added to a stirred suspension of compound 1 (252.6 mg, 0.59 mmol, 1 eq.) and methanol (5.05 ml, 20 volumes) at 50°C. The mixture was cooled to 25°C at a rate of 1°C/min and stirred at 25°C for 22 hours.

The solid was isolated by vacuum filtration and dried under vacuum.

Powder X-ray diffraction analysis confirmed the formation of a stable non-hygroscopic salt containing 4-5% water, having a water solubility measured at 1.9 mg/mL.

2.4.2. Maleic acid

Compound 1 (246.4 mg, 0.58 mmol, 1 eq.) was suspended in a solution of 5% water in acetone (4.95 ml, 20 volumes) at 25°C and the sample was heated to 50°C. Maleic acid (1 M solution in THF) (1.2 ml, 1.22 mmol, 2.1 eq.) was added to the stirred suspension at 50°C. The mixture was cooled to 25°C at a rate of 1°C/min and stirred at 25°C for 22 hours.

The solid was isolated by vacuum filtration and dried under vacuum.

Powder X-ray diffraction analysis confirmed the formation of a stable, non-hygroscopic salt having a water solubility measured at 0.4 mg/mL.

2.4.3. pTSA pTSA (1 M solution in EtOH) (1.3 ml, 1.3 mmol, 2.2 eq.) was added to a stirred suspension of compound 1 (250.7 mg, 0.59 mmol, 1 eq.) and THF ( 5 ml, 20 volumes) at 50°C. The mixture was cooled to 25°C at a rate of 1°C/min and stirred at 25°C for 22 hours.

The solid was isolated by vacuum filtration and dried under vacuum.

Powder X-ray diffraction analysis confirmed the formation of salt, which was found to be unstable.

2.5. Complementary salt analysis

Solids isolated using this protocol were also analyzed for purity, salt equivalent (IC), NMR (residual solvent), DSC, TGA (solvates) and stability evaluation (1 week at 40°C/75% RH) .

2.5.1. pKa definition

Data was collected on a Sirius GlpKa device with the D-PAS application. Measurements were made at 25°C in aqueous solution using UV and in methanol-water mixtures by potentiometry. The ionic strength of the titration medium was adjusted with 0.15 M KCl solution (aqueous solution). Values found in methanol-water mixtures were corrected to 0% co-solvent by extrapolation by Yasuda Shedlowski.

The data were refined using the Refinement Pro program.

Following this procedure, the following values were obtained: 1.58±0.01, 3.68±0.02 and 12.02±0.01.

2.5.2. pH determination

pH measurements were carried out on the test substance in the form of a suspension. Typically, 1 ml of demineralized water was added to about 300 mg of the test substance and the suspension was then stirred. If not enough liquid or too high a viscosity was obtained to measure the pH level using the pH electrode, then the amount of water was increased until it could be measured in 1 ml increments. The pH electrode was calibrated using a three-point calibration.

Finally, the pH was recorded at the start (=after 2 min to allow for relatively stable pH measurements).

2.5.3. Solubility study

2.5.3.1. Protocol

The solubility of the compound was determined in various solvents by equilibrating the excess product for at least 24 hours at 20° C. on a rotary shaker. The supersaturated solution was then filtered and the concentration of the product in the filtrate was determined using UV spectrometry.

In water, the pH was adjusted to pH 2, pH 7 and pH 9. HCl and NaOH were used to adjust the pH.

2.5.3.2. results

For example, using this protocol, the solubility values obtained for the free base and the corresponding HCl salt are shown in Table 1 below. IV. Surprisingly, going from compound 1 to compound 1.HC1.3H ₂ O does not systematically improve solubility.

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Table IV. Solubility of compound 1 as a free base and the corresponding HCl salt in various solvents

Solvent Compound 1 Solubility (mg/ml) Compound!.HC1.3H2O Solubility (mg/ml) Acetone 0.420 0.415 Acetonitrile 0.605 7.36 Acetonitrile/water (1/1) 1.57 25, 6 Acetonitrile/water (4/1) 3, 11 7.81 dichloromethane 5, 66 0.388 ethanol 0.151 0.764 0.005M HC1 0.252 3.10 0.01M HC1 0.288 2.34 0.01M HC1 + 2.0% Tween 0.313 2.79 80 ΗΡ-β-CD 40% pH2 0.577 7.88 ΗΡ-β-CD 40% pH3 0.550 9, 33 ΗΡ-β-CD 40% pH4 0.520 2.79 methanol 0.432 0.743 PEG400 1.53 36.6 Propanol 0.134 0.663 propylene glycol 0.464 11.7 tert-butylmethyl <0.00239 0.0263 ether Tetrahydrofuran 0.740 0.208 Purified water) 0.00384 3, 17 Water pH2 0.291 8.05 Water pH7 0.00432 2.26 Water pH9 0.00429 2, 68 Water/ethanol 1/1 0.327 8, 68

2.5.4 Conclusions

As demonstrated above, compound 1's solubility varies greatly depending on the solvent used, and salt formation does not necessarily occur with every acid, illustrating the difficulty of selecting a satisfactory solvent/acid combination that is specific to compound 1.

Example 3: Polymorphism Study

3.1. Compound 1

3.1.1. Formation of amorphous compound 1

Compound 1 (38 mg) was heated in a DSC analyzer under nitrogen atmosphere according to the following procedure:

1) Heated from 30°C to 250°C at a rate of 10°C/min;

2) Maintained isothermal mode for 1 min;

3) Cooled from 250 to 30°C at a rate of 100°C/min and

4) Maintained isothermal mode for 4 min.

Using powder X-ray diffraction analysis, it was confirmed that the resulting solid was amorphous.

3.1.2. Polymorphism research

The amorphous compound was exposed to forty different solvents (ethyl formate, TBME, acetone, methyl acetate, methanol, tetrahydrofuran, diisopropyl ether, ethyl acetate, ethanol, methyl ethyl ketone, acetonitrile, t-BuOH, 2-propanol, 1-2-dimethoxyethane, isopropyl acetate, 1-propanol , 2butanol, nitromethane, 1-4-dioxane, propyl acetate, 2-pentanone, 2-methyl-1-propanol, toluene, isobutyl acetate, methylisobutylketone, 1-butanol, 2-methoxyethanol, butyl acetate, methylbutylketone, 3-methyl-1butanol, 2 -ethoxyethanol, 1-pentanol, anisole, benzonitrile, nitrobenzene, IPA + 5% water, EtOH + 5% water, MeCN + 5% water, THF + 5% water and acetone + 5% water), temperature cycling against temperature environment up to 50°C for 4 hours in each state. After five days, the solids were collected by filtration and first analyzed by powder x-ray diffraction analysis.

3.1.3. Compound 1 polymorphism analysis results

Using this protocol, the resulting solids were analyzed by powder X-ray diffraction analysis and the next most stable samples are listed in

- 33 041895 tab. V, tab. VI and tab. VII below.

Table V. Sample 1 X-ray Powder Diffraction Pattern of Compound 1 (FIG. 1)

Angle (20°) Intensity (%) 16, 8 100.0 9, 4 30.4 9, 6 14.0 13, 1 26, 2 14.3 12.2 17.3 12.4 18.4 9.5 18.7 13.0 19.0 43, 8 20.6 9, 1

20, 8 15, 3 21.3 20.5 23, 8 18.0 24.6 14.3 25, 2 5, 8 29, 7 8.7

Table VI. Compound 1 powder X-ray diffraction pattern 3 (FIG. 2)

Angle (20°) Intensity (%) 8, 6 52 9, 7 24.6 10.6 35.5 13.0 39, 4 15, 3 61.7 16, 9 40 17.3 45.6 17.7 50.3 17.9 14.1 18.3 10, 9 18.9 100 19, 3 21.4 19.6 12.6 19, 8 28, 1 20.0 28.7 20.3 13, 9 20, 8 52.2 23.0 37 23.5 15, 4 23, 9 50.1 24.4 23, 8 25.0 14.1 29, 1 15 33.5 32.3 34.9 23, 6

- 34 041895

Table VII. Compound 1 Powder X-ray Diffraction Pattern 4 (FIG. 3)

3.2. Polymorphism of the HCl salt of compound 1 and its solvates

3.2.1. Formation of amorphous HCl salt of compound 1

Compound 1 (25.47 mg) was mixed with water (70 ml, -2750 v/v) and stirred at room temperature for 30 minutes. The mixture was filtered through a 0.45 mm syringe filter with a PVDF (polyvinylidene fluoride) membrane and frozen in a -78°C bath. The solvent was removed using a freeze dryer to give the mono-HCl salt of amorphous compound 1 (confirmed by XRPD, glass transition temperature 149° C. (modulated DSC analysis)).

3.2.2. Study protocols

Volumes of 25 different solvents (acetone, anisole, butanol, butyl acetate, TBME, DMSO, ethanol, ethyl acetate, heptane, isopropyl acetate, MEK, isopropyl acetate, MeCN, cyclohexane, DCM, dioxane, methanol, nitromethane, THF, methyl THF, toluene, water, 10% aqueous acetone, 10% aqueous THF and 10% methanol) were added to the amorphous mono-HCl salt of Compound 1, with the indicated volumes ranging from 125 μl (5 relative volumes) to 1 ml (40 relative volumes) , or until a mobile suspension or substantially complete dissolution is observed. The sample was stored in a shaker with a heat/cool cycle (50°C/room temperature, 4 h) for 23 h.

After 23 hours, when a solid formed, an aliquot was taken and the solid was isolated by vacuum filtration, and the remaining suspension was kept in a shaker with a heat/cool cycle (50°C/room temperature, 4 hours). The solid from the aliquot was dried under vacuum for 30 min and analyzed by powder x-ray diffraction, then dried further in a vacuum oven at 30° C. and 5 mbar for 34 h and re-analyzed by powder x-ray diffraction. Finally, the dried sample was stored at 40°C/75% relative humidity for 72 hours and re-analyzed using powder x-ray diffraction analysis.

After 47 hours, when a solid formed in the suspension, an aliquot was taken and the solid was isolated by vacuum filtration, and the remaining suspension was kept in a shaker with a heat/cool cycle (50°C/room temperature, 4 h). Aliquot solids were stored at ambient conditions for 72 hours and 14 days with XRPD analysis performed at each time point. The solid, which was stored at ambient conditions for 14 days, was then stored at 40°C/75% relative humidity for 43 h and re-analyzed using powder X-ray diffraction analysis.

After 143 h, when a solid formed, an aliquot was taken from the remaining suspension stored in a shaker with a heat/cool cycle (50°C/room temperature, 4 h) and the solid was isolated by vacuum filtration, dried under vacuum for 2 h, then analyzed by powder x-ray diffraction analysis. The isolated solid was dried in a vacuum oven at 30° C. and 5 mbar for 20 hours and re-analyzed by powder x-ray diffraction analysis. The dried sample was stored at 40°C/75% relative humidity for 114 hours and re-analyzed using powder X-ray diffraction analysis.

- 35 041895

3.2.3. results

As a result of this study, two stable forms were identified.

3.2.3.1. Compound 1.HCl

HCl (3.6 M solution in dioxane) (2.5 ml, 9.05 mmol 1.1 eq.) was added in portions to a stirred suspension of amorphous compound 1 (3.499 g, 8.22 mmol, 1 eq.) and methanol ( 70 ml, 20 relative volumes) at 50°C for 2 min. The mixture was cooled to 20°C at a rate of 0.1°C/min and stirred at 20°C for a further 10 hours. The mixture was cooled to 15°C at a rate of 0.5°C/min and stirred for 30 min. The resulting solid was isolated by vacuum filtration, washed with methanol (2x3.5 ml, 1x7 ml) and dried under vacuum to give compound 1.HC1.

3.2.3.2. Compound 1.HC1.3H2O

HCl (1 M solution in THF) (250 μl, 0.25 mmol, 1.05 eq.) was added to a suspension of amorphous compound 1 (100.37 mg, 0.24 mmol, 1 eq.) and DCM (2 ml, 20 relative volumes) at 40°C. The mixture was stored on a shaker with a heat/cool cycle (40°C/room temperature, 4 h) for 70 h. The solid was isolated by vacuum filtration and dried under vacuum to give compound 1.HC1.3H ₂ O.

3.2.3.3. Analysis

Diffraction patterns are disclosed below in table. VIII and tab. IX.

Table VIII. Powder X-ray diffraction pattern of compound 1.HC1 (Fig. 4)

Angle (20°) 7.4 8.9 12.4 14.8 15, 1 16, 9 17, 6 19, 4 20.7 21.1 22.8 24.9 26.0 28.6 29, 8 32.6

Intensity (%) 100.0 15, 6 21.3 61.1 31.7 15, 9 27.7 11.4 43.4 11.9 14.6 12.3 12.3 14.2 27.3 10.2

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Table IX. Powder X-ray diffraction pattern of compound 1.HC1.3H2O (Fig. 5)

Angle (20°) Intensity (%) 7.3 61.4 8.4 35, 3 8, 8 62.8 10, 7 26, 3 12.0 22.5 12.2 18.1 13.2 23, 6 13, 7 16.1 14.5 14.0 16, 3 31.0 16, 7 100.0 17, 6 11.3 19, 3 20.8 20.2 87.5 20.6 16, 4 21.0 18.0 21.4 52.0 21.8 58.8 22.8 45.0 23, 4 57.5 23, 9 10.6 24.5 10.8 25.2 21.7 25.7 35, 3 25, 9 33.1 26, 4 11.7 27.2 13.0 27.7 16.6 28.3 10.8 28.6 19, 3 28.9 17.2 29.2 20.2 29, 6 47.6 32.7 26.1

3.2.3.4. X-ray diffraction analysis of compound 1.HC1.3H ₂ O (Fig. 11)

Compound 1.HC1.3H ₂ O was recrystallized from acetone:water (1:1). The results are shown below in table. x.

Table X. Single-crystal structure of compound 1.HC1.3H ₂ O

Molecular formula C21H24N5O3S .01.3 (H ₂ O) Molecular mass 516.02 Crystal system Monoclinic Symmetry group crystal lattice R21/P A 13.1388(4) A A 102.089 (2)° b 8.9437(3) A β With 21, 6376(9) A γ V 2486, 24(15) A ³ Z 4 De 1.379 mg/ ^m3 μ 0.2 84 mm ^-1 Source Mo-Ka, 0.71073 A G (000) 1088 T 120(2) K Crystal colorless prism, 0.39x0.16x0.12 mm Θ range for data collection 2.982-27.483° completeness 99.3% Reflections 21028 Unique Reflections 5649 Hint 0.0307

- 37 041895

The data processing method is based on the least squares method in full-matrix approximation in F ² .

R[F ² >2σ(F ² ) ]=0.0377 and wR(F ² )=0.0837. Approximation Accuracy (S)=1.109. The data processing method used 5649 reflections, 339 parameters and 0 constraints. All hydrogen positions were identified using a difference map, and those atoms attached to C atoms & N atoms were then placed in the calculated positions and refined using the motion model. Those hydrogens that are associated with water oxygen and nitrogen were easily refined. Final values Apmax=0.314 e A' ³ and APm _in =-0.368 e A' ³ .

The crystal structure of the compound 1.HC1.3H2O (Fig. 11) shows the unexpected inclusion of water molecules in the crystal lattice, which can provide further stabilization of the system.

Example 4 Large-Scale Preparation of Compound 1.HC1.3H2O

4.1. Protocol 1

To compound 1 (44 kg, 1.0 equiv.) in an inert atmosphere was added water (15 relative volumes, 1000 l) and the mixture was stirred at 50°C. 3.5 eq. an aqueous solution of HCl (5 relative volumes) was added over 10-15 min, at a maximum temperature of 55°C. After the addition was complete, stirring was continued at 50°C for 15 minutes and the reaction mixture was then cooled to 15°C and stirred at this temperature for at least 12 hours, but not more than 24 hours.

The resulting solid was isolated by filtration and the filter layer was washed with water (2.0 relative volumes) and the filter layer was dried under nitrogen for at least 4 hours to obtain the desired product.

4.2. Protocol 2

DCM (675 ml) and methanol (225 ml) were added to Compound 1 (45 g, 106 mmol, 1 eq.) under an inert atmosphere. The resulting suspension was heated to 35°C with stirring, and a 15% aqueous solution of trimercaptotriazine trisodium salt (22.5 g, 14 mmol, 0.13 eq.) was added and the resulting solution was stirred for 5 h, after which the solution was filtered on filter paper 0 .45 µM under nitrogen atmosphere.

Water (50 ml) was added to the filtrate and the resulting biphasic mixture was stirred at 35°C for 15 min, after which the phases were separated and the organic layer was left to cool to 20°C and washed two more times with 50 ml of water.

The organic layer was cooled to 15-20° C., then a 10% solution of HCl in methanol (42.4 g, 116 mmol, 1.10 eq.) was added over 30 minutes causing precipitation of a solid. The suspension was further stirred at 20°C for 3 h, then the precipitate was isolated by filtration, the filter layer was washed with methanol (2x50 ml) to obtain the desired compound, which was dried under vacuum at 45°C for 3 h. The filter layer was then resuspended in water (220 ml) and stirred for 6 h at 50°C and then cooled to 15-20°C. The resulting solid was separated by filtration and the filter layer was washed with water (2x30 ml) and dried at 45°C for 3 hours to obtain the desired product.

4.3. Protocol 3

4.3.1. Step 1: Compound 1.HC1.MeOH

To compound 1 (100 g, 235 mmol, 1 eq.) suspended in DCM (1.5 L) was added MeOH (0.5 L) and the resulting solution was heated to 35°C. An 85% solution of trisodium trimercaptotriazine (8.7 g, 3 mmol, 0.13 eq.) in water (42 ml) was added and the resulting mixture was stirred at 35° C. for at least 5 hours. The solution was then filtered on 0 filter paper. .45 µM under nitrogen atmosphere.

Water (150 g) was added to the resulting solution, stirred at 35°C for 15-30 min, and the biphasic mixture was separated. The organic layer was again washed twice with water (2x150 g).

Finally, a solution of HCl in MeOH (10% w/w) (141 g) was added and the suspension was stirred at 20°C for 3 h and the resulting solid was isolated by filtration, the filter layer was washed with MeOH (2x118 g ), dried under vacuum for 3 h at 45° C. to give compound 1.HC1.MeOH, which was analyzed by powder X-ray diffraction analysis. (Table XI).

4.3.2. Step 2: Compound 1.HC1.3H2O

To formic acid (200 g, 1.6 equiv.) in water (36 g, 0.4 equiv.) was added the compound 1.HC1.MeOH (100 g, 1 equiv.) prepared in step 1 above. The resulting mixture was heated to 55°C with stirring and the solution was filtered through a 0.45 μm filter cartridge. An 85% aqueous formic acid solution (200 g) was added and the mixture was cooled to 28-32° C. with gentle stirring.

Water (100 g) was then added, followed by the addition of compound 1.HC1.3H2O (1 g), causing precipitation of compound 1.HC1.1.5HCO ₂ H, which was analyzed by powder X-ray diffraction analysis (Table XII).

While stirring at 28-32°C, water (2 L), 8 x 100 ml, 1 x 200 ml and 2 x 500 ml was added in portions.

- 38 041895

The resulting suspension was then filtered, the filter layer was washed with water (2x100 ml) and dried at 30-35°C to give compound 1.HC1.3H ₂ O, which was analyzed by powder x-ray diffraction analysis and DSC. (Fig. 5 and Fig. 7)

Table XI. Powder X-ray diffraction pattern of l.HCl.MeOH compound (Fig. 8)

Angle (20°) Intensity (%) 7.1 100 14.4 37.9 16.6 8.9 17.3 5, 1 18, 9 4.3 23, 4 4.8 24, 8 5, 1 29.0 10.4

Table XII. Powder X-ray diffraction pattern of compound 1.HC1.HCOOH (Fig. 9)

Angle (20°) Intensity (%) 7.1 100 14.4 76.4 14.8 17.1 16, 4 25, 1 17.4 42.8 18.6 20.6 20, 8 13.1 23.4 14.1 24.5 16, 1 24.9 13.4 29.0 39, 8

Example 5 Stability Test for Compound 1.HC1.3H ₂ O

5.1. Accelerated Stability Test

5.1.1. Protocol

Samples of compound 1.HC1.3H ₂ O were stored under conditions for evaluating chemical stability and physical stability as described in Table 1. XIII.

Table XIII. Conditions for determining stability

Conditions for determining chemical stability Conditions for determining physical stability 25°C/60% RH/Open recipient Room temperature/<5% RH/Open receptor 40°C/75% RH/Open recipient Room temperature/56% RH/Open receptor 50°C/Closed recipient RT/75%RH/Open receptor

Samples were taken at T0, then every month for three months and analyzed by FT-IR, DSC and XRPD.

5.2. Long term stability test

5.2.1. Protocol

Test compound samples were stored under chemical stability and physical stability conditions as described in Table 1 below. XIV:

Table XIV. Stability test conditions

Model Temperature (°C) / relative humidity (%) / recipient Cold conditions 5°С/_/Closed recipient Long term storage 25°C/60% RH/Open recipient Intermediate conditions 30°C/65% RH/Open recipient Accelerated Conditions 40°C/75% RH/Open recipient

- 39 041895

Samples were taken at T0, then every month for 12 months, then at time points 18, 24 and 36 months; and each of the aliquots was then analyzed by HPLC, Karl Fischer titration, FT-IR, DSC and XRPD.

5.3. GSP analysis of compound 1.HC1.3H2O

The GSP analysis was performed to evaluate the stability of the 1.HC1.3H2O compound and is shown in FIG. 6. The evolution of matter was tracked using XPRD at different points in time.

At elevated relative humidity, no change in shape was observed. Compound 1.HC1.3H2O underwent dehydration to anhydrous compound l.HCl when heated above 60° C. or at a relative humidity of less than 10%. Rehydration to compound 1.HC1.3H2O occurred upon cooling to ambient temperature or under the influence of a relative humidity of 20% or more.

5.3.1. conclusions

Surprisingly, Compound 1.HC1.3H2O can undergo dehydration to compound l.HCl, but is converted back to the more stable trihydrate form of compound 1.HC1.3H2O under normal conditions. This could not be predicted by a person skilled in the art.

Biological examples

Example 6 In Vitro Assays

6.1. JAKl kinase inhibition assay

The recombinant human JAKl kinase catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAKl kinase were incubated with 12.5 µg of polyGT substrate (Sigma, catalog number P0275) in kinase reaction buffer (final concentrations: 15 mM Tris-HCl pH 7.5, l mM DTT, 0.01% Tween- 20, 10 mM MgCl ₂ , 2 μM non-radioactive ATP), 0.25 μCi P3R-ΑΤΦ (GE Healthcare, catalog number AH9968)) with or without 5 μl containing test compound or vehicle (DMSO, 1% final concentration) , in a total volume of 25 µl, in a polypropylene 9b-well plate (Greiner, V-shaped bottom). After 45 minutes at 30° C., the reactions are stopped by adding 25 μl/well of 150 mM phosphoric acid. All stopped kinase reactions are transferred to pre-washed (75 mM phosphoric acid solution) 96-well filter plates (Perkin Elmer, cat. no. 6005177) using a cell harvester (Perkin Elmer). The plates are washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 µl/well of Microscint-20 scintillation fluid is added, the top of the plates is sealed and reading is performed using a Topcount scintillation counter (Perkin Elmer). Kinase activity is calculated by subtracting the counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from the counts per minute (cpm) obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as follows:

Inhibition expressed in %=((cpm determined from sample in presence of test compound - cpm determined from sample with positive control inhibitor)/(cpm determined in presence of vehicle - cpm determined from sample with positive control inhibitor))-100 %.

Serial dilutions are made to obtain compound doses that can be tested to determine dose-response effects in the JAKl kinase assay and calculate the IC ₅₀ for each compound. Each compound is typically tested at a concentration of 20 µM followed by a serial dilution of 1/3.8 points (20 µM - 6.67 µM - 2.22 µM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM) at the final concentration DMSO 1%. As activity increases, serial dilutions of the compound make further dilutions and/or lower the upper concentration (eg, 5 μM, 1 μM).

The following compounds were tested for their activity against JAKl kinase and the IC ₅₀ values determined in the assays described are shown in Table 1 below. XV.

Table XV. IC ₅₀ values for compounds with respect to JAKl kinase

G# Connection No. JAK1 1C ₅ o (nM) G146034 1 47, 07, 55, 66, 50, 1, 48.29

6.2. Analysis of the determination of Ki JAKl

To determine the Ki value, various amounts of the compound are mixed with the enzyme and then an enzymatic reaction follows as a function of ATP concentration. Ki is determined using a double inverse plot of Km versus compound concentration (Lineweaver and Burke plot). The assay uses 1 ng JAKl kinase (Invitrogen, PV4774). The substrate is 50 nM Ulight-JAK-l (Tyr1023) Peptide (Perkin Elmer, TRF0121). The reaction was carried out in 25 mM MOPS pH 6.8, 0.01%, 2 mM DTT, 5 mM MgCl ₂ Brij-35, with various concentrations of ATP and compound. Phosphorylated substrate is measured using Eu-labeled anti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068). Data reading was performed on an Envision instrument (Perkin Elmer) with excitation at 320 nm and emission at 615 and 665 nm.

- 40 041895

For example, when Compound 1 was tested in this assay, the Ki measured was nM.

6.3. JAK2 inhibition assay

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 honey. JAK2 is incubated with 2.5 μg of polyGT substrate (Sigma, catalog number P0275) in kinase reaction buffer (final concentrations: 5 mM MOPS (3-N-morpholine propane sulfonic acid) pH 7.5, 9 mM MgAc, 0.3 mM EDTA , 0.06% Brij (non-ionic surfactant) and 0.6 mM DTT, 1 µM non-radioactive ATP, 0.25 µCi P3P-ΑΤΦ (GE Healthcare, catalog number AH9968)) with or without 5 µl containing test compound or vehicle (DMSO, final concentration 1%), in a total volume of 25 μl, in a polypropylene 96-well plate (Greiner, V-shaped bottom). After 90 minutes at 30° C., the reactions are stopped by adding 25 μl/well of 150 mM phosphoric acid. All stopped kinase reactions are transferred to pre-washed (75 mM phosphoric acid solution) 96-well filter plates (Perkin Elmer, cat. no. 6005177) using a cell harvester (Perkin Elmer). The plates are washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 µl/well of Microscint-20 scintillation fluid is added, the top of the plates is sealed and reading is performed using a Topcount scintillation counter (Perkin Elmer). Kinase activity is calculated by subtracting the counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from the counts per minute (cpm) obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as follows:

Inhibition expressed in %=((cpm determined from sample in presence of test compound - cpm determined from sample with positive control inhibitor)/(cpm determined in presence of vehicle - cpm determined from sample with positive control inhibitor))-100 %.

Serial dilutions are made to obtain compound doses that can be tested to determine dose-response effects in the JAK2 kinase assay and calculate the IC ₅₀ for each compound. Each compound is typically tested at a concentration of 20 µM followed by a serial dilution of 1/3.8 points (20 µM - 6.67 µM - 2.22 µM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM) at the final concentration DMSO 1%. As activity increases, serial dilutions of the compound make further dilutions and/or lower the upper concentration (eg, 5 μM, 1 μM).

The following compounds were tested for their activity against JAK2 kinase and the IC ₅₀ values determined in the assays described are shown in Table 1 below. XVI.

Table XVI. IC ₅₀ values for compounds in relation to JAK2

G# Connection No. JAK2 1C ₅ o (nM) G146034 1 31.37, 41.16, 55.49, 167.34

6.4. Analysis of Kd determination of JAK2

JAK2 (Invitrogen, PV4210) is used at a final concentration of 5 nM. Binding assays were performed in 50 mM Hepes pH 7.5, 0.01% Brij-35, 10 mM MgCl ₂ , 1 mM EGTA using 25 nM kinase tag 236 (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) with varying compound concentrations. The detection of the label is carried out in accordance with the procedure specified by the manufacturer.

For example, when compound 1 was tested in this assay, the measured K _d value was 205 nM.

6.5. JAK3 inhibition assay

Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalog number PV3855) was purchased from Invitrogen. 0.025 honey. JAK3 kinases are incubated with 2.5 µg of polyGT substrate (Sigma, catalog number P0275) in kinase reaction buffer (final concentrations: 25 mM Tris-HCl pH 7.5, 0.5 mM EGTA (ethylene glycol tetraacetic acid), 0, 5 mM Na3VO4, 5 mM β-glyceryl phosphate, 0.01% Triton X-100, 1 µM non-radioactive ATP, 0.25 µCi P3P-γ-ΑΤΦ (GE Healthcare, cat. no. AH9968)) with or without 5 µl containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 µl, in a polypropylene 96-well plate (Greiner, V-shaped bottom). After 105 min at 30° C., the reactions are stopped by adding 25 μl/well of 150 mM phosphoric acid. All stopped kinase reactions are transferred to pre-washed (75 mM phosphoric acid solution) 96-well filter plates (Perkin Elmer, cat. no. 6005177) using a cell harvester (Perkin Elmer). The plates are washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 µl/well of Microscint-20 scintillation fluid is added, the top of the plates is sealed and reading is performed using a Topcount scintillation counter (Perkin Elmer). Kinase activity is calculated by subtracting the number of pulses per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM

- 41 041895 staurosporine), from the number of pulses per minute (cpm), obtained in the presence of a carrier. The ability of a test compound to inhibit this activity is determined as follows:

Inhibition expressed in %=((cpm determined for sample in presence of test compound - cpm determined for sample with positive control inhibitor)/(av determined in presence of vehicle - cpm determined for sample with positive control inhibitor)) P00% .

Serial dilutions are made to obtain compound doses that can be tested to determine dose-response effects in the JAK3 assay and calculate the IC ₅₀ for each compound. Each compound is typically tested at a concentration of 20 µM followed by a serial dilution of 1/3.8 points (20 µM - 6.67 µM - 2.22 µM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM) at the final DMSO concentration 1%. As activity increases, serial dilutions of the compound make further dilutions and/or lower the upper concentration (eg, 5 μM, 1 μM).

The following compounds were tested for their activity against JAK3 and the IC ₅₀ values determined in the assays described are shown in Table 1 below. XVII.

Table XVII. IC ₅₀ values for compounds in relation to JAK3

G# Connection No. JAK3 1C ₅ o (nM) G146034 1 149.35, 187.3, 189.3, 194.7

6.6. Analysis of determining the value of Ki for JAK3-kinase

To determine the Ki value, various amounts of the compound are mixed with the enzyme and then an enzymatic reaction follows as a function of ATP concentration. Ki is determined using a double inverse plot of Km versus compound concentration (Lineweaver and Burke plot). JAK3 (Carna Biosciences, 09CBS-0625B) was used at a final concentration of 10 ng/mL. The substrate is Poly(Glu, Tyr) sodium salt (4:1), MW 20000-50000 (Sigma, P0275). The reaction is carried out in 25 mM Tris pH 7.5, 0.01% Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na ₃ VO ₄ , 5 mM bglycerol phosphate, 10 mM MgCl ₂ with variation concentrations of ATP and compounds and stop the reaction by adding 150 mm phosphoric acid. The measurement of phosphate incorporation into the polyGT substrate is carried out by loading samples into a filter plate (using a harvester, Perkin Elmer) and subsequent washing. Incorporated 33P in polyGT was measured using a Topcount scintillation counter after adding scintillation fluid to filter plates (Perkin Elmer).

For example, when Compound 1 was tested in this assay, the measured Ki value was 353 nM.

6.7. TYK2 inhibition assay

The catalytic domain of recombinant human TYK2-(amino acids 871-1187; catalog number 08-147) was purchased from Carna Biosciences. 5 ng of TYK2 was incubated with 12.5 µg of polyGT substrate (Sigma, cat. no. P0275) in kinase reaction buffer (final concentrations: 25 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) pH 7.5, 100 mM NaCl, 0.2 mM Na3VO4, 0.1% NP-40, 0.1 µM non-radioactive ATP, 0.125 µCi 33P-y-ATP (GE Healthcare, catalog number AH9968)) with or without 5 µl containing test compound or vehicle (DMSO, final concentration 1%), in a total volume of 25 μl, in a polypropylene 96-well plate (Greiner, V-shaped bottom). After 90 minutes at 30° C., the reactions are stopped by adding 25 μl/well of 150 mM phosphoric acid. All stopped kinase reactions are transferred to pre-washed (75 mM phosphoric acid solution) 96-well filter plates (Perkin Elmer, cat. no. 6005177) using a cell harvester (Perkin Elmer). The plates are washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 µl/well of Microscint-20 scintillation fluid is added, the top of the plates is sealed and reading is performed using a Topcount scintillation counter (Perkin Elmer). Kinase activity is calculated by subtracting the counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from the counts per minute (cpm) obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as follows:

Inhibition expressed in %=((cpm determined from sample in presence of test compound - cpm determined from sample with positive control inhibitor)/(cpm determined in presence of vehicle - cpm determined from sample with positive control inhibitor)) 100 %.

Serial dilutions are made to obtain compound doses that can be tested to determine dose-response effects in the TYK2 assay and calculate the IC ₅₀ for each compound. Each compound is typically tested at a concentration of 20 µM followed by a serial dilution of 1/3.8 points (20 µM - 6.67 µM - 2.22 µM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM) at the final concentration DMSO 1%. As activity increases, serial dilutions of the compound make further dilutions and/or lower the upper concentration (eg, 5 μM, 1 μM).

The following compounds were tested for their activity against TYK2; and IC ₅₀ values,

- 42 041895 defined in the described analyzes are presented below in table. XVIII.

Table XVIII. IC ₅₀ values for compounds in relation to TYK2

G# Connection No. TYK2 IC ₅ o (nM) G146034 1 72.7, 73.75, 79.07, 86.77

6.8. Analysis of determining the Kd value for TYK2 kinase

TYK2 kinase (Carna Biosciences, 09CBS-0983D) was used at a final concentration of 5 nM. The binding assay was performed in 50 mM Hepes pH 7.5, 0.01% Brij-35, 10 mM MgCl ₂ , 1 mM EGTA using 25 nM 236 kinase label (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen , PV5594) with varying compound concentrations. The detection of the label is carried out in accordance with the procedure specified by the manufacturer.

For example, when compound 1 was tested in this assay, the measured Kd value was 376 nM.

Example 7 Cellular Assays

7.1. JAK-STAT signal transmission analysis.

Hela cells are maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Hela cells are used at 70% confluence for transfection. 20,000 cells in 87 µl cell culture medium are transiently transfected with 40 ng pSTAT1(2) luciferase reporter (Panomics), 8 ng LacZ reporter as internal control reporter, and 52 ng pBSK using 0.32 µl Jet-PEI (Polyplus) as transfection reagent per well in 9b-well plate format. After an overnight incubation at 37°C 10% CO ₂ the transfection medium is removed. Add 75 µl DMEM + 1.5% heat-inactivated fetal calf serum. 15 µl of the compound at a concentration of 6.7 x was added over 60 minutes followed by 10 µl of human OSM (cytokine oncostatin) (Peprotech) at a final concentration of 33 ng/ml.

All compounds were tested in duplicate starting at 20 µM followed by a 1/3 serial dilution for a total of 8 doses (20 µM - 6.6 µM - 2.2 µM - 740 nM - 250 nM - 82 nM - 27 nM - 9 nM ) at a final DMSO concentration of 0.2%.

After overnight incubation at 37° C. 10% CO _{2 ,} cells are lysed in 100 μl of lysis buffer/well (PBS (phosphate buffered saline), 0.9 mM CaCl 2 , 0.5 mM MgCl 2 , 5% trehalose, 0.025% Tergitol NP9, 0.15% BSA (bovine serum albumin)).

µl cell lysate is used to read β-galactosidase activity by adding 180 µl eGal solution (30 µl ONPG (o-nitrophenyl-β-D-galactopyranoside) at 4 mg/ml + 150 µl β-galactosidase buffer (0.06 M Na ₂ HPO ₄ , 0.04 M NaH ₂ PO ₄ , 1 mM MgCl ₂ )) for 20 min. The reaction is stopped by adding 50 µl of Na ₂ CO ₃ (1M). Absorption is read at a wavelength of 405 nm.

Luciferase activity is measured using 40 μl of cell lysate plus 40 μl of Steadylite® as described by the manufacturer (Perkin Elmer) on an Envision instrument (Perkin Elmer).

As a positive control (100% inhibition), 10 μM of panJAK inhibitor is used. 0.5% DMSO (0% inhibition) was used as a negative control. The positive control and negative control are used to determine numerical values (z) and percent inhibition (PIN).

Inhibition expressed as a percentage = ((fluorescence intensity determined in the presence of a carrier, fluorescence intensity determined on a sample in the presence of a test compound)/(fluorescence intensity, determined in the presence of a carrier, fluorescence intensity determined on a sample without a trigger)) 100%.

The PIN values are plotted for compounds tested in dose-response, the EC ₅₀ values are obtained, which are presented in Table 1 below. XIX.

Table XIX. JAK-STAT EU ₅₀ values

G# Connection No. ECbo (nM) G146034 1 922.5, 625.6, 987.7, 1767

7.2. OSM/IL-1e signal transmission analysis

OSM and IL-1e have been shown to synergistically increase levels of MMP13 metalloproteinase in the human chondrosarcoma cell line SW1353. Cells are seeded in 96-well plates at 15,000 cells/well in a volume of 120 µl of DMEM (Dulbecco's Modified Eagle's Medium, Invitrogen) containing 10% (v/v) FBS (fetal bovine serum) and 1% penicillin/streptomycin (Invitrogen ), incubated at 37°C, 5% CO2. Cells are pre-incubated with 15 µl of the compound in M199 medium with 2% DMSO 1 hour before activation with 15 µl of OSM and IL-1e to achieve an OSM concentration of 25 ng/ml and an IL-1e concentration of 1 ng/ml, and MMP13 levels are measured in conditioned medium 48 hours after activation. MMP13 activity is measured using an antibody capture activity assay. For this purpose, 384-well plates (NUNC, 460518, Max

- 43 041895 iSorb black) are coated with 35 µl of 1.5 µg/ml anti-human MMP13 (R&D Systems, MAB511) for 24 hours at 4°C. After washing the wells 2 times with PBS (phosphate buffered saline) + 0.05% Tween, the remaining binding sites are blocked with 100 µl of 5% non-fat dry milk (Santa Cruz, sc-2325, Blotto) in PBS for 24 h at 4°C. The wells are then washed 2 times with PBS + 0.05% Tween and 35 µl of a 1/10 dilution of culture supernatant containing MMP13 in 100-fold diluted blocking buffer is added and incubated for 4 hours at room temperature. The wells are then washed twice with PBS + 0.05% Tween followed by MMP13 activation by adding 35 µl of 1.5 mM 4-aminophenylmercury acetate (APMA) solution (Sigma, A9563) and incubating at 37°C for 1 hour. washed again with PBS + 0.05% Tween and 35 µl of MMP13 substrate (Biomol, P-126, OmniMMP fluorogenic substrate) is added. After incubation for 24 hours at 37° C., the fluorescence intensity of the converted substrate is measured on a Perkin Elmer Wallac Envision 2102 Multilabel Reader (excitation at wavelength: 320 nm, emission at wavelength: 405 nm).

Inhibition expressed as a percentage = ((fluorescence intensity determined in the presence of a carrier, fluorescence intensity determined on a sample in the presence of a test compound)/(fluorescence intensity, determined in the presence of a carrier, fluorescence intensity determined on a sample without a trigger)) 100%.

For example, when Compound 1 was tested in this assay, the measured EC ₅₀ was 2242.5 (±1098.5) nM.

7.3. PBL proliferation assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 and proliferation is measured using a BrdU (bromodeoxyuridine) incorporation assay. PBL is first stimulated for 72 hours with PHA (phytohemagglutinin) to induce IL-2 receptor, fixed for 24 hours to stop cell proliferation, followed by IL-2 stimulation for a further 72 hours (including 24 hours of BrdU labeling). Cells are pre-incubated with test compounds for 1 hour before adding IL-2. Cells are cultured in RPMI 1640 medium containing 10% (v/v) FBS.

Example 8 In Vivo Assays

8.1. Pharmacokinetic (PK)/Pharmacodynamic (PD) testing

8.1.1. Bioavailability Assay in Dogs

8.1.1.1. Experiment preparation

The purpose of this experiment is to compare PK in healthy male beagle dogs (3 dogs per group) after a single oral administration of Compound 1 or Compound 1.HCl.3H ₂ O formulated in capsules of two different dosages (25 and 100 mg).

Dogs were not fasted prior to compound administration and had free access to water. Each day of treatment, a half ration was provided after T0 blood draw, 8-17 min before treatment, and the other half ration was given immediately after administration or 1 hour after treatment for a period of 2. A washout period of 3 days was provided between treatments.

Compound 1 or compound 1.HCl.3H ₂ O was administered to a target dose of 10 mg/kg capsules (either 4x25 mg or 1x100 mg capsule). The composition of the capsules is described in table. XX below.

Table XX. Composition of capsules 25 and 100 mg

Component 25 mg capsule 100 mg capsule Compound 1 25.325 mg 101.3 mg Acdisol 4 mg 4 mg Aerosil 1 mg 1 mg Avicel 243.1 mg 71 mg magnesium stearate 1 mg 1 mg

The capsules were administered orally with water (5-10 ml) to ensure rapid passage through the esophagus. Each animal was monitored at least once a day.

Blood was taken from the jugular vein and collected in lithium heparin tubes at T0 (before meals) and then at 1, 2, 4, 6, 8, 10 and 24 hours after treatment.

Plasma was then obtained from blood by centrifugation (2500 g for 10 min at 4°C) and stored at -20°C until used in the assay.

8.1.1.2 Plasma analysis

Representative aliquots of plasma were diluted with canine control plasma, if necessary, to provide concentrations in the range of the calibration curve and extracted by protein precipitation with 2 volumes of acidified (0.1% formic acid) acetonitrile containing deuterated Compound 1 as internal standard (at 150 ng/ml ).

After vortexing and centrifugation at 4°C, the supernatants were diluted with 0.5 volumes of HPLC water in Eppendorf midi tubes in a 96-well plate. The plate was sealed and shaken to ensure sample homogeneity prior to analysis. Samples were analyzed for compound 1 by LC-MS/MS using a Waters TQS mass spectrometer against a range of matrix matched calibration and quality control standards.

The Waters TQS method includes a standard curve range of 100 ng/mL (lower limit of quantitation for undiluted samples) to a maximum of 4000 ng/mL for Compound 1.

Pharmacokinetic analysis was performed using the WinNonlin™ program version 5.3 using concentrations from individual animals. Non-compartmental analysis was used to determine PK parameters (C _max , T _max , AUC0-last, t _V 2 etc...)

Concentrations below detection limits were set to 0 for descriptive statistics and calculation of PK parameters.

The actual doses of Compound 1 administered to each dog were used to dose-normalize PK parameters (C _max and AUC). the results are presented in table. XXI below and in FIG. 10.

Table XXI. Results of bioavailability analysis in dogs

Connection form free base HC1 trihydrate Dose (single oral administration) 4x25 mg/kg 100 mg/kg 4x25 mg/kg 100 mg/kg Submission AUC (µg.hr/mL) 10.4 11.1 33, 6 21.7 Tmax (hour) 6.0 8.0 2.0 2.0 Stax (mcg/ml) 0.894 0.797 3, 05 2, 63 Τι/g (hour) Range 4.43-8.96

Compound 1 (as the free base) is administered orally and therefore passes through the HCl-containing acidic gastric environment where compound 1.HCl must be formed. Therefore, one skilled in the art would not expect any difference between the two forms administered.

However, as illustrated in FIG. 10, on average and at 2 doses in capsule form, Compound 1.HC1.3H ₂ O is more rapidly absorbed and exhibits better in vivo activity than Compound 1, resulting in reduced doses administered and thus better patient compliance. treatment regimen and potentially reduce toxicity or drug interaction problems.

Summary

Those skilled in the art will appreciate that the above description is illustrative and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will be able to determine obvious modifications and changes that can be made without departing from the spirit of the present invention. All such modifications are within the scope of the appended claims and are intended to be included therein. Thus, the invention is to be defined not by the description above, but by the following claims and their equivalents.

All publications, including, but not limited to, patents and patent applications, which are referenced in this application, are incorporated into this application by reference, as if each individual publication were specifically and individually indicated as being incorporated into this application by reference throughout completeness.

It should be understood that factors such as the differential cellular penetration capacity of various compounds can lead to a discrepancy between the activity of the compounds in in vitro biochemical and cellular assays.

At least some of the chemical names of the salts of the present invention, as indicated and described in this application, can be obtained automatically using a commercially available chemical naming program, and they have not been independently verified. Representative programs that perform this function include the Lexichem naming tool from Open Eye Software, Inc. and Autonom Software tool from MDL, Inc. In the event that the indicated chemical name and the presented structure differ, the presented structure takes precedence.

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Constantinescu, S. N., Girardot, M., & Pecquet, C. (2007). Mining for JAK-STAT mutations in cancer. Trends in Biochemical Sciences, 33(3), 122-131.

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Claims (8)

paradigm of IL-6: from basic science to medicine. Arthritis Res, 4 (suppl 3), S233-S242. O'Sullivan, L. A., Liongue, 0., Lewis, R. S., Stephenson, S. E., & Ward, A. C. (2007) . Cytokine receptor signaling through the Jak-Stat-Socs pathway in disease. Mol Immuno, 44(10), 2497-506. O'Dell, J. R. (2004). Therapeutic Strategies for Rheumatoid Arthritis. N Engl J Med, 350, 2591-602. Punwani, N., Scherle, P., Flores, R., Shi, J., Liang, J., Yeleswaram, S., et al. (2012). Preliminary clinical activity of a topical JAK1/2 inhibitor in the treatment of psoriasis. J Am Acad Dermatol, 67, 658-64. Smolen, J. S., & Steiner, G. (2003). Therapeutic strategies for rheumatoid arthritis. Nat Rev: Drug Disc, 2, 473-488. Tam, L., McGlynn, L. M., Traynor, P., Mukherjee, R., Bartlett, J. M., & Edwards, J. (2007). Expression levels of the JAK/STAT pathway in the transition from hormone-sensitive to hormone-refractory prostate cancer. British Journal of Cancer, 97, 378-383. Vainchenker, W., Dusa, A., & Constantinescu, S. N. (2008). JAKs in pathology: Role of Janus kinases in hematopoietic malignancies and immunodeficiencies. Seminars in Cell & Developmental Biology, 19, 385-393. Verstovsek, S. (2009). Therapeutic potential of JAK2 inhibitors. Hematology Am Soc Hematol Educ Program, 636-42. Xiang, Z., Zhao, Y., Mitaksov, V., Fremont, D. H., Kasai, Y., Molitoris, A., et al. (2008). Identification of somatic JAKI mutations in patients with acute myeloid leukemia. Blood, 111, 4809-4812. Zenz, R., Eferl, R., Florin, L., Hummerich, L., Mehic, D., Scheuch, H., et al. (2005). Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins. Nature, 437(7057), 369-75. Zhang, Q. (1996). Activation of JAK/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome. Proc. Natl. Acad. Sci., 93, 9148-9153. Zikherman, J., & Weiss, A. (2011). Unraveling the functional implications of GWAS: how T cell protein tyrosine phosphatase drives autoimmune disease. J Clin Invest, 121(12}, 4618-21. CLAIM 1. A pharmaceutically acceptable salt of a compound of formula (I) where the salt is a free base/HCl/H ₂ O 1:1:3 addition product in crystalline form, which is characterized by at least a diffraction peak in the powder x-ray pattern at any one or more of the following positions: 7.25, 8.31, 8.78, 10.69, 11.96, 12.25, 13.17, 13.74, 16.23, 16.71, 19.26, 19.64, 20.15, 20.57, 20, 95, 21.38, 21.69, 22.77, 23.37, 25.14, 25.71, 25.89, 27.66, 28.57, 28.81, 29.17, 29.58, 30.63, 30.94, 32.06, 32.63, 34.14, 36.22, 36.67 and 37.12° 2Θ ±0.2° 2θ. 2. A pharmaceutically acceptable salt according to claim 1, which is characterized by the following diffraction parameters: - 47 041895 Molecular formula C ₂₁ H ₂₄ N ₅ O ₃ S . Cl. ₃ (H ₂ O) Molecular mass 516.02 crystalline system Monoclinic Symmetry group of the crystal lattice R21/P A 13.1388(4) A A 102.089 (2)° b 8.9437(3) A β c 21, 6376(9) A At V 2486, 24(15) A ³ Z 4 De 1.379 mg / m ³ μ 0.2 84 mm ^-1 Source Μο-Κα, 0.71073 A F(000) 1088 T 120(2) K Crystal colorless prism, 0.39x0.16x0.12 mm Θ range for data collection 2.982-27.483° completeness 99.3% Reflections 21028 Unique reflections 5649 R-int 0.0307 3. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to claim 1 or 2 and a pharmaceutically acceptable carrier, excipient or diluent. 4. Pharmaceutical composition according to claim 3, including an additional therapeutic agent. 5. The use of a pharmaceutically acceptable salt according to claim 1 or 2 or a pharmaceutical composition according to claim 3 or 4 for the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with destruction and/or violation cartilage homeostasis, congenital cartilage defects and/or diseases associated with hypersecretion of IL6 or interferons. 6. Use according to claim 5, wherein the pharmaceutically acceptable salt according to claim 1 or 2 or the pharmaceutical composition according to claim 3 or 4 is administered in combination with another therapeutic agent. 7. The pharmaceutical composition according to claim 4 or the use according to claim 6, where the additional therapeutic agent is an agent for the prevention and/or treatment of inflammatory diseases, autoimmune diseases, proliferative diseases, allergies, transplant rejection, diseases associated with destruction and/or violation of cartilage homeostasis, congenital cartilage defects and / or diseases associated with hypersecretion of IL6 or interferons. 8. A method for obtaining a pharmaceutically acceptable salt according to claim 1 in solid crystalline form, including the steps: i) mixing the compound of formula (I) suspended in DCM with MeOH at 35°C, ii) adding water with stirring at 35°C for at least 15 min, iii) separating the organic layer, iv) adding 10 wt. ./wt. HCl solution in MeOH to the organic layer obtained in the previous step iii), v) isolating the solid obtained in the previous step iv) by filtration, vi) drying the solid obtained in the previous step v), vii) adding the solid obtained in the previous step vi) to a solution of 1.6/0.4 formic acid/water with stirring at 55°C, viii) adding water to the solution obtained in the previous step vii), ix) isolating by filtration the resulting solid obtained in the previous step viii), and x) drying the solid obtained in the previous step ix). -