EA045143B1 - METHODS AND INTERMEDIATES FOR PREPARATION OF JAK INHIBITOR - Google Patents

Description

This application claims priority to US Provisional Application No. 61/773,659, filed March 6, 2013, which is incorporated herein by reference in its entirety.

Field of technology

This invention relates to methods for preparing a compound of formula I:

which is useful in the treatment of diseases associated with Janus kinase (JAK) activity, including inflammatory diseases, autoimmune diseases, cancer and other diseases.

State of the art

Protein kinases (PKs) regulate various biological processes, including, but not limited to, cell growth, survival and differentiation, organ formation, morphogenesis, neovascularization, tissue repair and regeneration. In addition, protein kinases play a special role in a huge number of human diseases, including cancer. Cytokines, small molecule polypeptides or glycoproteins, regulate multiple pathways involved in the host inflammatory response to sepsis. Cytokines influence cell differentiation, proliferation and activation, and can modulate pro-inflammatory and anti-inflammatory responses, allowing the recipient to respond appropriately to pathogenic factors. Signal transduction of a wide range of cytokines includes the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and activators of transcription (STATs). There are four known mammalian JAKs: JAK1 (Janus kinase 1), JAK2, JAK3 (also known as Janus kinase, leukocyte; JAKL and L-JAK), and TYK2 (protein tyrosine kinase 2).

Cytokine-stimulated immune and inflammatory responses contribute to disease pathogenesis: pathologies such as severe combined immunodeficiency (SCID) that result from immune system suppression, while an overactive or inappropriate immune/inflammatory response contributes to the pathology of autoimmune diseases (eg, asthma, systemic red lupus, thyroiditis, myocarditis), and diseases such as scleroderma and osteoarthritis (Ortmann, R. A., T. Cheng, et al., (2000) Arthritis Res 2(1): 16-32).

Insufficient expression of JAK is associated with many pathological conditions. For example, Jak1-/- mice are born dwarfed, unable to suckle, and die during the perinatal period (Rodig, S. J., M. A. Meraz, et al., (1998) Cell 93(3): 373-83). Embryos of Jak2-/- mice are anemic and die around 12.5 days after coitus due to the lack of developed erythropoiesis.

The JAK/STAT pathway, and in particular all four JAKs, is believed to play a role in the pathogenesis of the asthmatic response, chronic obstructive pulmonary disease, bronchitis and other associated inflammatory diseases of the lower respiratory tract. A variety of cytokines that signal through JAK are associated with inflammatory diseases/conditions of the upper respiratory tract, such as those affecting the nose and paranasal sinuses (eg, rhinitis and sinusitis), whether they are classic allergic reactions or not. The JAK/STAT metabolic pathway is also involved in inflammatory eye diseases/conditions and chronic allergic reactions.

Activation of JAK/STAT in cancers can occur by stimulation of cytokines (eg, IL-6 or GM-CSF) or by reduction of endogenous suppressors of JAK signaling, such as SOCS (suppressor or cytokine signaling) or PIAS (protein inhibitor of activated STAT) (Boudny, V., and Kovarik, J., Neoplasm 49:349-355, 2002). Activation of STAT signaling, as well as other metabolic pathways downstream of JAKs (eg, Akt), has been correlated with poor prognosis in many types of cancer (Bowman, T., et al. Oncogene 19:2474-2488, 2000). Elevated levels of cytokines in the bloodstream that signal through JAK/STAT play a critical role in cachexia and/or chronic fatigue. As such, JAK inhibition may be beneficial for cancer patients due to additional enhancement of potential antitumor activity.

JAK2 tyrosine kinase may be beneficial for patients with myeloproliferative diseases, such as polycythemia vera (PV), idiopathic thrombocythemia (IT), myeloid metaplasia with myelofibrosis MMM (Levin, et al., Cancer Cell, vol. 7, 2005: 387-397) . Kinase inhibition

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JAK2V617F reduces hematopoietic cell proliferation, suggesting JAK2 as a potential target for pharmacological inhibition in patients with PV, IT, and MMM.

Inhibition of JAKs may benefit patients suffering from cutaneous immune disorders such as psoriasis, and skin sensitization. The maintenance of psoriasis is believed to depend on a number of inflammatory cytokines in addition to various chemokines and growth factors (JCI, 113:16641675), many of which signal through JAKs (Adv Pharmacol. 2000; 47:113-74).

JAK1 plays a central role in a number of cytokine and growth factor signaling pathways that, when dysregulated, can lead to, or contribute to, disease states. For example, IL-6 levels increase in rheumatoid arthritis, a disease in which it is believed to have detrimental effects (Fonesca, J.E. et al., Autoimmunity Reviews, 8:538-42, 2009). Because IL-6 signals, at least in part, through JAK1, antagonizing IL-6 directly or indirectly through inhibition of JAK1 is expected to provide beneficial clinical benefits (Guschin, D., N., et al. Embo J 14: 1421, 1995; Smolen, J. S., et al. Lancet 371:987, 2008). Additionally, in some cancers, JAK1 has mutated as a result, resulting in undesirable tumor cell growth and survival (Mullighan CG, Proc Natl Acad Sci U S A.106:9414-8, 2009; Flex E., et al. J Exp Med. 205:751-8, 2008). In other autoimmune diseases and cancer, systemic levels of inflammatory cytokines that activate JAK1 are increased, which may also contribute to the disease and/or associated symptoms. Therefore, patients with such diseases may benefit from JAK1 inhibition. Selective JAK1 inhibitors may be effective as long as the unnecessary and potentially undesirable effects of inhibition of other JAK kinases can be avoided.

Selective JAK1 inhibitors related to other JAK kinases may have several therapeutic advantages over less selective inhibitors. With regard to selectivity for JAK2, a number of important cytokines and growth factors signal through JAK2, including, for example, erythropoietin (Epo) and thrombopoietin (Tro) (Parganas E, et al. Cell. 93:385-95, 1998 ). Ero is a key growth factor for the production of red blood cells; therefore, a lack of EPO-dependent signaling can lead to a reduction in the number of red blood cells and anemia (Kaushansky K, NEJM 354:2034-45, 2006). Tro, another example of growth factor-dependent JAK2, plays a central role in controlling the proliferation and maturation of megakaryocytes, the cells from which platelets are produced (Kaushansky K, NEJM 354:2034-45, 2006). Thus, decreased Tro signaling will decrease the number of megakaryocytes (megakaryocytopenia) and reduce the number of circulating platelets (thrombocytopenia). This may result in unwanted and/or uncontrollable bleeding. Reduced inhibition of other JAKs, such as JAK3 and Tyk2, may also be desirable, since individuals lacking a functional version of these kinases have been shown to suffer from numerous diseases such as severe combined immunodeficiency or hyperimmunoglobulin E syndrome (Minegishi, Y , et al. Immunity 25:74555, 2006; Macchi P, et al. Nature 377:65-8, 1995). Therefore, a JAK1 inhibitor with reduced affinity for other JAKs would have significant advantages over a less selective inhibitor in reducing side effects, including immune suppression, anemia, and thrombocytopenia.

Due to the utility of JAK inhibitors, there is a need to develop new methods for producing JAK inhibitors. The present invention addresses this and other needs.

The essence of the invention

JAK inhibitors are described in US Pat. No. 2011/0224190, which is incorporated herein by reference in its entirety, including {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}- 3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, which is depicted below as Formula I.

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The present invention relates to a process for preparing a compound of formula I:

involving the interaction of a compound of formula VIII:

with a compound of formula IXa:

in the presence of a coupling agent to obtain a compound of formula VIIa:

and the reaction of a compound of formula VIIa with a compound of formula IVa:

under Suzuki coupling reaction conditions to produce a compound of formula I, wherein the Suzuki coupling reaction conditions include heating a reaction mixture containing a compound of formula VIIa, a compound of formula IVa, a Suzuki coupling reaction catalyst, a base, and a second solvent component.

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Detailed description of the invention

The methods described herein can be tested in accordance with any suitable method known in the art. For example, product formation can be monitored using spectroscopic techniques such as nuclear magnetic resonance spectroscopy (eg, 1H or ^13C ), infrared spectroscopy, or spectrophotometry (eg, UV-vis); or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) or other related techniques.

As used herein, the term contacting is used as is known in the art and generally refers to bringing reactants into contact in such a way as to allow them to interact at the molecular level to achieve a chemical or physical transformation. In some embodiments, the contacting involves two reagents, wherein one or more equivalents of the second reagent are used relative to the first reagent. The contacting steps of the methods described herein can be carried out at a time and under conditions suitable to obtain the identified product.

The preparation of compounds may involve protecting and deprotecting various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, Greene, et al., Protective Groups in Organic Synthesis, 4d. Ed., Wiley & Sons, 2007, which is incorporated herein by reference in its entirety. The selection of protecting groups and methods for their formation and cleavage described herein can be adjusted as necessary to accommodate different substituents.

The reactions of the methods described herein can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with the starting materials (reagents), intermediates, and intermediates at the temperatures at which the reactions occur, for example, temperatures that may range from the freezing point of the solvent to the boiling point of the solvent. Any selected reaction may be carried out in a single solvent or in a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for the particular reaction step may be selected. In some embodiments, the reactions can be carried out in the absence of a solvent, for example, when at least one of the reactants is a liquid or a gas.

Suitable solvents may include halogenated solvents such as carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane, tetrachlorethylene, trichlorethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1 ,1-dichloroethane, 2-chloropropane, α,α,α-trifluorotoluene, 1,2-dichloroethane, 1,2-dibromoethane, hexafluorobenzene, 1,2,4-trichlorobenzene, 1,2-dichlorobenzene, chlorobenzene, fluorobenzene, their mixtures and the like.

Suitable ether solvents include: dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, tert-butyl methyl ether , mixtures thereof, etc.

Suitable protic solvents may include, by way of example and without limitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2propanol, 2-methoxyethanol, 1-butanol , 2-butanol, isobutyl alcohol, tert-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, 3- or pentanol, neopentyl alcohol, tert-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol or glycerin.

Suitable aprotic solvents may include, by way of example and without limitation, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl3,4,5,6-tetrahydro- 2(1H)-pyrimidinone (DMPU, 1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP, formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethyl sulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate, sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane, nitrobenzene or hexamethylphosphoramide.

Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o- or p-xylene, octane, indane, nonane, or naphthalene.

The reactions of the methods described herein can be performed at appropriate temperatures, which can be readily determined by one skilled in the art. The reaction temperature will depend on, for example, the melting and boiling points of the reactants and the solvent, if present; reaction thermodynamics (for example, highly exothermic reactions may need to be carried out at low temperatures); and reaction kinetics (eg, elevated temperatures may be required to overcome a high activation energy barrier). Elevated temperature refers to temperatures above room temperature (about 22°C).

The reactions of the methods described herein can be carried out in air or in an inert atmosphere. In general, reactions that contain reagents or products that react significantly with air can be carried out using air-sensitive synthesis techniques that are well known to those skilled in the art.

When preparing compounds according to the methods described herein, conventional isolation and purification procedures, such as concentration, filtration, traction, solid phase extraction, recrystallization, chromatography, etc., can be used to isolate the product to be recovered.

In some embodiments, the compounds described herein, and their salts, are substantially isolated. By substantially isolated is meant that the compound is at least partially or substantially separated from the environment in which it is formed or detected. The partial separation may include, for example, a composition enriched with a compound of this invention. Substantial separation may include compositions that contain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of a compound of this invention, or a salt thereof. Methods for isolating the compounds and their salts are common in the art.

Methods for preparing certain intermediates can be found in US Provisional Patent Application No. 61/531896, filed September 7, 2011, US Patent Application No. 12/687623, filed January 14, 2010, and US Patent Application No. 13/043986, filed March 9, 2011, each of which is incorporated herein by reference in its entirety.

Methods and intermediates

The present invention provides methods for preparing a compound of Formula I. Accordingly, in one aspect, the present invention provides a method comprising:

interaction of a compound of formula VIII:

with a compound of formula IXa:

in the presence of a coupling agent to obtain a compound of formula VIIa:

and the interaction of a compound of formula Vila with a compound of formula IVa:

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Cl

IVa under Suzuki coupling reaction conditions to produce a compound of formula I, wherein the Suzuki coupling reaction conditions include heating a reaction mixture containing a compound of formula VIIa, a compound of formula IVa, a Suzuki coupling reaction catalyst, a base, and a second solvent component.

In some embodiments, the coupling agent is 1,8diazabicyclo[5,4,0]undecene.

In some embodiments, the interaction of a compound of formula VIII with a compound of formula IXa:

(a) carried out in a solvent component containing acetonitrile; and (b) carried out in a solvent component containing acetonitrile at a temperature of from 40 to 60°C.

In some embodiments, 1 to 1.2 equivalents of a compound of formula IXa are used based on a compound of formula VIII.

In some embodiments, the Suzuki coupling reaction catalyst is tetrakis(triphenylphosphine)palladium(0).

In some embodiments, the base is sodium bicarbonate.

In some embodiments, sodium bicarbonate is present in an amount of 4 or more equivalents based on the compound of Formula VIIa.

In some embodiments, the second solvent component contains 1,4dioxane and water.

In some embodiments, 1,4-dioxane and water are present in a 1:1 volume ratio.

In some embodiments, the compounds of formulas VIIa and IVa are present in a 1:1 molar ratio.

In some embodiments, the Suzuki coupling reaction conditions include heating a reaction mixture containing a compound of formula VIIa, a compound of formula IVa, tetrakis(triphenylphosphine)palladium(0), sodium bicarbonate, and a second solvent component, and wherein the second solvent component includes water and 1,4 -dioxane.

The Suzuki reaction in the methods described herein can be initiated using a number of different known Suzuki catalysts, including palladium(0) and palladium(II) based catalysts, and carried out under conditions known in the art (see, for example, Miyaura and Suzuki, Chem. Rev. 1995, 95, 2457-2483, which is incorporated herein in its entirety).

Application.

Compound of formula I, {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)- 1 H-pyrazol-1 -yl] azetidin-3 -yl } acetonitrile, is a JAK inhibitor (e.g. JAK1, JAK2). JAK inhibitors are used in the treatment of various JAK-associated diseases or disorders. Examples of JAK-associated diseases include diseases associated with the immune system, including, for example, organ transplant rejection (eg, allograft rejection and graft-versus-host disease). Additional examples of JAK-associated diseases include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, myasthenia gravis, immunoglobulin nephropathy, myocarditis, autoimmune thyroid diseases, chronic obstructive pulmonary disease (COPD), and the like. In some embodiments, the autoimmune diseases are bullous autoimmune skin diseases such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Additional examples of JAK-associated diseases include allergic conditions such as asthma, food allergies, eczematous dermatitis, contact dermatitis, atopic dermatitis (eczema) and rhinitis.

Additional examples of JAK-associated diseases include viral diseases such as Epstein-Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, varicella zoster virus (VZV), and human papillomavirus (HPV).

Additional examples of JAK diseases include diseases associated with cartilage turnover, for example, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze's syndrome, costal arthropathy, endemic deforming osteoarthritis, Mseleni's disease, Handigodu's disease, fibromyalgia which leads to

- 6 045143 degeneration, systemic lupus erythematosus, scleroderma, or ankylosing spondylitis.

Additional examples of JAK-associated diseases include congenital cartilage malformations, including hereditary chondrolysis, chondrodysplasia, and osteochondrodysplasia (eg, microtia, anotia, and metaphyseal chondrodysplasia).

Additional examples of JAK-associated diseases or conditions include skin diseases such as psoriasis (eg, psoriasis), atopic dermatitis, skin rash, skin irritation, skin sensitization (eg, contact dermatitis or allergic contact dermatitis). For example, certain substances, including some medications, may cause skin irritation when applied topically. In some embodiments, coadministration or sequential administration of at least one JAK inhibitor according to the invention with an agent that causes unwanted sensitization may be useful in treating such unwanted sensitization or dermatitis. In some embodiments, the skin disease is treated by topical administration of at least one JAK inhibitor of the invention.

Additional examples of JAK-associated diseases or conditions include those characterized by solid tumors (eg, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer , glioblastomas, Kaposi's sarcoma, Castleman's disease, uterine leiomyosarcoma, melanoma, etc.), hematological cancer (eg, lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or multiple myeloma), cancer skin and, for example, cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma. Example CTCLs include Sezary syndrome and mycosis fungoides. Other examples of JAK-associated diseases or conditions include pulmonary arterial hypertension.

Other examples of JAK-associated diseases or conditions include cancers associated with inflammation. In some embodiments, the cancer is associated with inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis. In some embodiments, the inflammatory bowel disease is Crohn's disease. In some embodiments, the inflammation-associated cancer is a colitis-associated cancer. In some embodiments, the cancer associated with inflammation is colon cancer or colorectal cancer. In some embodiments, the cancer is gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), adenocarcinoma, small bowel cancer, or rectal cancer.

JAK-associated diseases may further include diseases characterized by the expression of: JAK2 mutants, such as those that have at least one mutation in the pseudo-kinase region (eg, JAK2V617F); JAK2 mutants having at least one mutation outside the pseudo-kinase region; JAK1 mutants; JAK3 mutants; erythropoietin receptor (Epor) mutants; or dysregulated expression of CRLF2.

JAK-associated diseases may additionally include myeloproliferative disorders (PDS), such as polycythemia vera (PV), ether thrombocythemia (ET), myelofibrosis with myeloid metaplasia (MMM), primary myelofibrosis (PMI), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), systemic mastocytosis (SMCD), and the like. In some embodiments, the myeloproliferative disorder is myelofibrosis (eg, primary myelofibrosis (PMF) or post-polycythemia vera/major thrombocythemia myelofibrosis (Post-PV/Post-ET MF)). In some embodiments, the myeloproliferative disorder is post-core thrombocythemia myelofibrosis (Post-ET MF). In some embodiments, the myeloproliferative disorder is post-polycythemia vera myelofibrosis (Post-PV MF).

Other examples of JAK-associated diseases or conditions include the relief of dermatological side effects from other drugs by administering a compound of this invention. For example, numerous pharmaceutical agents lead to unwanted allergic reactions, which may manifest as acne or are associated with dermatitis. Examples of pharmaceutical agents that have such unwanted side effects include anti-cancer drugs such as gefitinib, cetuximab, erlotinib and the like. The compounds of this invention can be administered systemically or locally (eg, localized to the dermatitis) in combination with (eg, simultaneously or sequentially) a pharmaceutical agent that has unwanted dermatological side effects. In some embodiments, the compounds of this invention may be administered topically along with one or more pharmaceuticals where other pharmaceuticals, when administered systemically in the absence of a compound of this invention, cause contact dermatitis, allergic contact sensitization, or similar skin conditions. Respectively,

- 7 045143 compositions of this invention include present compositions that contain the compounds of this invention and additionally a pharmaceutical agent that can cause dermatitis, skin diseases or have associated side effects.

Additionally, JAK-associated diseases include inflammation and inflammatory diseases. Examples of inflammatory diseases include sarcoidosis, inflammatory diseases of the eye (eg, iritis, uveitis, scleritis, conjunctivitis, or similar diseases), inflammatory diseases of the respiratory tract (eg, upper respiratory tract, including the nose and sinuses, such as rhinitis or sinusitis, or lower respiratory tract including bronchitis, chronic obstructive pulmonary disease, and the like), inflammatory myopathy such as myocarditis, and other inflammatory diseases. In some embodiments, the inflammatory eye disease is blepharitis.

Additionally, JAK-associated diseases include ischemia-reperfusion injury or disease or condition related to inflammatory ischemic events such as stroke or cardiac arrest, endotoxin-driven disease states (eg, complications after bypass surgery or chronic endotoxin conditions contributing to chronic heart failure), anorexia, cachexia, fatigue that results from or is associated with cancer, restenosis, sclerodermatitis, fibrosis, conditions associated with hypoxia or astrogliosis, such as, for example, diabetic retinopathy, cancer, or neurodegeneration and other inflammatory diseases such as systemic inflammatory syndrome response (SIRS) and septic shock.

Other JAK-associated diseases include gout and enlarged prostate due to, for example, benign prostatic hypertrophy or benign prostatic hyperplasia, as well as bone resorption diseases such as osteoporosis or osteoarthritis, bone resorption diseases associated with: hormonal imbalance and/or hormonal therapy, autoimmune diseases (eg, sarcoidosis of the bone), or cancer (eg, myeloma).

Additionally, JAK-associated diseases include dry eye disorders. As used herein, dry eye disorder encompasses the disease conditions summarized in the recent white paper of the Dry Eye Workshop (DSE), which defined dry eye as a multifactorial disease of the tears and ocular surface, resulting in symptoms of discomfort, visual disturbances, and instability of the tear film with the potential for damage to the ocular surface. This is accompanied by an increase in tear film osmolarity and inflammation of the ocular surface. Lemp, The Definition and Classification of Dry Eye Disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop, The Ocular Surface, 5(2), 75-92 April 2007, which is incorporated in its entirety herein by links. In some embodiments, the dry eye disorder is selected from aqueous deficiency dry eye (ADD) or evaporative dry eye disorder, or corresponding combinations thereof. In some embodiments, the dry eye disorder is Sjögren's dry eye syndrome (SDS). In some embodiments, the dry eye disorder is non-Sjögren's dry eye syndrome (NSDS).

Additionally, JAK-associated diseases include conjunctivitis, uveitis (including chronic uveitis), chorioditis, retinitis, cyclitis, scleritis, episcleritis, or iritis. Other JAK-associated illnesses include respiratory dysfunction or failure associated with viral infections such as influenza and colds.

Examples

The present invention will be described in more detail with the help of specific examples. The following examples are presented for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily understand various non-critical parameters that can be changed or modified to achieve substantially the same results.

Example 1. Synthesis of 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2 (trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile adipate (9)

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Scheme I

tert-Butyl 3-(cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-1- carboxylate (3).

Isopropanol (IPA, 200 mL), 1,8-diazabicyclo[5,4,0]undecene (DBU, 9.8 g, 64.4 mmol) were added sequentially to a 1 L flask equipped with a nitrogen inlet, thermocouple, and mechanical stirrer. , 0.125 eq), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.101 g, 520.51 mmol, 1.01 eq) and tert-butyl-3-(cyanomethylene)azetidine-1-carboxylate (2.100 g, 514.85 mmol) at room temperature to generate the reaction mixture as a suspension. The resulting reaction mixture was heated at reflux for 30 minutes to obtain a homogeneous solution, and the mixture was kept at reflux for an additional 2-3 hours. After the reaction was completed, monitoring was carried out by HPLC, n-heptane (400 ml) was gradually added to the reaction mixture over 45 min while maintaining the mixture at reflux. The dry residue precipitated during the addition of n-heptane. After the addition of n-heptane was completed, the mixture was gradually cooled to room temperature and stirred at ambient temperature for an additional 1 hour. The solid residue was collected by filtration, washed with n-heptane (200 ml), and dried under vacuum at 50°C to equilibrate with nitrogen to constant weight to give tert-butyl-3-(cyanomethyl)-3-(4-( 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-1-carboxylate (3, 181 g, 199.9 g theoretical, 90, 5%) as a white to pale yellow solid. For 3: Ή NMR (400 MHz, DMSO- _d6 ) δ 8.31 (s 1H), 7.74 (s, 1H), 4.45-4.23 (m, 2H), 4.23-4 .03 (m, 2H), 3.56 (s, 2H), 1.38 (s, 9H), 1.25 (s, 12H) ppm; ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 155.34, 145.50, 135.88, 116.88, 107.08 (br), 83.15, 79.36, 58.74 (br) , 56.28, 27.96, 26.59, 24.63 ppm; C19H29BN4O4 (Mol. weight 388.27), HPMS (EI) m/e 389 (M++H).

tert-Butyl-3-(4-(7H-pyrrolo[2,3-d]pyrumidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)azetidine-1carboxylate (5). 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine (4, 39.6 g, 257.6 mmol), tert-butyl-3 (cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin-1-carboxylate (3, 100 g, 257.6 mmol, 1.0 eq), cesium fluoride (136.9 g, 901.4 mmol, 3.5 eq), tert-butanol (250 ml), water (250 ml), and [1, 1'-bis(di-cyclohexylphosphino)ferrocene] dichloropalladium (II) (Pd-127, 351.4 mg, 0.46 mmol, 0.0018 equiv) at room temperature. The resulting reaction mixture was degassed and flushed with nitrogen 3 times before being heated to reflux under nitrogen for 20-24 hours. When HPLC indicated the reaction was complete, the reaction mixture was cooled to 45-55°C. After 30 min, separation into two phases occurred and the aqueous phase was discarded. n-heptane (125 ml) was added to the organic phase over 30 min at 4555°C. The resulting mixture was slowly cooled to ambient temperature over one hour and stirred at ambient temperature for an additional 2 hours. The solid residue was collected by filtration, washed with n-heptane (100 ml), and dried under vacuum at 50°C to equilibrate with nitrogen to constant weight to obtain tert-butyl-3-(4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(cyanomethyl)-azetidin-1- carboxylate (5, 96.8 g, 97.7 g theoretical, 99%) as a pale yellow solid. For 5: 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.89 (s, 1H), 8.68 (s, 1H), 8.44 (s, 1H), 7.60 (d, J=3, 5 Hz, 1H), 7.06 (d, J=3.6 Hz, 1H), 4.62-4.41 (m, 2H), 4.31-4.12 (m, 2H), 3.67 ( s, 2H), 1.39 (s, 9H) ppm; ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 155.40, 152.60, 150.63, 149.15, 139.76, 129.53, 127.65, 122.25, 116.92, 113 .21, 99.71, 79.45, 58.34 (US), 56.80, 27.99, 26.83 ppm; C19H21N7O2

- 9 045143 (Mol. weight 379.4), VZhMS (EI) m/e 380 (M++H).

2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1 -yl)azetidin-3-yl)acetonitrile dihydrochloride (6). Tert-butyl-3-(4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H was added to a 0.5-liter flask equipped with a nitrogen inlet, a thermocouple, an additional funnel, and a mechanical stirrer -pyrazol-1-yl)-3-(cyanomethyl)azetidine-1-carboxylate (5.15 g, 39.5 mmol), water (7.5 ml, 416 mmol) and dichloromethane (75 ml) at room temperature. The mixture was stirred at ambient temperature until a suspension was obtained. A solution of 5 M hydrogen chloride (HCl) in isopropanol (55 mL, 275 mmol, 7.0 eq) was added to the suspension over 5 min. The resulting reaction mixture was heated to low reflux and kept at reflux for 3-4 hours. After the reaction was complete, as monitored by HPLC, tert-butyl methyl ether (TBME, 45 ml) was added to the reaction suspension. The mixture was gradually cooled to room temperature and stirred for another hour. The solid residue was collected by filtration, washed with tert-butyl methyl ether (TBME, 45 ml) and dried under vacuum at 50°C to equilibrate with nitrogen to constant weight to obtain 2-(3-(4-(7H-pyrrolo[2,3d) dihydrochloride salt ]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile (6, 13.6 g, 13.9 g theoretical, 98%) as an off-white to light yellow solid substances. For 6: 1H NMR (400 MHz, D ₂ O) δ 8.96 (s, 1H), 8.81 (s, 1H), 8.49 (s, 1H), 7.78 (d, J=3 .8 Hz, 1H), 7.09 (d, J=3.7 Hz, 1H), 4.93 (d, J=12.8 Hz, 2H), 4.74 (d, J=12.5 Hz, 2H), 3.74 (s, 2H) ppm; ¹³ C NMR (101 MHz, D2O) δ 151.35, 143.75, 143.33, 141.33, 132.03, 131.97, 115.90, 114.54, 113.85, 103.18, 59.72, 54.45 (2C), 27.02 ppm; C ₁₄ H ₁₅ Cl ₂ N ₇ (C ₁₄ H ₁₃ N ₇ for free base, mass 279.30), HPLM (EI) m/e 280 (M ⁺ +H).

2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1 -(3-fluoro-2-(trifluoromethyl )isonicotinoyl)pipyridin-4-yl)azetidin-3-yl)acetonitrile (8, Free Base). 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl) dihydrochloride salt was added to a 0.5-liter flask equipped with a nitrogen inlet, a thermocouple, an additional funnel, and a mechanical stirrer. )-1Hpyrazol-1-yl)azetidin-3-yl)acetonitrile (6.20 g, 5 6.78 mmol), dichloromethane (200 ml) and triethylamine (TEA, 16.62 ml, 119.2 mmol, 2. 1 equiv) at room temperature. The mixture was stirred at ambient temperature for 30 minutes before 1-(3-fluoro-2-(trifluoromethyl)-isoisonicotinoyl)piperidin-4one (7, 17.15 g, 57.91 mmol, 1.02 eq) was added to the mixture. The mixture was then treated with sodium triacetoxyborohydride (25.34 g, 113.6 mmol, 2.0 eq) for 5 min at ambient temperature (below 26°C). The resulting reaction mixture was stirred at ambient temperature for two hours. After the reaction was complete, as monitored by HPLC, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (200 ml). The two phases were separated and the aqueous phase was extracted with methylene chloride (200 ml). The combined organic phase was washed with 4% brine (100 ml) followed by changing the solvent from methylene chloride to acetone by distillation. The resulting solution of the desired crude product (8) in acetone was used directly for the subsequent preparation of the adipic acid salt. A small portion of the solution was purified by column chromatography (SiO ₂ , 0-10% MeOH in EtOAc gradient elution) to give analytically pure 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl )-1 H-pyrazol-1 -yl)-1-(1 -(3-fluoro-2(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (8 free base) as almost white solid matter. For 8: 1H NMR (400 MHz, DMSO-d ₆ ) δ 12.17 (d, J=2.8 Hz, 1H), 8.85 (s, 1H), 8.70 (m, 2H), 8 .45 (s, 1H), 7.93 (t, J=4.7 Hz, 1H), 7.63 (dd, J=3.6, 2.3 Hz, 1H), 7.09 (dd, J=3.6, 1.7 Hz, 1H), 4.10 (m, 1H), 3.78 (d, J=7.9 Hz, 2H), 3.61 (t, J=7.9 Hz, 1H), 3.58 (s, 2H), 3.46 (m, 1H), 3.28 (t, J=10.5 Hz, 1H), 3.09 (ddd, J=13.2 , 9.5, 3.1 Hz, 1H), 2.58 (m, 1H), 1.83-1.75 (m, 1H), 1.70-1.63 (m, 1H), 1, 35-1.21 (m, 2H) ppm; ¹³ C NMR (101 MHz, DMSO-d^) δ 160.28, (153.51, 150.86), 152.20, 150.94, 149.62, (146.30, 146.25), 139 ,48, (134.78, 134.61), (135.04, 134.92, 134.72, 134.60, 134.38, 134.26, 134.03, 133.92), 129.22 , 127.62, 126.84, 121.99, 122.04, (124.77, 122.02, 119.19, 116.52), 117.39, 113.00, 99.99, 61.47 , 60.49, 57.05, 44.23, 28.62, 27.88, 27.19 ppm; C ₂₆ H ₂₃ F ₄ N ₉ O (Mol. weight 553.51), HPMC (EI) m/e 554.1 (M ⁺ +H). 2-(3-(4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1 -(3-fluoro-2-(trifluoromethyl)isonicotinoyl )piperidin-4-yl)azetidin-3-yl)acetonitrile adipate (9). A solution of crude 2(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 1 H-pyrazol-1 -yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (8 free base, 31.38 g, 56.7 mmol) in acetone (220 ml) and adipic acid (8.7 g, 59.53 mmol, 1.05 eq) at room temperature. The reaction mixture was then refluxed to obtain a solution, n-Heptane (220 ml) was gradually added to the reaction mixture at 40-50°C over one hour. The resulting mixture was gradually cooled to room temperature over one hour and stirred at ambient temperature for an additional 16 hours. The solid residue was collected by filtration, washed with n-heptane (2x60 ml), and dried under vacuum at 50°C to equilibrate with nitrogen to constant weight to obtain 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro- 2(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile adipate (9, 34.0 g, 39.7 g theoretical, 85.6% for two steps) as a white to off-white solid substances. 9: ^1H NMR

- 10 045143 (400 MHz, DMSOC) δ 12.16 (s, 1H), 12.05 (ush, 2H), 8.85 (s, 1H), 8.72 (s, 1H), 8.69 ( d, J=4.7 Hz, 1H), 8.45 (s, 1H), 7.93 (t, J=4.7 Hz, 1H), 7.63 (dd, J=3.6, 2 ,3 Hz, 1H), 7.09 (dd, J=3.6, 1.7 Hz, 1H), δ 4.11 (dt, J=11.0, 4.4 Hz, 1H), 3, 77 (d, J=7.8 Hz, 2H), 3.60 (t, J=7.8 Hz, 2H), 3.58 (s, 2H), 3.44 (dt, J=14.4 , 4.6 Hz, 1H), 3.28 (t, J=10.4 Hz, 1H), 3.09 (ddd, J=13.2, 9.6, 3.2 Hz, 1H), 2 .58 (tt, J=8.6, 3.5 Hz, 1H), 2.28-2.17 (m, 4H), 1.83-1.74 (m, 1H), 1.67 (d , J=11.0 Hz, 1H), 1.59-1.46 (m, 4H), 1.37-1.21 (m, 2H) ppm; ¹³ C NMR (101 MHz, DMSO-b ₆ ) δ 174.38, 160.29, (153.52, 150.87), 152.20, 150.94, 149.63, (146.30, 146, 25), 139.48, (134.79, 134.62), (135.08, 134.97, 134.74, 134.62, 134.38, 134.28, 134.04, 133.93) , 129.21, 127.62, 126.84, 122.05, (124.75, 122.02, 119.29, 116.54), 117.39, 113.01, 99.99, 61.47 , 60.50, 57.06, 44.24, 33.42, 30.70, 28.63, 27.89, 27.20, 24.07 ppm; C ₃₂ H ₃₃ F ₄ N ₉ O ₅ (Mol. weight 699.66; C ₂₆ H ₂₃ F ₄ N ₉ O for free base, Mol. weight, 553.51), HPMS (EI) m/e 554.0 (M++N).

Example 2: Alternative synthesis of 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimudin-4-yl)-1H-pyrazol-1-yl)-1(1 -(3-fluoro-2 -(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile.

Scheme II

2-(Azetidin-3-ylidene)acetonitrile hydrochloride (2a). To a 0.5 L flask equipped with a nitrogen inlet, thermocouple and mechanical stirrer, tert-butyl 3(cyanomethylene)azetidine-1-carboxylate (2.30 g, 154.46 mmol) and methylene chloride (300 mL) were added ) at ambient temperature. The solution was then treated with a solution of 5 M hydrogen chloride (HCl) in isopropanol solution (294.2 ml, 1.54 mol, 10 eq) at ambient temperature and the resulting reaction mixture was stirred at ambient temperature for 18 hours. After completion of the reaction, what was determined using HPLC, a suspension of tert-butyl methyl ether (TBME, 150 ml) was added and the mixture was stirred at ambient temperature for 2 hours. The solid residue was collected by filtration, washed with n-heptane (2x100 ml), and dried on a filter funnel at environment for 3 hours to obtain 2-(azetidin-3-ylidene)acetonitrile hydrochloride (2a, 13.7 g, 20.2 g theoretical, 67.8%) as a white solid. For 2a: 1H NMR (500 MHz, DMSO-a ₆ ) δ 9.99 (s, 2H), 5.94 (p, J=2.5 Hz, 1H), 4.85-4.80 (m, 2H), 4.77-4.71 (m, 2H) ppm; ¹³ C NMR (126 MHz, DMSO-a6) δ 155.65, 114.54, 94.78, 55.26, 54.63 ppm; C5H7ClN2 (MW 130.58; C5H6N2 for free base, MW 94.11), HPMC (EI) m/e 95 (M ⁺ +H). 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-ylidene)acetonitrile (10). 2-(azetidin-3ylidene)acetonitrile hydrochloride (2a, 4.5 g, 34.46 mmol), 1-(3-fluoro- 2-(trifluoromethyl)isonicotinoyl)piperidin-4-one (7.10 g, 34.46 mmol, 1.0 eq), and methylene chloride (100 ml) at ambient temperature and the resulting mixture was then treated with sodium triacetoxyborohydride (14.6 g, 68.93 mmol, 2.0 equiv) at ambient temperature. The reaction mixture was stirred at ambient temperature for two hours before quenching with a saturated solution of sodium bicarbonate (NaHCO3) in water (50 ml). The two phases were separated and the aqueous phase was extracted with dichloromethane (200 ml). The combined organic phase was washed with water (50 ml) and brine (50 ml) and concentrated under reduced pressure to give the crude desired product (10), which was purified by column chromatography (SiO ₂ , 0-10% ethyl acetate in hexanes gradient elution) to give 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3

- 11 045143 ylidene)acetonitrile (10, 9.5 g, 12.7 g theoretical, 74.8%) as a white solid. For 10: 1H

NMR (400 MHz, CDCl ₃ ) δ 8.57 (d, J=4.7 Hz, 1H), 7.54 (t, J=4.6 Hz, 1H), 5.29 (p, J=2 .4 Hz, 1H), 4.18-4.08 (m, 1H), 4.08-4.03 (m, 2H), 3.98-3.94 (m, 2H), 3.57- 3.39 (m, 2H), 3.17-3.04 (m, 1H), 2.56 (tt, J=7.4, 3.5 Hz, 1H), 1.86-1.77 ( m, 1H), 1.75-1.64 (m, 1H), 1.54-1.43 (m, 1H), 1.43-1.31 (m, 1H) ppm; ¹³ C NMR (101 MHz, CDCl ₃ ) δ 161.34, 160.73, 152.62 (d, J=269.1 Hz), 145.75 (d, J=6.1 Hz), 136.73 (qd, J=36.1, 12.0 Hz), 134.56 (d, J=16.9 Hz), 126.89, 120.58 (qd, J=275.0, 4, 9 Hz), 115.11, 92.04, 62.05, 60.57 (2C), 44.47, 39.42, 29.38, 28.47 ppm; C ₁₇ H ₁₆ F ₄ N ₄ O (Mol. weight 368.33), HPMC (EI) m/e 369 (M++H).

2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2 -yl)-1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile (11). 4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.210 mg) was added to a 25 mL flask equipped with a nitrogen inlet, thermocouple, and magnetic stirrer. , 1.08 mmol, 1.08 equiv), 2-(1-(1-(3-fluoro-2(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-ylidene)acetonitrile (10, 370 mg, 1.0 mmol) and acetonitrile (3 ml) at ambient temperature. The solution was then treated with 1,8diazabicyclo[5,4,0]undec-ene (DBU, 173 mg, 0.17 ml, 1.12 mmol, 1.12 eq) at ambient temperature and the resulting reaction mixture was heated to 50°C and stirred at 50°C overnight. When the reaction was complete, as determined by HPLC, the reaction mixture was transferred directly to a silica gel column for purification chromatography (0-2.5% MeOH in ethyl acetate gradient elution) to give 2-(1-(1-(3-fluoro-2 -(trifluoromethyl)isonicotinoyl)piperidin-4yl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidin- 3-yl)acetonitrile 11, 263 mg, 562.4 mg theoretical, 46.7%) as a white solid. For 11: 1H NMR (400 MHz,

DMSO-d6) δ 8.64 (d, J=4.7 Hz, 1H), 8.22 (d, J=0.6 Hz, 1H), 7.88 (dd, J=4.7 Hz, 1H), 7.69 (s, 1H), 4.10-3.99 (m, 1H), 3.58 (d, J=7.8 Hz, 2H), 3.52-3.42 (m , 2H), 3.44 (s, 2H), 3.41-3.33 (m, 1H), 3.28-3.15 (m, 1H), 3.03 (ddd, J=12.9 , 9.2, 3.2 Hz, 1H), 2.51-2.44 (m, 1H), 1.77-1.66 (m, 1H), 1.64-1.54 (m, 1H ), 1.28-1.17 (m, 2H), 1.24 (s, 12H) ppm; ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 160.22, 152.13 (d, J=265.8 Hz), 146.23 (d, J=5.7 Hz), 145.12, 135 .41, 134.66 (d, J=16.9 Hz), 134.43 (qd, J=35.0, 11.7 Hz), 127.58, 120.61 (qd, J=274.4 , 4.6 Hz), 117.35, 106.59 (US), 83.10, 61.40, 60.53 (2C), 56.49, 44.17, 38.99, 28.55, 27 .82, 27.02, 24.63 ppm; C ₂₆ H ₃₁ BF ₄ N ₆ O ₃ (Mol. weight 562.37), HPMS (EI) m/e 563 (M ⁺ +H).

2-(3-(4-(7H-pyrrolo[2,3-d]nirimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl) isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (8). 2-(1-(1-(3-fluoro-2(trifluoromethyl)-isonicotinoyl)piperidin-4-yl)-3-(4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1Hpyrazol-1-yl)azetidin-3-yl)acetonitrile (11, 307 mg, 0.546 mmol), 4-chloro -7H-pyrrolo[2,3-d]pyrimidine (4, 84.8 mg, 0.548 mmol, 1.0 eq), sodium bicarbonate (NaHCO ₃ , 229 mg, 2.72 mmol, 5.0 eq), water (1.6 ml), and 1,4-dioxin (1.6 ml) at room temperature. The mixture was then treated with tetrakis(triphenylphosphine)palladium(0) (12.8 mg, 0.011 mmol, 0.02 eq) at ambient temperature and the resulting reaction mixture was degassed and nitrogen filled 3 times before heating to 85°C. The reaction mixture was stirred at 85°C under nitrogen atmosphere overnight. When the reaction was complete, as determined by HPLC, the reaction mixture was concentrated to dryness under reduced pressure to yield the desired product, 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl)-1-(1-(3-fluoro-2(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (8 free base, 135 mg, 302.2 mg theoretical, 44.6%), was obtained as an off-white solid by direct purification on a silica gel (SiO ₂ ) column chromatography (0-10% ethyl acetate in hexane gradient elution) of the dried reaction mixture.

The compound obtained by this synthesis method is identical in all comparable respects to compound 8 obtained using the synthesis method described in Example 1 above.

Example 3. yl)methanone.

Scheme III

Synthesis of (3-fluoro-2-(trifluoromethyl)pyridin-4-yl) (1,4-dioxa-8-azaspiro[4,5]decane-8N HCI aq. NaOH

N N.

12a c ₇ h ₁₄ cino ₂

Mol. weight: 179.64

CF ₃

C7H3F4NO2 Mol. weight: 209.10

VOR, TEA DMF

_{C7H13NO2 _ _}

Mol. weight: 143.18

Mol. mass: 334.27 aq. HCI

CH ₃ CN

Mol. mass: 290.21 (3-fluoro-2-(trifluoromethyl)pyridin-4-yl) (1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (14). A 30L reactor equipped with a mechanical stirrer, dropping funnel and septum was charged with sodium hydroxide (NaOH, 1.4 kg, 35 mol, 2.0 eq) and water (7 L) and the resulting solution was treated with 1,4-dioxa-8

- 12 045143 azaspiro[4.5]decane hydrochloride (3.13 kg, 17.43 mol) at ambient temperature. The resulting mixture was then stirred at ambient temperature for 30 min before being saturated with solid sodium chloride (1.3 kg) and extracted with 2-methyltetrahydrofuran (3x7 L). The combined organic phase was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 1.3 kg) and concentrated under reduced pressure (70 mmHg) at 50°C after removal of the drying reagent, sodium sulfate (Na ₂ SO ₄ ), filtering. The yellow oil thus obtained was distilled under reduced pressure (80 mmHg, boiling point 115 to 120°C) to give 1,4-dioxa-8-azaspiro[4,5]decane (2.34 kg, 2.496 kg theoretical, 93.8%) as a clear oil, which is directly used in the subsequent cross-coupling reaction.

A dry 100 L reactor equipped with a mechanical stirrer, dropping funnel, thermometer and vacuum outlet was charged with 3-fluoro-2-(trifluoromethyl)isonicotinic acid (13, 3.0 kg, 14.35 mol), benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate (BOP reagent, 7.6 kg, 17.2 mol, 1.2 eq), 1,4-dioxa-8-azaspiro[4.5]decane (2.34 kg, 16.36 mol , 1.14 equiv) and N,Ndimethylformamide (DMF, 18 L) at room temperature. The resulting solution was stirred at ambient temperature for 20 min before cooling from 5 to 10°C. Triethylamine (Et ₃ N, 4 L, 28.67 mol, 2.0 eq) was then added to the reaction mixture over 1 hour and the internal temperature was maintained between 5 and 10° C. during the addition of triethylamine. The dark brown solution thus obtained was stirred for 12 hours at ambient temperature (about 20°C) and then cooled to about 10°C. With vigorous stirring, 18 L of saturated aqueous sodium bicarbonate (NaHCO ₃ ) solution and 36 L of water were successively added to the cooled reaction mixture and the internal temperature was maintained at 15°C. The precipitate (filter cake) thus obtained is collected by filtration. The aqueous phase is then saturated with 12 kg of solid sodium chloride (NaCl) and extracted with EtOAc (2x18 L). The combined organic layer was washed successively with saturated sodium bicarbonate (NaHCO ₃ ) aqueous solution (18 L) and water (2x18 L). The collected filter cake was then dissolved back into the organic phase and the resulting dark brown solution was washed with water (2x18 L) and then concentrated under reduced pressure (40-50°C, 30 mmHg) to give approximately 5.0 kg crude target product (14) in the form of a viscous brown oil. The crude product obtained above was then dissolved in EtOH (8.15 L) at 50°C and the resulting solution was treated with water (16.3 L) for 30 minutes at approximately 50°C. The brown solution was collected before being gradually cooled to ambient temperature (about 20°C) over 3 hours with stirring and stirred at ambient temperature for 12 hours. Solids were collected by filtration, washed with a mixture of ethanol and water (EtOH :H ₂ O=1:20, 2 l) and dried under reduced pressure (50 mmHg) at a temperature of approximately 60°C for 24 hours to obtain (3-fluoro-2-(trifluoromethyl) pyridine-4 -yl) (1,4-dioxa-8azaspiro[4,5]decan-8-yl)methanone (14, 3.98 kg, 4.797 kg theoretical, 83.0%) as a white solid. For 14: 1H NMR (300 MHz, DMSO-d ₆ ) δ 8.64 (d, ³ JHH=4.68 Hz, 1H, NCH in pyridine), 7.92 (dd, ³ Jhh=4.68 Hz, ⁴ JHf=4.68 Hz, 1H, NCCH in pyridine), 3.87-3.91 (m, 4H, OCH2CH2O), 3.70 (us s, 2H, one NCH ₂ proton in the piperidine ring, one proton of the other NCH ₂ in the piperidine ring, both in the axial position), 3.26 (t, ³ JHH=5.86 Hz, 2H, one NCH2 proton in the piperidine ring, one proton of the other NCH2 in the piperidine ring, both in the equatorial position), 1 .67 (d, ³ JHH=5.86 Hz, 2H, one NCCH2 proton in the piperidine ring, one proton of the other NCCH2 in the piperidine ring, both in the equatorial position), 1.58 (ush s, 2H, one NCCH2 proton in the piperidine ring , one proton of the other NCCH2 in the piperidine ring, both in axial position) ppm; ¹³ C NMR (75 MHz, DMSO-d6) δ 161.03 (NC=O), 151.16 (d, 1JCF=266.03 Hz, CF), 146.85 (d, ⁴ JCF=4.32 Hz , NCH in pyridine), 135.24 (d, ² Jcf=11.51 Hz, s-s=°), 135.02 (quartet, ² Jcf=34.57 Hz, nccf3), 128.24 (d, ⁴ Jcf=7.48 Hz, in pyridine), 119.43 (dχquartet, %f=274.38 Hz, ³ Jcf=4.89 Hz, CF3), 106.74 (OSO), 64.60 (OSSO) , 45.34 (NC in piperidine ring), 39.62 (NC in piperidine ring), 34.79 (NCC in piperidine ring), 34.10 (NCC in piperidine ring) ppm; ¹⁹ F NMR (282 MHz, DMSO-d6) δ -64.69 (d, ⁴ JFF=15.85 Hz, F3C), -129.26 (dχkquartet, ⁴ JFF=15.85 Hz, ⁴ JFH=3, 96 Hz, FC) ppm; C ₁₄ H ₁₄ F ₄ N ₂ O ₃ (Mol. weight, 334.27), HPMS (EI) t/e 335.1 (M++H).

(3-fluoro-2-(trifluoromethyl)pyridin-4-yl) (1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (7). A 5 L 4 neck round bottom flask equipped with a mechanical stirrer, thermocouple, dropping funnel and nitrogen inlet was charged with (3-fluoro-2-(trifluoromethyl)pyridin-4-yl) (1,4-dioxa-8azaspiro[4,5 ]decan-8-yl)methanone (14, 100 g, 0.299 mol) in acetonitrile (ACN, 400 ml) at ambient temperature. The resulting solution was cooled to below 10°C before treatment with 6.0 N. aqueous hydrochloric acid (HCl) solution (450 ml, 2.70 mol, 9.0 eq) and the internal temperature was maintained below 10°C. The resulting reaction mixture was then gradually warmed to room temperature and an additional amount of 6.0 N. aqueous hydrochloric acid (HCl) solution (1050 mL, 6.30 mol, 21.0 eq) was added slowly to the reaction mixture at ambient temperature over 8 h through a dropping funnel. When the reaction was complete, as monitored by HPLC, the reaction mixture was then cooled to 0°C before being treated with 30%

- 13 045143 aqueous sodium hydroxide solution (NaOH, 860 ml, 8.57 mmol, 28.6 eq) while the internal temperature was maintained below 10°C. The resulting reaction mixture was warmed to room temperature before adding solid sodium bicarbonate (NaHCO ₃ , 85.0 g, 1.01 mol, 3.37 eq) over 1 hour. The mixture was then extracted with ethyl acetate (2x1.2 L) and the combined organic phase was washed with 16% aqueous sodium chloride (2x800 ml) and concentrated to approximately 1.0 L by vacuum distillation. n-Heptane (2.1 L) was added to the residue and the resulting mixture was concentrated to 1.0 L by vacuum distillation. n-heptane (2.1 L) was added to the concentrated mixture. The resulting white suspension was concentrated to 1.0 L by vacuum distillation. Tert-butyl methyl ether (MTBE, 1.94 L) was then added to the white suspension. The white cloudy solution was heated to 40°C to obtain a clear solution. The resulting solution is concentrated to about 1.0 L by vacuum distillation. The mixture was stirred at ambient temperature for 1 hour. The white precipitate was collected by filtration, washed with n-heptane (400 ml) and filter dried under a nitrogen atmosphere with a vacuum pull to give (3-fluoro-2-(trifluoromethyl)pyridin-4yl) (1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (7, 78.3 g, 86.8 g theoretical, 90.2%) as an off-white solid. For 7: ^1H NMR (300 MHz, DMSO-d6) δ 8.68 (d, ³ Jhh=4.69 Hz, 1H, NCH in pyridine), 7.97 (dd, ³ Jhh=4.69 Hz, ⁴ Jhf=4.69 Hz, 1H, NCCH in pyridine), 3.92 (ush s, 2H, one proton NCH2 in the piperidine ring, one proton of the other NCH2 in the piperidine ring, both in axial position), 3.54 (t , ³ Jhh=6.15 Hz, 2H, one NCH2 proton in the piperidine ring, one proton of the other NCH2 in the piperidine ring, both in the equatorial position), 2.48 (t, ³ Jhh=6.44 Hz, 2H, NCCH2) , 2.34 (t, ³ Jhh=6.15 Hz, 2H, NCCH2) ppm; ¹³ C NMR (75 MHz, DMSO-66) δ 207.17 (C=O), 161.66 (NC=O), 151.26 (d, 1JCF=266.89 Hz, CF), 146.90 ( d, ⁴ Jcf=6.05 Hz, NCH in pyridine), 135.56 (C-C=O), 134.78-135.56 (m, NCCF3), 128.27 (d, ³ Jcf=7, 19 Hz, NCCH in pyridine), 119.52 (dhquartet, 1J _CF = 274.38 Hz, ³ Jcf = 4.89 Hz, CF3), 45.10 (NC in piperidine ring) ppm, one carbon ( NCC in the piperidine ring) is absent due to overlap with (CD3)2SO; ¹⁹ F NMR (282 MHz, DMSO-66) δ -64.58 (d, ⁴ Jff=15, 8 5 Hz, F3C), -128.90 (d x quartet, ⁴ Jff=15.85 Hz, ⁴ Jfh =4.05 Hz, FC) ppm; C ₁₂ H ₁₀ F4N ₂ O ₂ (Mol. mass, 290.21), HPMS (EI) t/e 291.1 (M++H).

Example 4. Synthesis of tert-butyl-3-(cyanomethylene)azetidine-1-carboxylate.

Example IV

1-Benzhydrylazetidin-3-ol hydrochloride (16). A solution of diphenylmethanamine (2737 g, 15.0 mol, 1.04 eq) in methanol (MeOH, 6 L) was treated with 2-(chloromethyl)oxirane (1330 g, 14.5 mol) using a dropping funnel at ambient temperature. A small endotherm was observed during the initial addition. The resulting reaction mixture was stirred at ambient temperature for 3 days before being heated to reflux for an additional 3 days. When TLC indicated that the reaction was considered complete, the reaction mixture was first cooled to room ambient and then to 0-5°C in an ice bath. The solids were collected by filtration and washed with acetone (4 L) to obtain a first batch of crude title product (1516 g). The filtrate was concentrated under reduced pressure and the resulting semi-solid was diluted with acetone (1 L). This solid was then collected by filtration to obtain a second portion of crude title product (221 g). The crude product, 1-benzhydryl azetidin-3-ol hydrochloride (1737 g, 3998.7 g theoretical yield 43.4%), was found to be sufficiently pure to be used without further purification in the subsequent reaction. 1H NMR (300 MHz, DMSO-66) δ 12.28 (br.d, 1H), 7.7 (m, 5H), 7.49 (m, 5H), 6.38 (d, 1H), 4 .72 (br.s, 1H), 4.46 (m, 1H), 4.12 (m, 2H), 3.85 (m, 2H) ppm; C ₁₆ H ₁₈ ClNO (Molecular weight 275.77; C ₁₆ H ₁₇ NO for free base, Mol. weight 239.31), HPLM (EI) m/e 240 (M ⁺ +H).

tert-Butyl-3-hydroxyazetidine-1-carboxylate (17). A suspension of 1-Benzhydrylazetidin-3-ol hydrochloride (625 g, 2.27 mol) in a 10% solution of aqueous sodium carbonate (Na2CO3, 5 L) and dichloromethane (CH2Cl2, 5 L) was stirred at ambient temperature until all solids were completely dissolved

- 14 045143 substances. The two layers were separated, and the aqueous layer was extracted with dichloromethane. (CH2C12, 2 l). The combined organic extracts were dried over sodium sulfate (Na ₂ SO ₄ ) and concentrated under reduced pressure. The resulting crude 1-benzhydrylazetidin-3-ol free base was then dissolved in THF (6 L) and the solution was placed in a large Parr bomb. Di-tert-butyl dicarbonate (BOC2O, 545 g, 2.5 mol, 1.1 eq) and 20% palladium (Pd), on carbon (125 g, 50% moisture) were added to the Parr bomb. The flask was charged with 30 psi of hydrogen gas (H ₂ ) and stirred at constant pressure under a hydrogen atmosphere (the flask was charged three times to maintain a pressure of 30 psi) at ambient temperature for 18 hours. When HPLC indicated Once the reaction was complete (no more hydrogen was consumed), the reaction mixture was filtered through a pad of celite and the celite pad was washed with THF (4 L). The filtrates were concentrated under reduced pressure to remove the solvent and the residue was loaded onto a Biotage 150 column with a minimal amount of dichloromethane (CH ₂ Cl ₂ ). The column was eluted with 20-50% ethyl acetate in n-heptane and fractions containing the pure title product, tert-butyl-3-hydroxyazetidine-1-carboxylate, were collected and pooled. Solvents were removed under reduced pressure to give tert-butyl 3-hydroxyazetidine 1-carboxylate (357 g, 393.2 g theoretical yield, 90.8%) as a colorless oil which solidified on standing at ambient temperature under vacuum. ^1H NMR (300 MHz, _CDCl3 ), δ 4.56 (m, 1H), 4.13 (m, 2H), 3.81 (m, 2H), 1.43 (s, 9H) ppm . tert-Butyl-3-oxoazetidine-1-carboxylate (18). A solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (50 g, 289 mmol) in ethyl acetate (400 ml) was cooled to 0°C. The resulting solution was then treated with solid TEMPO (0.5 g, 3.2 mmol, 0.011 eq) and a solution of potassium bromide (KBr, 3.9 g, 33.2 mmol, 0.115 eq) in water (60 ml) at 0-5 °C. While maintaining the reaction temperature between 0-5°C, saturated aqueous sodium bicarbonate (NaHCO ₃ , 450 ml) and aqueous sodium hypochlorite (NaClO, 10-13% active chlorine, 450 ml) were added. After the sodium hypochlorite solution was added, the color of the reaction mixture instantly changed. When additional sodium hypochlorite solution was added, the color of the reaction mixture gradually disappeared. When TLC showed that all starting material had been consumed, the color of the reaction mixture no longer changed. The reaction mixture was then diluted with ethyl acetate (EtOAc, 500 ml) and the two layers were separated. The organic layer was washed with water (500 ml) and saturated aqueous sodium chloride (500 ml) and dried over sodium sulfate (Na ₂ SO ₄ ). The solvent was then removed under reduced pressure to yield the crude product, tert-butyl 3-oxoazetidine-1-carboxylate (48 g, 49.47 g theoretical, 97% yield), which was found to be sufficiently pure and was used directly in the subsequent reaction without further cleaning. 1H NMR ( _CDCl3 , 300 MHz) δ 4.65 (s, 4H), 1.42 (s, 9H) ppm.

tert-Butyl-3-(cyanomethylene)azetidine-1-carboxylate (2).

Diethylcyanomethylphosphate (745 g, 4.20 mol, 1.20 eq) and anhydrous tetrahydrofuran (THF, 9 L) were added to a four-neck flask equipped with a thermowell, addition funnel, and nitrogen protection tube at ambient temperature. The solution was cooled in an ice bath with methanol to -14°C and a 1.0 M solution of potassium tert-butoxide (t-BuOK) in anhydrous tetrahydrofuran (THF, 3.85 L, 3.85 mol, 1.1 eq) was added to for 20 minutes, maintaining the reaction temperature below -5°C. The resulting reaction mixture was stirred for 3 hours at -10°C and a solution of 1-tert-butoxycarbonyl-3azetidinone (600 g, 3.50 mol) in anhydrous tetrahydrofuran (THF, 2 L) was added over 2 hours, maintaining the internal temperature below - 5°C. The reaction mixture was stirred at -5 to -10°C for over an hour and then slowly warmed to room temperature and stirred at ambient temperature overnight. The reaction mixture was then diluted with water (4.5 L) and saturated aqueous sodium chloride (NaCl, 4.5 L) and extracted with ethyl acetate (EtOAc, 2x9 L). The combined organic layers were washed with brine (6 L) and dried over anhydrous sodium sulfate (Na ₂ SO ₄ ). The solvent was removed under reduced pressure and the residue was diluted with dichloromethane (CH ₂ Cl ₂ , 4 L) before it was absorbed onto silica gel (SiO ₂ , 1.5 kg). The crude product, which was absorbed onto silica gel, was purified by flash column chromatography (SiO ₂ , 3.5 Kg, 0-25% EtOAc/hexane gradient elution) to give tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate ( 2, 414.7 g, 679.8 g theoretical, 61% yield) as a white solid. For 2: 1H NMR (300 MHz, CDCl ₃ ) δ 5.40 (m, 1H), 4.70 (m, 2H), 4.61 (m, 2H), 1.46 (s, 9H) m. d.; C ₁₀ H ₁₄ N ₂ O ₂ (Mol. mass, 194.23), HPMS (EI) m/e 217 (M ⁺ +Na).

Example 5. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole.

Example V

env. environment temp. env. pace. env.

Wednesday 20 Wednesday 21 1

C ₃ H ₄ N ₂ C3H3IN2 CgHnlNzSi C9H.15BN2O2

Mol. weight: 68.08 Mol. weight: 193.97 Mol. mass: 266.15 Mol. weight: 194.04

4-iodopyrazole (20). A flask equipped with a nitrogen inlet, dropping funnel, thermowell, and mechanical stirrer was charged with pyrazole (1.450 g, 6.62 mol) and tetrahydrofa

- 15 045143 wounds (THF, 5 l) at ambient temperature. The mixture was then cooled to 10°C and Niodosuccinimide (NIS, 1490 g, 6.62 mol, 1.0 eq) was added to the mixture portionwise as a solid at approximately 10°C. The resulting reaction mixture was then stirred at ambient temperature for 1 hour (longer reaction times may be necessary depending on ambient temperature). The mixture was then filtered and THF was removed under reduced pressure. The residue was suspended in ethyl acetate (6 L) and insoluble materials were filtered off. The dark filtrate was washed successively with saturated aqueous sodium thiosulfate (2x3 L) (the organic layer cleared to pale yellow), water (2x3 L) and brine (2 L). The resulting organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 4-iodopyrazole (1138 g, 1284.1 g theoretical, 88.6%) as a white to pale yellow solid after drying in a vacuum oven at about 30°C overnight. 1H NMR (400 MHz, DMSO-d ₆ ) δ 13.17 (br.s, 111), 7.93 (br.s, 111), 7.55 (br.s, 1H) ppm; C3H3IN2 (Mol.wt, 193.97) HPLM (EI) m/e 195 (M++H).

1-Trimethylsilyl-4-iodopyrazole (21). A flask equipped with a reflux condenser, a nitrogen inlet valve, a mechanical stirrer, and a steam chamber was charged with 4-iodopyrazole (200 g, 1.03 mol) and THF (2 L) at room temperature. Triethylamine (TEA, 158 ml, 1.13 mol, 1.1 equiv) was added to this solution and the resulting solution was cooled to 0° C. in an ice salt bath. To this solution was added chlorotrimethylsilane (TMC-Cl, 137 mL, 1.08 mol, 1.05 eq) with vigorous stirring, allowing the temperature to reach 18°C. (The reaction mixture becomes very thick and difficult to stir, but becomes mobile over time.) When the exothermic process subsided, the cold bath was removed and the reaction mixture was warmed to room temperature. The reaction progress was monitored by GC and was found to be complete after about 1 hour (sampling from the reaction should be done without air when diluted with a dry solvent to prevent TMS hydrolysis). The reaction mixture was dissolved in n-heptane (2 L) before filtering under nitrogen. The solvent was removed from the filtrate under reduced pressure by a vented rotary evaporator under a nitrogen atmosphere. The residual oil was diluted with n-heptane (1 L) and re-concentrated. If solids formed when n-heptane was added, then a second filtration was necessary. The residue was then distilled under reduced pressure (70-90°C about 0.5 Torr) using a Kugelrof still to obtain 1-trimethylsilyl-4-iodopyrazole (263 g, 274.1 g theoretical, 96%) as a colorless oils This material must be stored under nitrogen at all times since the TMS group hydrolyzes rapidly. It was subsequently found that 1trimethylsilyl-4-iodopyrazole could be prepared by heating iodopyrazole with 2 equivalents of hexamethyldisilazane for 1 hour.

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1). A flask equipped with a mechanical stirrer, nitrogen inlet valve, dropping funnel, and thermowell was charged with 1-trimethylsilyl-4-iodopyrazole (225.1 g, 0.85 mol) and THF (2200 mL) at room temperature. The mixture was cooled to about -6°C in an ice salt bath before a solution of isopropyl magnesium chloride in tetrahydrofuran (2 M solution in THF, 510 ml, 1.02 mol, 1.2 eq) was added at such a rate that the internal temperature did not exceed 0° C. The extent of metal/halogen exchange was monitored by GC and the reaction was found to be complete in about 10 minutes. 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (isopropyl pinacolborate, 347 mL, 1.7 mol, 2.0 eq) was then added slowly to the orange-brown solution, initially while maintaining the temperature below 0 °C, and then faster after about half the amount of compound has been added, allowing the temperature to reach 5 °C (the reaction mixture becomes quite viscous and then liquefies slowly). The reaction mixture was then stirred at 0°C for 10 minutes, before it was warmed to room temperature over 1 hour and stirred at ambient temperature for an additional 1 hour. The reaction mixture was cooled to approximately 6°C and saturated aqueous ammonium chloride (NH ₄ Cl, 2.2 L) was added while raising the temperature to 25°C. The mixture was stirred for 5 minutes and then diluted with toluene (10 L). The layers were separated (large amounts of solid present in the aqueous layer) and the organic layer was washed successively with water (6 x 2.2 L) and brine (2 x 2.2 L) and then dried over sodium sulfate (Na ₂ SO ₄ ). The drying agent, sodium sulfate (Na ₂ SO ₄ ), was removed by filtration and the solution was concentrated under reduced pressure. The residual toluene was co-evaporated with n-heptane to give 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazole (1, 90.3 g, 164.9 g theoretical , 54.8%) in the form of a white solid. For 1: ^1H NMR (400 MHz, DMSO- _d6 ) δ 13.8 (br.s, 1H), 7.94 (s, 1H), 7.62 (s, 1H), 1.23 (s , 12H) ppm; C9Hi ₅ BN ₂ O ₂ (Mol. weight, 194.04), VZhMS (EI) m/e 195 (M ⁺ +H).

Example 6 Alternative synthesis of 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole.

- 16 045143

Scheme VI

NBS nV -----►

H ₂ O, temp. 19 approx. environment C ₃ H ₄ N ₂

Mol. weight: 68.08

C ₃ H ₃ BrN ₂ Mol. weight: 146.97 _______o _D J m / ^Br

NS1/CH ₂ C1 ₂ ^Νΐ:/

_{C7HiiBrN2O _}

Mol. mass: 219.08 pV

-О-В V ο-χ /PrMgCI/THF

SchzNgzV^Oz Mol. weight: 266.14

CgHi5BN ₂ O ₂ Mol. weight: 194.04

4-Bromopyrazole (22). Pyrazole (19, 34.0 g, 0.5 mol) and NBS (89.0 g,

0.5 mol, 1.0 equiv) were suspended in water (625 ml) at ambient temperature. The resulting suspension was stirred at ambient temperature overnight. The reaction mixture was then extracted with EtOAc (2x100 ml). The combined EtOAc extracts were washed with aqueous Na ₂ S ₂ O ₃ and brine, dried over Na ₂ SO ₄ , and concentrated under reduced pressure to give crude 4-bromopyrazole (72.0 g, 73.5 g theoretical, 98% yield ) as a white solid (GC purity: >98%), which was directly used in the subsequent reaction without further purification.

4-Bromo-1-(ethoxyethyl)-1H-pyrazole (23). To a solution of 4-bromopyrazole (70.0 g, 0.476 mol) in CH ₂ Cl ₂ (60 0 ml) was added a solution of 3.1 M HCl in dioxane (4 ml) and ethyl vinyl ether (41 g, 0.569 mol, 1. 2 eq) at ambient temperature. The resulting reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was quenched with an aqueous solution of NaHCO ₃ and the two layers were separated. The organic layer was washed with water, dried over Na2SO4, and concentrated under reduced pressure to dryness to give 4-bromo-1-(ethoxyethyl)-N-pyrazole (113 g, 104.3 g theoretical, 97% yield) as an oil (GC grade : 89%), which was directly used in the subsequent reaction without further purification.

1-(Ethoxyethyl)-4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-pyrazole (24). To a 100 mL solution of iPrMgCl.LiCl (50 mmol, 1.8 equiv) in THF was added 4-bromo-1-(ethoxyethyl)-1H-pyrazole (6.15 g, 28 mmol) at ambient temperature. The resulting reaction mixture was stirred at ambient temperature for 12 hours, then cooled to -20°C. Methoxypina coluorate (10.6 g, 67 mmol, 2.4 equiv) was added to the reaction mixture at -20°C. The resulting mixture was stirred at 0-10°C for 1 hour. An aqueous solution of NH ₄ Cl was added to quench the reaction. The mixture was then extracted with petroleum ether (PE). The combined PE extracts were washed with saturated NaHCO ₃ , dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was crystallized from PE to give 1-(ethoxyethyl)-4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)1H-pyrazole (24, 4.2 g, 7 .45 g theoretical, 56.4% yield) as a white-yellowish solid (GC purity: 99%). For 24: ^1H NMR (DMSO^, 400 MHz) 8.09 (s, 1H), 8.58 (s, 1H), 7.62 (s, 1H), 5.55 (q, 1H, J= 6.1 Hz), 3.37 (b.kv., 1H, J=7.l, 9.6 Hz), 3.12 (b.kv., 1H, J=7.0, 9.7 Hz), 1 .56 (d, 3H, J=6, 0 Hz), 1.24 (s, 12H), 1.00 (t, 3H, J=7.0 Hz) ppm; C13H23BN2O3 (Mol. wt. 266.14), HPMS (EI) m/e 267 (M++H).

4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1). To a mixture of 2,3-dimethylbutane-2,3diol (25.0 kg, 211.6 mol) and 1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1Hpyrazole (24, 55.0 kg, 206.7 mol) in 1,2-dichloroethane (750 kg) a solution of HCl in MTBE (25.0 kg, 20-30% HCl) was slowly added at 0 -5°C. The resulting reaction mixture was stirred at 10-20°C for 3-5 hours. After selective deprotection was completed, as monitored by HPLC (1: below 1%), the reaction mixture was degassed and filled with nitrogen, then cooled to -15 °C. Triethylamine (TEA, 30.0 kg, 296.5 mol) was then added to the cooled reaction mixture to adjust the pH to 7-8. The mixture was then gradually warmed to ambient temperature before being treated with water (150 kg). The two phases were separated and the organic layer was washed with brine (60 kg) and dried over sodium sulfate (Na ₂ SO ₄ ). The drying agent, sodium sulfate (Na ₂ SO ₄ ), was removed by filtration and the resulting solution was concentrated under reduced pressure at 40-50°C to a thick oil. The residue was heated to 60-70°C and diluted with petroleum ether (100 kg) at the same temperature. The resulting mixture was then gradually cooled to room temperature and then to -5°C and stirred at the same temperature for 3 hours. The solids were collected by centrifugation and dried at 50-60°C under vacuum to obtain the crude desired product (1, 33 .75 kg, 40.11 kg theoretical, 84.1%). The crude desired product was then suspended in 1,2-dichloroethane (30 kg) and the resulting mixture was refluxed to obtain a clear solution. Petroleum ether (150 kg) was then added to the hot solution at the same temperature. The resulting mixture is then gradually cooled to room temperature, and then to - 5°C and stirred at that

- 17 045143 at the same temperature for 3 hours. The solids were collected by centrifugation and dried under vacuum at 50-60°C to obtain 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2- yl)-1H-pyrazole (1, 31.0 kg,

40.11 kg theoretical, 77.3%) as an off-white solid that is identical in all comparable respects to the material synthesized using the synthesis method described in Example 5 above.

Example 7. Synthesis of 4-chloro-7H-[pyrrolo[2,3-d]pyrimidine.

Scheme VII

POS1 ₃ DMF

C4H4N2O2

Mol. weight: 112.09 reflux

NH ₃ in MeOH toluene, 55 - 60°C

C5H2CI2N2O C ₅ H ₄ CIN ₃ O

Mol. weight: 176.99 Mol. weight: 157.56

Ph ₃ P ⁺ CH ₂ OMe SG *ViOK

THF, 20-25 °C

C ₇ H ₈ CIN ₃ O

Mol. mass: 185.61 conc. aq. HCI

THF, reflux

C ₆ H ₄ CIN ₃

Mol. weight: 153.57

4,6-Dichloropyrimidine-5-carbaldehyde (26). A 5 L 4-neck flask equipped with a mechanical stirrer, dropping funnel, condenser, thermocouple, and N ₂ sweep into an aqueous NaOH wash solution was charged and cooled in an ice salt bath of phosphorus oxychloride (POCl ₃ , 1 L, 10.572 mol, 4.82 eq). N,N-Dimethylformamide (DMF, 320 ml, 4.138 mol, 1.85 eq) was added dropwise to the flask at

0±2°C. After adding approximately 100 ml DMF for about 0.5 h, crystallization occurred and the reaction temperature was increased from 0 to 10°C. The addition was stopped and the mixture was allowed to cool again to approximately 2°C. The residual amount of DMF was added over 2.5 hours at a temperature below 8°C. The suspension became very thick, making stirring difficult. When the addition of DMF was complete, the mixture was stirred at 3-5°C for 0.5 hours. 4,6 Dihydroxypyrimidine (250 g, 2.232 mol) was added portionwise as a solid. After adding about one-third of 4,6-dihydroxypyrimidine, the reaction mixture became thinner and a slow exothermic process was observed with the reaction temperature increasing to approximately 12°C over 0.5 hours. The residual amount of 4,6-dihydroxypyrimidine was added in portions over 0.25 hours. increasing the reaction temperature from 12 to 27°C. The reaction temperature was maintained at 25-27°C with alternating cooling during which the yellow suspension became thinner and then thickened again. After the exothermic process ceased after about 1 hour, the reaction mixture was slowly heated. Around 55°C the reaction mixture became very thick and a second mild exothermic process was observed. The heating grid was removed while the reaction temperature continued to rise to approximately 63°C and remained at this temperature for several minutes before falling. Heating of the mixture was resumed until a slight boil was reached (about 100°C). At about 95°C, there was a constant, fairly rapid evolution of HCl gas and the reaction mixture gradually became liquefied and darkened. After about 0.5 h, a clear brown solution was obtained when the heating temperature was slowly increased to 115°C over 1.25 h. After a total of 2.5 h at reflux, the reaction mixture was cooled to ambient temperature and stirred overnight at ambient temperature. Excess POCl ₃ (as much as possible) was removed under reduced pressure (bath temperature 45-50°C). The residual thick brown oil was poured very slowly into cold H ₂ O (5 L) in a 20 L separatory funnel, adding ice to keep the aqueous mixture around ambient temperature. The aqueous mixture was then extracted with EtOAc (2x3 L followed by 1x2 L). The combined EtOAc extracts were washed with H ₂ O (2x2.5 L), NaHCO ₃ saturated aqueous solution (1 L), concentrated saline (1 L), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure (bath temperature at 35°C) to give crude 4,6-dichloropyrimidine-5-carbaldehyde (270 g, 395 g theoretical, 68.4%) as a yellow-orange solid. A 20 g sample of this crude material was purified by Kugelrohr distillation (oven temperature 90-100°C, 225 mTorr) to yield 15.3 g of pure 4,6-dichloropyrimidine-5-carbaldehyde as a white solid which turned yellow on storage at ambient temperature. ^1H NMR (300 MHz, CDCl ₃ ) 10.46 (s, 1H), 8.89 (s, 1H) ppm.

4-Amino-6-chloropyrimidine-5-carbaldehyde (27). A solution of 7 M NH ₃ in MeOH (265 ml, 1.855 mol, 2.0 eq) was added over 1.25 h to a solution of 4,6-dichloropyrimidine-5-carbaldehyde (163.7 g, 0.9301 mol) in toluene (3 l) at ambient temperature. The temperature of the reaction mixture gradually increased from 20 to 26°C and a yellow suspension was formed. Gentle cooling was used to maintain the temperature of the reaction mixture below 26°C. The suspension was stirred at ambient temperature for 3.5 hours before solids were separated by filtration. Solids

- 18 045143 washed with EtOAc (1 l). The filtrate was concentrated under reduced pressure and the solids were triturated with toluene and n-heptane (2:1 v/v, 600 ml), filtered and dried to give 71.1 g of 4-amino6-chloropyrimidine-5-carbaldehyde as a yellow solid substances. The starting solid was filtered from the reaction mixture, which contained additional 4-amino-6chloropyrimidine-5-carbaldehyde. The product was extracted from the filtered solid by stirring in EtOAc (1.25 L) for 1.5 hours, filtered, then stirred in THF (750 ml) for 1 hour and filtered again. Both the EtOAc and THF filtrates were concentrated under reduced pressure and the resulting solids were triturated with toluene and n-heptane (2:1 v/v, 450 ml), filtered and dried to yield an additional 44.1 g of 4-amino-6-chloropyrimidine- 5-carbaldehyde as a yellow solid. The total yield of 4-amino-6-chloropyrimidine-5-carbaldehyde (115.2 g, 146.5 g theoretical) was 78.6%. ^1H NMR (300 MHz, DMSO- _d6 ) δ 10.23 (s, 1H), 8.71 (br.s, 1H), 8.55 (br.s, 1H), 8.39 (s, 1H) ppm, C5H4QN3O (Mol. weight, 157.56), HPMS (EI) m/e 158 (M++H).

6-Chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine (28). A suspension of (methoxymethyl)triphenylphosphonium chloride (276.0 g, 0.807 mol, 1.1 eq) in THF (1.5 L) was cooled in an ice salt bath to -2°C and 1 M potassium tert-butoxide (KOtBu) in THF ( 807 ml, 0.807 mol, 1.1 equiv) was added over 1.5 h at - 2 to - 3°C. The dark red-orange mixture was stirred at -2 to -3°C for 1 hour. 4-Amino-6chloropyrimidine-5-carbaldehyde (115.2 g, 0.7338 mol, 1.0 eq) was then added to the reaction mixture in portions as a solid using THF (200 ml) to rinse the container and separatory funnel. During addition, the temperature of the reaction mixture increased from -3 to 13°C and the color changed to brown. When the temperature of the reaction mixture dropped to 10°C, the cooling bath was removed and the reaction mixture was allowed to warm to room temperature and stirred at ambient temperature for 42 hours. The reaction mixture was cooled to -2°C before quenching by slowly adding saturated aqueous NH4Cl ( 750 ml). The mixture was concentrated under reduced pressure to remove most of the THF. The residue was partitioned between EtOAc (3 L) and H ₂ O (1 L). The organic phase was filtered to remove insoluble material at the interface, then extracted with 2N. HCl (4x250 ml), then 3 N. HCl (2x250 ml). The combined HCl extracts were again extracted with ethyl acetate (500 ml), then filtered through celite to remove insoluble material. The filtrate was cooled in an ice salt bath and adjusted to pH 8 with 6N. aqueous NaOH solution and extracted with EtOAc (3x1 L). The combined EtOAc extracts were washed with brine (1 L), dried over Na ₂ SO ₄ , stirred with activated carbon (10 g) and silica gel (10 g) for 1 hour. The mixture was filtered through celite, washing the celite with ethyl acetate (1 L). . The filtrate was concentrated by co-evaporating the residual EtOAc with n-heptane (500 ml). The resulting brown solid was pumped under high vacuum for 2 hours to give crude 6-chloro-5-(2methoxyvinylyl)pyrimidin-4-ylamine (72.3 g, 136.2 g theoretical, 53.1%). The crude desired product was used in the next reaction without further purification. A sample of the crude product (2.3 g) was purified by silica gel column chromatography eluting with 0-35% EtOAc/n-heptane to provide 1.7 g of pure 6-chloro-5-(2-methoxyvinyl)pyrimidine-4- ylamine as a white solid, which was a mixture of 1 to 2 E/Z isomers. 1H NMR (300 MHz, DMSO-d ₆ ) for E-isomer: δ 8.02 (s, 1H), 7.08 (br.s, 2H), 6.92 (d, 1H, J=13.1), 5.35 (d, 1H, J=13.0 Hz), 3.68 (s, 3H) ppm. and for the 2-isomer: δ 8.06 (s, 1H), 7.08 (br.s, 2H), 6.37 (d, 1H, J=6.8 Hz), 5.02 (d, 1H , J=6.7 Hz), 3.69 (s, 3H) ppm; C7H8ClN3O (Mol. mass, 185.61), HPMS (EI) m/e 186/188 (M ⁺ +H).

4-Chloro-7H-[pyrrolo[2,3-d]pyrimidine (4). Concentrated HCl (5 ml) was added to a solution of crude 6-chloro-5-(2-methoxyvinylyl)pyrimidin-4-ylamine (70.0 g, 0.3784 mol) in THF (700 ml) and the resulting reaction mixture was heated to reflux. refrigerator for 7.5 hours. When heated, a thin suspension formed, which gradually re-dissolved. When the reaction was complete, as confirmed by HPLC monitoring, the reaction mixture was cooled to ambient temperature and stirred at ambient temperature overnight.

Solid NaHCO ₃ (15 g) was added to the reaction mixture and the resulting mixture was stirred at ambient temperature for 1 hour. Activated carbon (7 g), silica gel (7 g) and Na ₂ SO ₄ (20 g) were added to the mixture and heated to 40°C for 1 hour. The mixture was then cooled to room temperature and filtered through celite, washing the celite with THF (1 L). The filtrate was concentrated under reduced pressure and the resulting solid was dried under reduced pressure to obtain crude 4-chloro-7H-[pyrrolo[2,3-d]pyrimidine (4, 58.1 g, 58.1 g theoretical, 100%) in as a yellow-brown solid. This crude target product was dissolved in EtOAc (1 L) at 50-55°C and treated with activated carbon (3 g). The mixture was filtered through celite while heating and the celite layer was washed with warm EtOAc (250 ml). The filtrate was concentrated to about 500 ml and the suspension was kept at ambient temperature overnight. The suspension was then cooled to 0-5°C for 2 hours before collecting the solids by filtration. The solids were dried to give pure 4-chloro-7H-[pyrrolo[2,3-d]pyrimidine (4, 54.5 g, 58.1 g theoretical, 94%) as tan crystals. 1H NMR (400 MHz, DMSO-d ₆ ) δ 12.58 (br.s, 1H), 8.58 (s, 1H), 7.69 (d, 1H,J=3.5 Hz), 6.59 (d, 1H, J=3.5 Hz) ppm; VZhMS (EI) m/e 154/156 (M ⁺ +H).

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Example A: In vitro JAK Kinase assay.

The compound of formula I was tested for inhibitory activity against JAK entities according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (aa 837-1142) and JAK2 (aa 828-1132) with N-terminal tags were expressed by baculovirus in insect cells and purified. The catalytic activities of JAK1 and JAK2 were analyzed by measuring the phosphorylation of the biotinylated peptide.

The phosphorylated peptide was detected by uniform time-resolved fluorescence (HTRF). IC ₅₀ compounds were measured for each kinase in 40 μl reactions that contained enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT and 0.1 mg/ml (0.01%) BSA. For 1 mM IC ₅₀ measurements, the ATP concentration in the reactions was 1 mM. Reactions were performed at ambient temperature for 1 h and then stopped with 20 μl of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Massachusets). Binding to the europium-labeled antibody occurred for 40 min and the HTRF signal was measured on a Fusion plate counter (Perkin Elmer, Boston, Massachusets). The compound of Formula I and the adipic acid salt had an IC ₅₀ in JAK1 of <5 nM (measured at 1 mM ATP) with a JAK2/JAK1 ratio of >10 (measured at 1 mM ATP).

Example B: Cellular studies.

Cancer cell lines dependent on cytokines and therefore JAK/STAT signaling for growth were seeded at 6000 cells per well (96-well plate format) in RPMI 1640 medium, 10% FBS, and 1 ng/ml of the appropriate cytokine. the compound can be added to cells in DMSO/vehicle (final DMSO concentration 0.2%) and incubated for 72 h at 37°C, 5% CO ₂ . The effect of the compound on cell viability was assessed using the CellTiter-Glo Luminescent Cell Cell Viability Assay (Promega) followed by TopCount (Perkin Elmer, Boston, Massachusets) quantitation. Potential off-target effects of the compounds are measured in parallel using non-JAKs that drive the cell line with the same readout assay. All experiments were usually performed in duplicate.

The cell lines mentioned above can also be used to study the effects of compounds on phosphorylation of JAK kinases or potential substrates downstream in the biochemical pathway, such as STAT, Akt, Shp2 or Erk proteins. These experiments can be performed overnight with cytokine starvation, followed by a short preincubation with compound (2 hours or less) and cytokine stimulation for about 1 hour or less. Proteins were then extracted from the cells and analyzed by methods familiar to those skilled in the art, including Western blotting or ELISA using antibodies that can differentiate between phosphorylated and total protein. These experiments may use normal or cancer cells to examine the effects of compounds on tumor cell survival biology or mediators of inflammatory disease. For example, relative to the latter, cytokines such as IL-6, IL-12, IL23 or IFN can be used to stimulate JAK activation, leading to phosphorylation of STAT protein(s) and potentially transcriptional profiles (as assessed by array or qPCR technology ) or the production and/or secretion of proteins such as IL-17. The ability of compounds to inhibit these cytokine-mediated effects can be measured using methods well known to those skilled in the art.

The compounds of this invention may further be tested in cellular models designed to evaluate their efficacy and activity against mutant JAKs, such as the JAK2V617F mutation found in myeloid proliferative disorders. Such experiments often use cytokine-dependent hematological cell lines (eg BaF/3) that ectopically express wild-type or mutant JAK kinases (James, S., et al. Nature 434:1144-1148; Staerk, J., et al. al JBC 280:41893–41899). Endpoints include the compounds' effects on cell survival, cell proliferation, and phosphorylated JAK, STAT, Akt, or Erk proteins.

Certain compounds of this invention may be evaluated for their activity in inhibiting T cell proliferation. Such an assay may include a second cytokine (ie, JAK)-triggered proliferation assay, as well as a simplified assay for immunosuppression or inhibition of immune activation. Below is a short outline of how these experiments can be performed. Peripheral blood mononuclear cells (PBMCs) are obtained from human whole blood samples using the Ficoll Hypaque separation method, and T cells (fraction 2000) can be obtained from PBMCs by decantation. Freshly isolated human T cells can be maintained in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μ g/ml streptomycin) at a density of 2x106 cells/ml at 37°C for up to 2 days. To assay IL-2 stimulated cell proliferation, T cells are first treated with phytohemagglutinin (PHA) at a final concentration of 10 μ μg/ml for 72 h. After washing once with PBS, 6000 cells/well are plated on 96-well plates and treated with compounds in various concentrations in the nutrient medium in the presence of 100 U/ml human IL-2 (ProSpec-Tany TechnoGene;

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Rehovot, Israel). The plates are incubated at 37°C for 72 hours, and the proliferation index is assessed using CellTiter-Glo fluorescent reagents following the manufacturer's suggested protocol (Promega; Madison, Wisconsin).

Example C. In Vivo Antitumor Efficacy.

The compounds of this invention can be evaluated in human tumor xenograft models in immunocompromised mice. For example, a carcinogenic variant of the plasmacytoma cell line INA-6 can be used for subcutaneous inoculation in SCID mice (Burger, R., et al. Hematol J. 2:42-53, 2001). Tumor-bearing animals can then be randomized to drug or vehicle treatment, and various doses of the compounds can be administered by any number of conventional routes, including oral, intraperitoneal, or continuous infusion using implantable pumps. Tumor growth is monitored over time using a caliper. Subsequently, tumor samples can be collected for analysis at any time after the start of treatment, as outlined above (Example B), to assess the effect of the compound on the activity of JAK and downstream signaling pathways. Additionally, the selectivity of the compound(s) can be assessed using tumor xenograft models that are driven by other known kinases (eg Bcr-Abl), such as the K562 tumor model.

Example D: Delayed contact hypersensitivity skin reaction test in mice.

The compounds of this invention can further be tested for their potency (inhibition of JAK targets) in a T cell-triggered delayed hypersensitivity test model in mice. The cutaneous delayed contact hypersensitivity reaction (DTH) in mice is considered a useful model of clinical contact dermatitis and other T-lymphocyte-mediated immune skin disorders such as psoriasis (Immunol Today. 1998 Jan;19(1):37-44). Numerous characteristics are common to HRT in mice with psoriasis, including an immune infiltrate, a concomitant increase in inflammatory cytokines, and hyperproliferation of keratinocytes. In addition, many classes of agents effective in treating psoriasis in the clinic are also effective inhibitors of the DTH response in mice (Agents Actions. 1993 Jan;38(12):116-21).

On days 0 and 1, Balb/c mice were sensitized by topical application of 2,4-dinitro-fluorobenzene (DFB) antigen to the shaved abdomen. On day 5, ear thickness was measured using an engineering micrometer. The measurement results were recorded and used as a baseline. Both ears of the animals were then loaded with a topical application of DNFB at a total dose of 20 μl (10 μl for the inner part and 10 μl for the outer part of the auricle) with a concentration of 0.2%. The ears were measured again 24-72 hours after exercise. Treatment with test compounds was carried out during the sensitization and loading phases (from day -1 to day 7) or before and during the loading phase (usually from the second half of day 4 to day 7). Treatment with test compounds (in different concentrations) was carried out systemically or locally (local application of the drug to the ears). The effectiveness of the study compounds was indicated by a decrease in ear swelling compared to no treatment. Compounds that cause a reduction of 20% or more are considered effective. In some experiments, mice were loaded without sensitization (negative control).

The inhibitory effect (suppression of JAK-STAT pathway activation) of the test compounds can be confirmed by immunohistochemical analysis. Activation of the JAK-STAT pathway(s) leads to the production and translocation of functional transcription factors. Further, the influx of immune cells and increased proliferation of keratinocytes should also provide unique changes in the expression profile in the ear that can be examined and quantified. Formalin-fixed, paraffin-embedded ear sections (sampled after the loading phase of the DTH model) were subjected to immunohistochemical analysis using an antibody that specifically interacts with phosphorylated STAT3 (clone 58E12, Cell Signaling Technologies). Mouse ears are treated with test compounds, vehicle, or dexamethasone (a clinically effective treatment for psoriasis), or left untreated in a HRT model for comparison. The test compounds and dexamethasone may produce similar transcriptional changes, both qualitatively and quantitatively, and the test compounds and dexamethasone may reduce the number of infiltrating cells. Systemic and local use of the test compounds may cause an inhibitory effect, i.e. reduction in the number of infiltrating cells and inhibition of transcriptional changes.

Example E. In vivo anti-inflammatory activity.

The compounds of this invention can be evaluated in rodent or non-rodent models designed to reproduce a single or complex inflammatory response. For example, rodent models of arthritis can be used to evaluate the therapeutic potential of compounds administered preventively or therapeutically. These models include, but are not limited to, collagen-induced arthritis in mice or rats, adjuvant-induced arthritis in rats, and anti-collagen antibody-induced arthritis. In addition, autoimmune diseases, including, but not limited to, multiple sclerosis, type I diabetes mellitus, uveoretinitis, thyroiditis, myasthenia gravis, immunoglobulin nephropathies, myocarditis, respiratory sensitization (asthma), lupus, or colitis, may be used to assessing the therapeutic potential of the compounds of this invention.

These models are well studied in the research community and are familiar to those skilled in the art (Current Protocols in Immunology, Vol. 3., Coligan, J.E. et al., Wiley Press.; Methods in Molecular Biology: Vol. 225, Inflammation Protocols., Winyard, P.G. and Willoughby, D.A., Humana Press, 2003).

Example F: Animal Models for the Treatment of Dry Eyes, Uveitis, and Conjunctivitis.

The agents may be evaluated in one or more preclinical dry eye models known to those skilled in the art, including, but not limited to, the concanavalin A (ConA) rabbit lacrimal gland model, the scopolamine (subcutaneous or transdermal) mouse model, mouse lacrimal gland model using botulinum toxin or any of a number of spontaneous autoimmune rodent models that lead to ocular gland dysfunction (eg NOD-SCID, MRL/lpr or NZB/NZW) (Barabino et al., Experimental Eye Research 2004, 79, 613-621 and Schrader et al., Developmental Opthalmology, Karger 2008, 41, 298-312, each of which is incorporated herein by reference in its entirety). Endpoints in these models may include histopathology of the ocular glands and eye (cornea, etc.), as well as the classic Schirmer test or modified versions (Barabino et al.), which measure tear production. Activity is assessed by multiple dosing routes (eg, systemically or locally), which may begin before or after the onset of measurable disease.

The agents can be evaluated in one or more preclinical models of uveitis known to those skilled in the art. These include, but are not limited to, experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (IEU) models. EAU experiments can be performed in rabbit, rat or mouse, and can involve passive or active immunization. For example, any of a variety of retinal antigens can be used to sensitize the invention of animals to the corresponding immunogenic agent, after which the eyes of the animals can be loaded with the same antigen. The IED model is more acute and involves local or systemic administration of lipopolysaccharides in sublethal doses. Endpoints of IED and EAU models may include, but are not limited to, fundoscopic examination and histopathology. These models are reviewed by Smith et al. (Immunology and Cell Biology 1998, 76, 497-512, which is incorporated herein by reference in its entirety). Activity is assessed by multiple dosing routes (eg, systemically or locally), which may begin before or after the onset of measurable disease. In addition, some of the models listed above may develop scleritis/episcleritis, choroiditis, cyclitis or iris inflammation, and are thus suitable for studying the potential activity of compounds for the therapeutic treatment of these diseases.

Additionally, the agents can be evaluated in one or more preclinical models of conjunctivitis known to those skilled in the art. These include, but are not limited to, rodent models using the guinea pig, rat or mouse. Guinea pig models include models using active or passive immunization and/or immune protocols of loading antigens such as egg white or ragweed (reviewed in Groneberg, D.A., et al., Allergy 2003, 58, 1101-1113, which is incorporated herein document by reference in its entirety). The rat and mouse models are similar in general design to the guinea pig models (also reviewed by Groneberg). Activity is assessed by multiple dosing routes (eg, systemically or locally), which may begin before or after the onset of measurable disease. Endpoints for such a study may include, for example, histological, immunological, biochemical or molecular analysis of ocular tissues such as the conjunctiva.

Example G. In vivo bone protection.

The compounds can be evaluated in various preclinical models of osteopenia, osteoporosis or bone resorption known to those skilled in the art. For example, ovariectomized rodents can be used to evaluate the ability of compounds to affect traits and markers of bone turnover and/or density (W.S.S. Jee and W. Yao, J Musculoskel. Nueron. Interact., 2001, 1(3), 193-207 , which is incorporated herein by reference in its entirety). Alternatively, bone density and architecture can be assessed in control or compound-treated rodents in models of treatment (eg, glucocorticoid)-induced osteopenia (Yao, et al. Arthritis and Rheumatism, 2008, 58(6), 3485-3497; and ibid. 58 (11), 1674-1686; both of which are incorporated herein by reference in their entirety). In addition, the effects of the compounds on bone resorption and density can be assessed in the rodent models of arthritis discussed above (Example E). Endpoints for all of these models may vary, but often include histological and radiological assessment, as well as immunohistology and suitable biochemical markers of bone turnover.

A number of embodiments of the invention have been described. However, it should be understood that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments of the invention are within the scope of the accompanying

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Claims (12)

CLAIM claims. 1. Method for preparing a compound of formula I: involving the interaction of a compound of formula VIII: with a compound of formula IXa: in the presence of a coupling agent to obtain a compound of formula VIIa: and the reaction of a compound of formula VIIa with a compound of formula IVa: under Suzuki coupling reaction conditions to produce a compound of formula I, wherein the Suzuki coupling reaction conditions include heating a reaction mixture containing a compound of formula VIIa, a compound of formula IVa, a Suzuki coupling reaction catalyst, a base, and a second solvent component. 2. The method according to claim 1, wherein the coupling agent is 1,8-diazabicyclo[5,4,0]undecene. 3. The method according to claim 1 or 2, in which from 1.05 to 1.2 equivalents of a binding agent are used, based on - 23 045143 from a compound of formula VIII. 4. Method according to any one of claims 1-3, in which the reaction of a compound of formula VIII with a compound of formula 1Xa: (a) carried out in a solvent component containing acetonitrile; and (b) carried out in a solvent component containing acetonitrile at a temperature of from 40 to 60°C. 5. The method according to any one of claims 1 to 4, in which from 1 to 1.2 equivalents of a compound of formula 1Xa based on a compound of formula VIII are used. 6. A method according to any one of claims 1 to 5, wherein the catalyst for the Suzuki coupling reaction is tetrakis(triphenylphosphine)palladium(0). 7. Method according to any one of claims 1 to 6, wherein the base is sodium bicarbonate. 8. The method of claim 7, wherein the sodium bicarbonate is present in an amount of 4 or more equivalents based on the compound of formula Vila. 9. Method according to any one of claims 1 to 8, wherein the second solvent component contains 1,4-dioxane and water. 10. The method according to claim 9, in which 1,4-dioxane and water are present in a volumetric ratio of 1:1. 11. Method according to any one of claims 1 to 10, in which the compounds of formulas Vila and IVa are present in a molar ratio of 1:1. 12. The method according to any one of claims 1 to 11, wherein the Suzuki coupling reaction conditions include heating a reaction mixture containing a compound of formula Vila, a compound of formula IVa, tetrakis(triphenylphosphine)palladium (0), sodium bicarbonate and a second solvent component, and wherein the second solvent component includes water and 1,4-dioxane. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2