EA036970B1 - USE OF {1-{1-[3-FLUORO-2-(TRIFLUOROMETHYL)ISONICOTINOYL]PIPERIDINE-4-YL}-3-[4-(7H-PYRROLO[2,3-d]PYRIMIDINE-4-YL)-1H-PYRAZOL-1-YL]AZETIDINE-3-YL}ACETONITRILE IN TREATING JAK1-ASSOCIATED DISEASES - Google Patents

Abstract

The invention provides methods of use of 1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidine-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)-1H-pyrazol-1-yl]azetidine-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof which modulates the activity of Janus kinase 1 (JAK1) and is useful in the treatment of diseases related to the activity of JAK1 selected from allograft rejection and graft-versus-host disease in a patient in need thereof.

Description

Technology area

The present invention provides the use of a compound of {1- {1- [3fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidine- 4-yl) -1Hpyrazol-1-yl] azetidin-3-yl} acetonitrile and a method of treatment using said compound that modulates Janus kinase (JAK) activity and is useful for the treatment of diseases associated with JAK1 activity, including, for example , allograft rejection, and graft versus host disease.

State of the art

Protein kinases (PKs) regulate a variety of important biological processes, including, but not limited to, cell growth, survival and differentiation, organ formation and morphogenesis, neovascularization, tissue repair and regeneration. Protein kinases also play special roles in the carrier of human diseases, including cancer. Cytokines, low molecular weight polypeptides, or glycoproteins, regulate many pathways involved in the host's inflammatory response to sepsis. Cytokines affect cell differentiation, proliferation and activation, and can modulate pro-inflammatory and anti-inflammatory responses to ensure an appropriate host response to pathogens. Signaling of a wide variety of cytokines includes the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and transcriptional activators (STAT). Four mammalian JAKs are known: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as Janus kinase, leukocyte; JAKL; and L-JAK) and TYK2 (protein tyrosine kinase 2).

New or improved agents that inhibit kinases such as JAK constantly require the development of new and more effective drugs designed to enhance or suppress immune and inflammatory pathways (such as immunosuppressive agents for organ transplants). The compound of the present invention, as well as its compositions and methods described herein, are directed to meeting these needs and other purposes.

The essence of the invention

The present invention, inter alia, provides a method for treating a disease selected from allograft rejection and graft versus host disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound that is {1- {1- [3-fluoro-2 - (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof.

The present invention further provides compositions comprising {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidine- 4-yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The present invention also provides the use of {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] πiperidin-4-yl} -3- [4- (7H-πyrrolo [2,3-d] πyrimidine-4- yl) -1H-pirazol1-yl] azetidin-3-yl} acetonitrile described herein, or pharmaceutically acceptable salts thereof, for the preparation of medicaments for the treatment of a disease selected from allograft rejection and graft versus host disease.

Details of one or more embodiments of the present invention are set forth in the accompanying figures and descriptions. Other features, objects and advantages of the present invention are clear from the description and figures, as well as from the claims.

Brief description of figures

FIG. 1 shows a DSC thermogram for the product of Example 3.

FIG. 2 is a TG A thermogram for the product of Example 3.

FIG. 3 presents an XRPD chart for the product of Example 3.

Detailed description

The present invention provides methods for treating a JAK-related disease or disorder in a subject (e.g., a patient) by administering to a subject in need of such treatment a therapeutically effective amount or dose of a compound that is {1- {1- [3-fluoro-2 ( trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt. Examples of JAK-related diseases include diseases that affect the immune system, including, for example, organ transplant rejection (eg, allograft rejection and graft versus host disease).

In one embodiment, the disease is allograft rejection.

In one embodiment, the disease is a graft versus host disease.

In one embodiment, the pharmaceutically acceptable salt of adipic acid is {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-1 036970 pyrrolo [ 2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile.

In some embodiments, the salt is the salt described in Example

358.

In some embodiments, the salt has a melting point of about 178 ° C. In some embodiments, the salt has a differential scanning calorimetry thermogram that has an endothermic peak with an onset temperature of about 176 ° C. In some embodiments, the salt has a differential scanning calorimetry thermogram substantially as shown in FIG. one.

In some embodiments, the salt has a thermogravimetric analysis generally as shown in FIG. 2. In some embodiments, the salt has a powder x-ray diffraction pattern including a characteristic peak expressed in degrees 2θ at about 10.4. In some embodiments, the salt has a powder X-ray diffraction pattern including a characteristic peak, expressed in degrees 2θ, at about 6.9. In some embodiments, the salt has a powder x-ray diffraction pattern including a characteristic peak expressed in degrees 2θ at about 21.0. In some embodiments, the salt has a powder X-ray diffraction pattern including a characteristic peak, expressed in degrees 2θ, at about 23.3. In some embodiments, the salt has a powder x-ray diffraction pattern including a characteristic peak expressed in degrees 2θ at about 6.9, 10.4, 21.0, and 23.3. In some embodiments, the salt has a powder X-ray diffraction pattern substantially as shown in FIG. 3.

XRPD reflections (peaks) are generally considered to be an imprint of a particular crystal form. It is well known that the relative intensities of XRPD peaks can vary widely depending, among other things, on the method of sample preparation, crystal size distribution, different filters used, sample clamping procedure, and the particular instrument used. In some cases, new peaks may be observed or existing peaks may disappear depending on the type of device or settings (for example, depending on whether a Ni filter is used or not). As used herein, the term peak refers to a reflection having a relatively high height / intensity of at least 4% of the height / intensity of the maximum peak. In addition, instrumental biases and other factors can influence 2-theta values. Therefore, the assignment of peaks like those provided herein can vary by plus or minus about 0.2 ° (2-theta), and the term generally used in the description in the context of XRPD is intended to cover any of the above. ...

Likewise, temperatures due to DSC, TGA, or other thermal experiments can vary around ± 3 ° C depending on instrument, specific settings, sample preparation, and so on. Accordingly, a crystalline form given herein, which has a DSC thermogram substantially as shown in any of the figures, is intended to encompass such variations.

The compound of the invention and its pharmaceutically acceptable salts can be prepared together with other materials such as water and solvents (eg hydrates and solvates), or can be isolated.

In some embodiments, the compound of the present invention, or salts thereof, are substantially isolated. The term generally isolated means that a compound is at least partially or largely separated from the environment in which it was formed or found. Partial separation may include, for example, a composition enriched in the compounds of the present invention. Substantial separation can include compositions containing at least about 50 wt.%, At least about 60 wt.%, At least about 70 wt.%, At least about 80 wt.%, At least about 90 wt. %, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of a compound of the present invention or a salt thereof. Methods for isolating compounds and their salts are conventional in the art.

The expression pharmaceutically acceptable, as used herein, refers to such compounds, materials, compositions and / or dosage forms that are, according to the results of careful medical clinical evaluation, suitable for use in contact with tissues of the human and animal body, without excessive toxicity, irritation , allergic reactions or other problems or complications, commensurate with the ratio of acceptable benefit and risk.

The expressions ambient temperature and room temperature, as used herein, are understood in the art and denote generally a temperature, such as a reaction temperature, which is about the temperature in a room in which the reaction is performed, such as a temperature from about 20 to about 30 ° C.

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Also included in the present invention are pharmaceutically acceptable salts of the compounds described herein. As used herein, the term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acidic or basic group into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, salts with mineral or organic acids and basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic group by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the corresponding base or acid in water or an organic solvent or a mixture of both; in general, non-aqueous media such as ether, ethyl acetate, alcohols (eg, methanol, ethanol, isopropanol or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts are presented in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. In some embodiments, the compounds described herein include N-oxide forms.

Synthesis.

The compound of the present invention, including its salts, can be prepared by known synthetic procedures and can be synthesized by one of numerous possible synthetic routes, such as those shown in the schemes below. The reactions to prepare the compound of the present invention can be carried out in suitable solvents which are readily selected by one skilled in the art of organic synthesis. Suitable solvents can be solvents that are largely non-reactive with starting materials (reagents), intermediates or products at reaction temperatures, for example temperatures that can range from the freezing point of the solvent to the boiling point of the solvent. This reaction can be performed in one solvent or in a mixture of more solvents. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by a person skilled in the art.

The preparation of the compound of the present invention can include protecting and deprotecting various chemical groups. The need for protection or deprotection, as well as the selection of appropriate protecting groups, can be easily determined by the person skilled in the art. The chemistry of protecting groups is presented, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: New Jersey, (2007), which is incorporated herein by reference in its entirety.

Reactions can be controlled in accordance with any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g. 1H or ¹³ C), infrared spectroscopy, spectrophotometry (e.g. UV-visible), mass spectrometry, or chromatographic techniques such as high performance liquid chromatography ( HPLC) or thin layer chromatography (TLC).

The compound of the invention can be prepared by the methods outlined in Schemes 1-4. Intermediates of Formula 1-5 can be synthesized by the methods described in Scheme 1. Commercially available starting material pyrrolo [2,3-d] pyrimidine-4-halide or 5-substituted pyrrolo [2,3-b] pyridine-4- the halide (1-1) can be converted to SEM (2 (trimethylsilyl) ethoxymethyl), a protected intermediate of formula 1-2, by treatment with sodium hydride followed by 2- (trimethylsilyl) ethoxymethyl chloride. Suzuki coupling of compound 1-2 with pyrazole boronic acid such as 1- (1-ethoxyethyl) -4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2yl) -1H-pyrazole (1 -3), using a palladium reagent such as tetrakis (triphenylphosphine) palladium (0), gives intermediate 1-4, which can be converted in situ to desired product 1-5 after continued reaction.

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Scheme 1

Intermediates of Formula 2-3 can be synthesized according to the sequence depicted in Scheme 2. SEM-protected intermediate 1-2 undergoes Suzuki coupling with a protected pyrrole boronic acid such as 1 (triisopropylsilyl) pyrrole-3-boronic acid (2 -1) using a palladium reagent such as tetrakis (triphenylphosphine) palladium (0) in the presence of a base. The coupling product of Formula 2-2 can be converted to the desired product of Formula 2-3 in situ by performing the reaction overnight in the same medium.

Scheme 2

Intermediates of formula 3-7 can be prepared by the procedures outlined in Scheme 3. The Boc-protected azetidinone of formula 3-1 is Wittig reacted with a phosphonate such as diethyl cyanomethylphosphonate in the presence of a base such as sodium hydride to form a cyano derivative of formula 3- 2. Michael addition of intermediates of formula 1-5 or 2-3 to a derivative of formula 3-2 in the presence of a base such as DBU gives an adduct of formula 3-3. After removal of the Boc group (for example, using an acid such as 4N HCl in dioxane) reductive amination of the resulting azetidine of formula 3-4 with an N-Boc protected piperidinone of formula 3-5 using a reducing agent such as sodium triacetoxyborohydride gives the compound Formulas 3-6. Removal of the Boc group in a compound of Formula 3-6 (eg using an acid such as 4N HCl in dioxane) provides the desired intermediates of Formula 3-7.

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Scheme 3

Vos

3-6 3-7

Intermediates of formula 3-7 can be derivatized with piperidine nitrogen to give a number of compounds of formula I, as shown in Scheme 4. Reaction of a compound of formula 3-7 with sulfonyl chloride followed by treatment first with TPA and then with ethylenediamine to remove the SEM group gives sulfonamide derivatives of formula 4-1. Coupling of a compound of formula 3-7 with a carboxylic acid using a coupling agent such as BOP or an acyl chloride followed by removal of the SEM group affords amide compounds of formula 4-2. Reductive amination of a compound of formula 3-7 with an aldehyde using a reducing agent such as sodium triacetoxyborohydride followed by removal of the SEM group gives the N-alkyl derivatives of formula 4-3. Reaction of a compound of formula 3-7 with an isocyanate followed by removal of the SEM group provides urea compounds of formula 4-4.

Scheme 4

Ways.

The compound of the present invention is a JAK inhibitor and is a selective JAK1 inhibitor. A selective JAK1 inhibitor is a compound that inhibits JAK1 activity preferentially over other Janus kinases. For example, a compound of the present invention preferentially inhibits JAK1 over one or more of JAK2, JAK3, and TYK2. In some embodiments, the compound inhibits JAK1 preferentially over JAK2 (eg, have a JAK1 / JAK2 IC50> 1).

In some embodiments, the compound is about 10 times more selective for JAK1 than for JAK2. In some embodiments, the compound is about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold more selective for JAK1 than JAK2, as calculated by measuring IC ₅₀ at 1 mM ATP (eg see Example A).

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Selective JAK1 inhibitors over other JAK kinases may have multiple therapeutic benefits over less selective inhibitors. In terms of selectivity against 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). Uro is a key growth factor for red blood cell production; therefore, the lack of Epo-dependent signaling can lead to a reduced number of red blood cells and anemia (Kaushansky K., NEJM 354: 2034-45, 2006). Tro, another example of a JAK2-dependent growth factor, 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). Therefore, decreased Tro signaling decreases the number of megakaryocytes (megakaryocytopenia) and decreases the number of circulating platelets (thrombocytopenia). This can lead to unwanted and / or uncontrolled bleeding. Decreased inhibition of other JAKs, such as JAK3 and Tyk2, may also be desirable, as individuals with a functional version of these kinases have been shown to suffer from many diseases such as complex combined immunodeficiency or hyperimmunoglobulin E syndrome (Minegishi, Y., et al Immunity 25: 745-55, 2006; Macchi P., et al. Nature 377: 65-8, 1995). Therefore, a JAK1 inhibitor with a reduced affinity for other JAKs has significant advantages over a less selective inhibitor in terms of a reduced number of side effects including immune suppression, anemia, and thrombocytopenia.

In some embodiments, embodiments of the present invention provides the use of a compound that is {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7Hpyrrolo [2,3-d ] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a disease selected from allograft rejection and graft versus host disease.

As used herein, the term interaction refers to the connection of these groups in in vitro systems or in vivo systems. For example, interaction of a JAK with a compound of the present invention includes administering a compound of the present invention to a subject or patient, such as a human, having JAK, as well as, for example, administering a compound of the present invention to a sample containing a cellular or purified preparation containing JAK.

As used herein, the terms subject or patient, as used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and most preferably humans ...

As used herein, the expression therapeutically effective amount refers to that amount of active compound or pharmaceutical agent that elicits a biological or medical response expected by a researcher, veterinarian, treating physician or other clinician in a tissue, system, animal, subject, or human body. In some embodiments, the therapeutically effective amount is from about 5 to about 1000 mg, or from about 10 to about 500 mg.

As used herein, the term treatment or therapy refers to one or more of (1) preventing disease; for example, preventing a disease, condition, or disorder in a subject who is possibly predisposed to the disease, condition or disorder, but is not yet sick or exhibiting pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition, or disorder in a subject who is already ill or exhibiting a pathology or symptomatology of that disease, condition or disorder (ie, arresting further development of the pathology and / or symptomatology); and (3) relief of disease; for example, amelioration of a disease, condition, or disorder in a subject who is already ill or exhibiting a pathology or symptomatology of a disease, condition or disorder (ie, reversal of pathology and / or symptomatology), such as a decrease in the severity of the disease.

Pharmaceutical compositions and dosage forms.

When used as medicaments, the compounds of the present invention can be administered in the form of pharmaceutical compositions. These compositions can be prepared by methods well known in the art of pharmacy and can be administered in a variety of ways depending on whether local or systemic treatment is required, as well as the area to be treated. Administration can be local (including transdermal, epidermal, ophthalmic and mucosal administration, including intranasal, vaginal and rectal delivery), pulmonary (eg, by inhalation or insufflation of powders or aerosols, including through a nebulizer; intratracheally or intranasally), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or intraperitoneal injection or infusion, or intracranial, for example, intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus administration or, for example, via a continuous perfusion pump. Pharmaceutical compositions and formulations for topical use may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories,

- 6 036970 sprays, liquids and powders. Standard pharmaceutical carriers, aqueous, powdery or oily bases, thickeners, and the like may be necessary or desirable.

Also included in the present invention are pharmaceutical compositions which contain, as an active ingredient, a compound of the present invention, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carrier (excipients). In some embodiments, the composition of the present invention is suitable for topical administration. In preparing the compositions of the present invention, the active ingredient is usually mixed with an excipient, diluted with an excipient, or incorporated into such a carrier in the form of, for example, a capsule, sachet, paper or other package. When the excipient is a diluent, it can be a solid, semi-solid or liquid material that acts as a filler, carrier or medium for the active ingredient. Thus, the compositions of the present invention can be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 wt.% active compound, soft or hard gelatin capsules, suppositories, sterile injectable solutions or sterile packaged powders.

When preparing the composition, the active compound may be ground to an appropriate particle size before being mixed with other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially soluble in water, then the particle size can be adjusted by milling to obtain a substantially uniform distribution in the composition, for example, about 40 mesh.

The compounds of the present invention can be milled by known milling methods such as wet milling to obtain the appropriate particle size for tabletting and for formulating other types of pharmaceutical compositions. Finely divided (nano-sized) compositions of the compounds of the present invention can be prepared by methods known in the art, see, for example, International Patent Application No. WO 2002/000196.

Some examples of suitable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The compositions may additionally include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents, emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoates, sweeteners and flavorings. The compositions of the present invention may be formulated to provide rapid, continuous or sustained release of the active ingredient after administration to a patient using methods known in the art.

In some embodiments, the pharmaceutical composition comprises silicated microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicated microcrystalline cellulose comprises about 98% w / w. microcrystalline cellulose and about 2% wt./wt. silicon dioxide.

In some embodiments, the composition is a sustained release composition comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose, and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and hydroxypropyl methylcellulose. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102 ™. In some embodiments, the lactose monohydrate is Fast-flo 316 ™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (eg, Methocel K4 M Premier ™) and / or hydroxypropyl methylcellulose 2208 K100LV (eg, Methocel K00LV ™). In some embodiments, the polyethylene oxide is WSR 1105 polyethylene oxide (eg, Polyox WSR 1105 ™).

In some embodiments, a wet granulation process is used to form the composition. In some embodiments, a dry granulation process is used to form the composition.

The compositions can be formulated in unit dosage forms, each form containing from about 5 to about 1000 mg (1 g), more often from about 100 to about 500 mg, of the active ingredient. In some embodiments, each dose contains about 10 mg of the active ingredient. In some embodiments, each dose contains about 50 mg of the active ingredient. In some embodiments, each dose contains about 25 mg of the active ingredient. The term unit dosage form refers to a physically separate unit suitable as a unit dosage for humans or other mammals, each unit containing a predetermined amount of active material calculated to provide the desired therapeutic effect in combination with a suitable pharmaceutical excipient.

In some embodiments, the compositions of the present invention contain from about 5 mg to about 50 mg of the active ingredient. One of ordinary skill in the art will understand that the compounds or compositions of these embodiments comprise about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 mg of the active ingredient.

In some embodiments, the compositions of the present invention contain from about 50 to about 500 mg of the active ingredient. One of ordinary skill in the art will understand that the compounds or compositions of these embodiments comprise about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 350 to about 400, or from about 450 to about 500 mg of active ingredient.

In some embodiments, the compositions of the present invention contain from about 500 to about 1000 mg of the active ingredient. One of ordinary skill in the art will understand that the compounds or compositions of these embodiments comprise about 500 to about 550, about 550 to about 600, about 600 to about 650, about 650 to about 700, about 700 to about 750, about 750 to about 800, about 800 to about 850, about 850 to about 900, about 900 to about 950 mg, or about 950 to about 1000 mg of the active ingredient.

The active compound can be effective over a wide range of dosages and is usually administered in a pharmaceutically effective amount. However, it should be understood that the actual amount of compound administered is usually determined by a physician in accordance with existing circumstances, including the condition to be treated, the mode of administration chosen, the actual compound administered, the age, weight and response of the particular patient, the severity of the patient's symptoms, and the like.

To obtain solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to obtain a pre-formulated solid composition containing a homogeneous mixture of a compound of the present invention. In such pre-formulated homogeneous compositions, the active ingredient is usually dispersed uniformly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. Such solid pre-formulated compositions are then subdivided into unit dosage forms of the types described above containing, for example, from about 0.1 to about 1000 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise compounded to form a dosage form that allows for a sustained release. For example, tablets or pills can include an internal or external drug component, the latter being in the form of a shell around the excipient. The two components can be separated by an enteric layer, which serves to prevent breakdown in the stomach and passage of the intact internal component into the duodenum or to delay its release. Various materials can be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

Liquid forms in which the compounds and compositions of the present invention can be used for oral or injectable administration include aqueous solutions, suitable flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil. as well as elixirs and similar pharmaceutical carriers.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable carriers, as previously described. In some embodiments, the compositions are administered by the oral or nasal respiratory route for a local or systemic effect. The compositions can be sprayed with inert gases. The nebulized solutions can be inhaled directly from the nebulization device, or the nebulization device may be connected to a face mask, tarpaulin or intermittent positive pressure breathing apparatus. Compositions in solutions, suspensions or powders can be administered orally or nasally through devices that deliver the composition

- 8 036970 as appropriate.

Topical compositions can contain one or more conventional carriers. In some embodiments, the ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. Carrier compositions in creams can be water-based in combination with glycerin and other component (s), for example glycerol monostearate, PEG-glycerol monostearate, and cetylstearyl alcohol. Gels can be prepared using isopropyl alcohol and water, in appropriate combination with other components such as, for example, glycerin, hydroxyethyl cellulose, and the like. In some embodiments, topical compositions comprise at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 wt% of a compound of the present invention. Topical compositions can be packaged in tubes of, for example, 100 g each, optionally accompanied by instructions for treating the selected indication, for example psoriasis or other skin condition.

The amount of a compound or composition administered to a patient varies depending on what is being administered, the purpose of the administration such as prophylaxis or therapy, the condition of the patient, the route of administration, and the like. In therapeutic applications, the compounds can be administered to a patient suffering from a disease in an amount effective to treat or at least partially arrest the symptoms of the disease and its complications. Effective doses depend on the condition of the disease to be treated, as well as on the decision of the attending physician, depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

Compositions to be administered to a patient may be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or can be sterilized by filtration. Aqueous solutions can be packaged for use in the original or lyophilized form, the lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the formulations of the compounds of the present invention is generally from 3 to 11, more preferably from 5 to 9, and more preferably from 7 to 8. It should be understood that the use of certain of the aforementioned carriers, excipients or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention may vary according to, for example, the use for the treatment of a particular disease, the route of administration of the compound, the health and condition of the patient, and the judgment of the treating physician. The proportion or concentration of a compound of the present invention in a pharmaceutical composition may vary depending on a number of factors, including dosage, chemical properties (eg, hydrophobicity), and route of administration. For example, the compounds of the present invention can be prepared in an aqueous physiological buffer solution containing from about 0.1 to about 10% w / v. compounds for parenteral administration. Some standard dosage levels range from about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage level is from about 0.01 to about 100 mg / kg body weight per day. The dosage is likely to depend on variables such as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative bioavailability of the selected compound, the composition of the carrier, and the route of administration. Effective dosages can be extrapolated from dosage response curves obtained from in vitro test systems or animal models.

The compositions of the present invention may further comprise one or more additional pharmaceutical agents such as chemotherapeutic, steroidal, anti-inflammatory compounds, or immunosuppressants, examples of which are listed above herein.

Examples of

Unless otherwise indicated, the compounds from the examples below containing one or more chiral centers were obtained in enantiomerically pure form or in the form of scalemic mixtures.

Unless otherwise indicated, the compounds of the examples were purified by preparative HPLC using basic conditions (method B) and were obtained as free bases.

Method B.

Column: Waters X Bridge C18, 5 μm particle size, 30 x 100 mm;

Mobile phase: water (0.15% NH ₄ OH) / acetonitrile.

Example 1. {1- {1- [3-Fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H -pyrazol-1-yl] azetidin-3-yl} acetonitrile

- 9 036970

Stage A. tert-Butyl 3-oxoazetidine-1-carboxylate. Wax Oxidation Swern l --- l noo

To a mixture of tert-butyl 3-hydroxyazetidine-1-carboxylate (10.0 g, 57.7 mmol), dimethyl sulfoxide (24.0 ml, 338 mmol), triethylamine (40 ml, 300 mmol) and methylene chloride (2.0 ml ) in portions added a complex of sulfur trioxide and pyridine (40 g, 200 mmol) at 0 ° C. The mixture was stirred for 3 h, quenched with brine and extracted with methylene chloride. The combined organic extracts were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified on a silica gel column (0-6% ethyl acetate (EtOAc) in hexanes) to give tert-butyl 3-oxoazetidine-1-carboxylate (5.1 g, 52% yield).

Step B. tert-Butyl 3- (cyanomethylene) azetidine-1-carboxylate

Sodium hydride (5.6 g, 0.14 mol) and tetrahydrofuran (THF) (140 ml) were placed in an oven-dried 1 L 4-neck round-bottom flask equipped with a magnetic stirrer, septum, nitrogen supply, 250 ml dropping funnel and thermocouple. ) in a nitrogen atmosphere. The mixture was cooled to 3 ° C and then diethyl cyanomethylphosphonate (22.4 mL, 0.138 mol) was charged dropwise with a syringe over 20 minutes. The solution turned into a light yellow suspension. The reaction mixture was then stirred for 75 minutes while heating to 18.2 ° C. In an oven-dried round bottom flask, a solution of tert-butyl 3-oxoazetidine-1 carboxylate (20 g, 0.1 mol) in tetrahydrofuran (280 ml) was prepared, loaded into an addition funnel through a cannula, and then added to the reaction mixture dropwise over 25 min. The color of the reaction solution turned red. The reaction mixture was left stirring overnight. After 24 h, the reaction mixture was checked by TLC (70% Hexane / EtOAc) and found to be complete. The reaction mixture was diluted with 200 ml 20% brine and 250 ml EtOAc. The solution was separated and the aqueous phase was extracted with 250 ml EtOAc. The combined organic phase was dried over MgSO ₄ and filtered, evaporated under reduced pressure and purified by flash chromatography (0 to 20% EtOAc / hexanes, 150 g flash column) to afford the desired product, tert-butyl 3- (cyanomethylene) azetidine- 1-carboxylate (15 g, 66.1% yield).

Step C. 4-Chloro-7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidine

A dark solution of 4-chloropyrrolo [2,3-0] pyrimidine was slowly added to a suspension of sodium hydride (36.141 g, 903.62 mmol) in N, N-dimethylacetamide (118 ml) at -5 ° C (ice-salt bath) (119.37 g, 777.30 mmol) in N, N-dimethylacetamide (237 ml). The flask and addition funnel were washed with N, N-dimethylacetamide (30 ml). A large amount of gas was released immediately. The mixture became slightly cloudy and turned orange. The mixture was stirred at 0 ° C for 60 min until it turned light brown and cloudy. To the mixture was slowly added [2- (trimethylsilyl) ethoxy] methyl chloride (152.40 g, 914.11 mmol) and the reaction mixture was stirred at 0 ° C for 1 hour. The reaction was quenched by the slow addition of 12 mL H ₂ O. Additional amount was added water (120 ml), and then methyl tert-butyl ether (MTBE) (120 ml). The mixture was stirred for 10 minutes. The organic layer was separated. The aqueous layer was extracted with another portion of MTBE (120 ml). The organic extracts were combined, washed with brine (120 mlx2) and concentrated under reduced pressure to give the crude product, 4-chloro-7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2.30] pyrimidine in the form of a dark oily substance. Yield: 85.07 g (97%); LCMS: 284.1 (M + H) +. It was used for the next reaction without purification.

Stage D. 4- (1H-Pyrazol-4-yl) -7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-0] pyrimidine

- 10 036970

In a 1000 ml round bottom flask was charged 4-chloro-7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3 ^] pyrimidine (10.00 g, 35.23 mmol), 1-butanol (25 , 0 ml), 1- (1-ethoxyethyl) -4- (4,4,5,5tetramethyl-1,3,2-dioxoborolan-2-yl) -1Н-pyrazole (15.66 g, 52.85 mmol ), water (25.0 ml) and potassium carbonate (12.17 g, 88.08 mmol). This solution was degassed 4 times, each time filling with nitrogen. To the solution was added tetrakis (triphenylphosphine) palladium (0) (4.071 g, 3.523 mmol). The solution was degassed 4 times, each time filling with nitrogen. The mixture was stirred overnight at 100 ° C. After cooling to room temperature, the mixture was filtered through a pad of celite and the celite was washed with ethyl acetate (42 ml). The filtrate was combined and the organic layer was separated. The aqueous layer was extracted with ethyl acetate. The organic extracts were combined and concentrated under reduced pressure with a bath temperature of 3070 ° C to give the final compound, 4- (1H-pyrazol-4-yl) -7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3 -b] pyrimidine. Yield: 78%. LCMS: 316.2 (M + H) +.

Stage E. tert-Butyl 3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3b] pyrimidin-4-yl) -1 H- pyrazol-1-yl] azetidine-1-carboxylate

In a 2 L round-bottom flask, equipped in the upper part with a stirrer, septum and nitrogen supply, was charged tert-butyl 3- (cyanomethylene) azetidine-1-carboxylate (9.17 g, 0.0472 mol), 4- (1H-pyrazole -4-yl) -7 {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-b] pyrimidine (14.9 g, 0.0472 mol) and acetonitrile (300 ml). The resulting solution was heterogeneous. To this solution, 1,8-diazabicyclo [5.4.0] undec-7-ene (8.48 ml, 0.0567 mol) was added with a syringe over 3 min at room temperature. The solution slowly became homogeneous and yellow in color. The reaction mixture was left stirring at room temperature for 3 hours. The reaction was complete by HPLC and LC / MS and was concentrated on a rotary evaporator to remove acetonitrile (~ 150 ml). EtOAc (100 ml) was added followed by 100 ml of 20% brine. The two phases were separated. The aqueous phase was extracted with 150 ml EtOAc. The combined organic phases were dried over MgSO ₄ , filtered and concentrated to give an orange oily substance. Purification by flash chromatography (150 g silica, 60% EtOAc / hexanes, CH ₂ Cl ₂ charged) afforded the title compound tert-butyl 3- (cyanomethyl) -3- [4- (7 - {[ 2 (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1 H-pyrazol-1-yl] azetidine-1 carboxylate as a yellow oily substance (21.1 g, yield 88%). LCMS: [M + H] + = 510.3.

Step F. {3- [4- (7- {[2- (Trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3 -d] pyrimidin-4-yl) -1 Npyrazol-1-yl] azetidine- 3-yl} acetonitrile dihydrochloride

To a solution of tert-butyl 3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazole- 1-yl] α-zetidine-1-carboxylate (2 g, 3.9 mmol) in 10 ml of THF was added 10 ml of 4N hydrochloric acid. HCl in dioxane. The solution was stirred at room temperature for 1 hour and concentrated in vacuo to give 1.9 g (99%) of the title compound as a white solid powder, which was used for the next reaction without purification. LCMS: [M + H] + = 410.3.

Step G. tert-Butyl 4- {3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-d] pyrimidin-4-yl) - 1 N-pyrazol-1-yl] azetidin-1-yl} piperidine-1-carboxylate

- 11 036970

Vos

Into a solution of {3- [4- (7- {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1Hpyrazol-1-yl] azetidin-3- yl} acetonitrile dihydrochloride (2.6 g, 6.3 mmol), tert-butyl 4-oxo-1piperidinecarboxylate (1.3 g, 6.3 mmol) in THF (30 ml) added X, X-diisopropylethylamine (4, 4 ml, 25 mmol) and sodium triacetoxyborohydride (2.2 g, 10 mmol). The mixture was stirred at room temperature overnight. After adding 20 ml of brine, this solution was extracted with EtOAc. The extract was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified on a combined flash column eluting with 30-80% EtOAc in hexanes to give the desired product, tert-butyl 4- {3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) 1H-pyrazol-1-yl] azetidin-1-yl} piperidine-1-carboxylate. Yield: 3.2 g (86%); LCMS: [M + H] + = 593.3.

Stage H. {1-Piperidin-4-yl-3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazole -1 -yl] azetidin-3-yl} acetonitrile trihydrochloride

To a solution of tert-butyl 4- {3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1H -pyrazol-1-yl] azetidin-1-yl} piperidine-1-carboxylate (3.2 g, 5.4 mmol) in 10 ml of THF was added 10 ml of 4N hydrochloric acid. HCl in dioxane. The reaction mixture was stirred at room temperature for 2 h. Removing the solvents under reduced pressure gave 3.25 g (100%) {1-piperidin-4-yl-3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1Hpyrazol-1-yl] azetidin-3-yl} acetonitrile trihydrochloride as a white solid powder which was used directly in the next reaction. LCMS: [M + H] ⁺ = 493.3. Ή NMR (400 MHz, DMSO-d6): δ 9.42 (s, 1H), 9.21 (s, 1H), 8.89 (s, 1H), 8.69 (s, 1H), 7, 97 (s, 1H), 7.39 (d, 1H), 5.68 (s, 2H), 4.96 (d, 2H), 4.56 (m, 2H), 4.02-3.63 (m, 2H), 3.55 (s, 2H), 3.53 (t, 2H), 3.49-3.31 (3, 3H), 2.81 (m, 2H), 2.12 ( d, 2H), 1.79 (m, 2H), 0.83 (t, 2H), -0.10 (s, 9H).

Stage I. {1- {1- [3-Fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7 - {[2 (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidine-3yl} acetonitrile

Ν-Ν

A mixture of {1-piperidin-4-yl-3 - [4- (7- {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin4-yl) -1H-pyrazole-1 -yl] azetidin-3-yl} acetonitrile trihydrochloride (1.22 g, 2.03 mmol), 3-fluoro-2 (trifluoromethyl) isonicotinic acid (460 mg, 2.2 mmol), benzotriazole-3yloxytris (dimethylamino) phosphonium hexafluorophosphate (1.07 g, 2.42 mmol) and triethylamine (2.0 ml, 14 mmol) in dimethylformamide (DMF) (20.0 ml) were stirred at room temperature overnight. LCMS indicated the reaction was complete. EtOAc (60 ml) and saturated aqueous NaHCO ₃ solution (60 ml) were added to the reaction mixture. After stirring at room temperature for 10 min, the organic phase was separated and the aqueous layer was extracted with EtOAc three times. The combined organic phase was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. Purification by flash chromatography yielded the desired product, {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7 - {[2 (trimethylsilyl) ethoxy ] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-12 036970 yl} acetonitrile. LCMS: 684.3 (M + H) +.

Step J. {1- {1- [3-Fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H -pyrazol-1-yl] azetidin-3-yl} acetonitrile solution

{1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7 - {[2 (trimethylsilyl) ethoxy] methyl} -7Н-pyrrolo [2, 3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidine-3yl} acetonitrile (56 mg, 0.1 mmol) in methylene chloride (1.5 ml) trifluoroacetic acid (1.5 ml) was added ... The mixture was stirred at room temperature for 2 hours. After removal of the solvents in vacuo, the residue was dissolved in a methanol solution containing 20% ethylenediamine. After stirring at room temperature for 1 hour, the solution was purified using HPLC (Method B) to give the title compound. LCMS: 554.3 (M + H) ⁺ ; 1H NMR (400 MHz, CDCl ₃ ): 9.71 (s, 1H), 8.82 (s, 1H), 8.55 (d, J = 4.6 Hz, 1H), 8.39 (s, 1H), 8.30 (s, 1H), 7.52 (t, J = 4.6 Hz, 1H), 7.39 (dd, J1 = 3.4 Hz, J ₂ = 1.5 Hz, 1h ), 6.77 (dd, J1 = 3.6 Hz, J ₂ = 0.7 Hz, 1H), 4.18 (m, 1H), 3.75 (m, 2H), 3.63 (dd, J1 = 7.8 Hz, J2 = 3.7 Hz, 2H), 3.45 (m, 2H), 3.38 (s, 2H), 3.11 (m, 1H), 2.57 (m, 1H), 1.72 (m, 1H), 1.60 (m, 1H), 1.48 (m, 1H), 1.40 (m, 1H).

Example 2.3 - [(3- {3 - (Cyanomethyl) -3 - [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1 H-pyrazol-1 yl] azetidine-1 -yl} -8-azabicyclo [3.2.1] oct-8-yl) carbonyl] -5-fluorobenzonitrile

Stage A. tert-Butyl 3- {3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl ) -1Н-pyrazol-1-yl] azetidin-1 -yl} -8-azabicyclo [3.2.1] octane-8-carboxylate

To a solution of 3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1Hpyrazol-1-yl] azetidin-3-yl } acetonitrile dihydrochloride (2.6 g, 6.3 mmol) in THF (30 ml) was added tert-butyl 3-oxo-8-azabicyclo [3.2.1] octane-8-carboxylate (1.3 g, 6.3 mmol), N, N-diisopropylethylamine (4.4 ml, 25 mmol) and sodium triacetoxyborohydride (2.2 g, 10 mmol). The mixture was stirred at room temperature overnight and quenched by the addition of 20 ml of brine. The solution was extracted with EtOAc. The extract was dried over anhydrous Na ₂ SO ₄ . After removal of solvent, the residue was purified on a combined flash column eluting with 30-80% EtOAc in hexanes to afford the desired product, tert-butyl 3- {3- (cyanomethyl) -3- [4- (7- {[2- (trimethylsilyl ) ethoxy] methyl} -7H-pyrrolo [2,3d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -8-azabicyclo [3.2.1] octane-8-carboxylate. LCMS: 619.3 (M + H) ⁺ .

Stage B. {1 - (8-Azabicyclo [3.2.1] oct-3-yl) -3 - [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-d] pyrimidin-4-yl) -1 N-pyrazol-1-yl] azetidin-3-yl} acetonitrile trihydrochloride

- 13 036970

To a solution of tert-butyl 3- {3- (cyanomethyl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Hpyrrolo [2,3-b] pyrimidin-4-yl) -1 N-pyrazol-1-yl] azetidin-1-yl} -8-azabicyclo [3.2.1] octane-8-carboxylate (123 mg, 0.2 mmol) in THF (3 ml) was added 4N hydrochloric acid. HCl solution in dioxane (3 ml). After stirring at room temperature for 2 hours, the solution was concentrated. The resulting residue was used for the next reaction. LCMS: 519.3 (M + H) +.

Stage C. 3 - [(3- {3- (Cyanomethyl) -3- [4- (7H-pyrrolo [2,3-b] pyrimidin-4-yl) -1H-pyrazol-1yl] azetidin-1-yl } -8-azabicyclo [3.2.1] oct-8-yl) carbonyl] -5-fluorobenzonitrile.

A mixture of {1- (8-azabicyclo [3.2.1] oct-3-yl) -3- [4- (7 - {[2- (trimethylsilyl) ethoxy] methyl} -7Н-pyrrolo [2,3b] pyrimidine- 4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile (100.0 mg, 0.193 mmol), 3-cyano-5fluorobenzoic acid (31.8 mg, 0.193 mmol), benzotriazole-1- yloxitris (dimethylamino) phosphonium hexafluorophosphate (93.8 mg, 0.212 mmol) and triethylamine (0.108 ml, 0.771 mmol) in DMF (3.0 ml) was stirred at room temperature for 2 h. Purification by HPLC gave the coupling product as white powder. Found LCMS: 666.3 (M + 1) ⁺ . The white powder was dissolved in trifluoroacetic acid (2 ml) and methylene chloride (2 ml). The resulting solution was stirred at room temperature for 1 hour. The solvents were evaporated to dryness. The residue was treated with methanol (3 ml) and ethylenediamine (0.3 ml, 4 mmol) for 1 h at room temperature. Purification using HPLC Method A gave the title compound, 3 - [(3- {3 (cyanomethyl) -3 - [4- (7H-pyrrolo [2,3-b] pyrimidin-4-yl) - 1H-pyrazol-1-yl] azetidin-1-yl} -8-azabicyclo [3.2.1] oct-8-yl) carbonyl] -5-fluorobenzonitrile in the form of TPA salt. Found LCMS: 536.3 (M + H) ⁺ . 1H NMR (400 MHz, DMSO-66): δ 8.93 (s, 1H), 8.81 (s, 1H), 8.51 (s, 1H), 7.97 (s, 1H), 7, 81 (s, 1H), 7.72 (m, 3H), 7.18 (s, 1H), 4.53 (m, 2H), 3.80 (m, 1H), 3.57 (m, 6H ), 1.55-2.08 (m, 8H).

Example 3. {1- {1- [3-Fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3b] pyrimidin-4-yl) -1H -pyrazol-1-yl] azetidin-3-yl} acetonitrile adipino acid salt

Screening.

Free base {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7Hpyrrolo [2,3-b] pyrimidin-4-yl) -1H- pyrazol-1-yl] azetidin-3-yl} acetonitrile (Example 1) is an amorphous substance. A salt screening study was performed using pharmaceutically acceptable acids to form the crystalline salts of Example 1. Adipic acid was determined to give the crystalline adipic acid salt of Example 1. The initially obtained solid form (Form I) of the adipic acid salt of Example 1 with a melting point of 17 8 was selected for optimization. ° C. This form is a crystalline form, as confirmed by X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The stoichiometry of this salt was found to be 1: 1 (free base to adipic acid) by ¹ H NMR spectroscopy and elemental analysis.

Polymorphic screening study was performed on the adipate salt of Example 1. Phase equilibrium studies were performed by suspending crystals of Form I in various solvents (MeCN, CHCl ₃ , CH ₂ Cl ₂ , MIBK, MEK, acetone, toluene, hexane, heptane, THF, MTBE, EtOH, i-PrOH, n-BuOH, EtOAc, i-PrOAc at 25 or 50 ° C. At 25 ° C all solvents tested gave the same crystalline Form I after suspension. At 50 ° C, the same results were observed, except for ethanol. XRPD diagram The solid obtained from the ethanol suspension showed the presence of a free base, which can be explained by simple dissociation of the salt.The results of the phase equilibrium studies suggest that Form I is a stable crystalline form. In addition, multiple batches (gram to kilogram) of adipino acid salt Example 1 obtained to date are set to be batches of the same crystalline form (Form I) To trigger the formation of Form I during crystallization, seeds were used. However, so

- 14 036970 it was observed that Form I was obtained even by crystallization without seeding.

Receiving.

Adipic acid (790 g, 5.406 mol) was dissolved in methanol (29 L) at 16 ° C. 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 free base (2.85 kg, 5.149 mol) was added to a solution of adipic acid in methanol at 16 ° C. The reaction mixture was heated under reflux for 2 hours. The resulting reaction mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure to obtain the crude adipic acid salt. The crude adipic acid salt was dissolved in acetone (14 L) at ambient temperature. To a solution of the crude adipic acid salt in acetone, n-heptane (20 L) was added for 2 h at 18 ° C to precipitate the salt. The resulting slurry was stirred at 18 ° C for 1 hour. The salt was isolated by filtration. The wet filter cake was washed with n-heptane (6 L). The product was dried on a filter funnel under suction for 18 h to obtain the crude adipic acid salt 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 (3.572 kg, 5.105 mol, 99.2% yield) as a white crystalline solid.

The crude adipic acid salt can be further purified by recrystallization. The crude adipic acid salt (3.378 kg, 4.828 mol) was suspended in acetone (24 L) at ambient temperature. The resulting suspension was heated to 55 ° C and stirred at 50-60 ° C to obtain a clear solution. The solution was filtered through an in-line filter to remove particulate matter. To the solution was added n-heptane (24 L) at 55 ° C for 2 h to precipitate the salt. After completion of the n-heptane addition, the slurry was cooled to 30 ° C for 3 hours. The pure adipic acid salt was isolated by filtration. The wet filter cake was washed with a mixture of n-heptane and acetone (2: 1, v / v, 6.8 L). The product was dried on a filter funnel under suction for 15 h and additionally dried in a vacuum oven at 55 ° C for 42 h to obtain pure adipic acid salt 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 (3.116 kg, yield 92.2%) as a white crystalline substance.

For the adipic acid salt: So pl. 178 ° C; ¹ H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 12.02 (brs, 2H), 8.81 (s, 1H), 8.69 (s, 1H), 8.66 (d, J = 4.7 Hz, 1H), 8.42 (s, 1H), 7.90 (dd, J = 4.7, 4.7, 1H), 7.60 (dd, J = 2.3, 3.5 Hz, 1H), 7.06 (dd, J = 1.8, 3.6 Hz, 1H), 4.08 (m, 1H), 3.74 (m, 2H ), 3.57 (m, 2H), 3.55 (m, 2H), 3.39 (m, 1H), 3.25 (m, 1H), 3.07 (m, 1H), 2.56 (m, 1H), 2.19 (m, 4H), 1.77 (m, 1H), 1.62 (m, 1H), 1.48 (m, 4H), 1.36-1.12 ( m, 2H); ¹³ C NMR (100 MHz, DMSO-de) δ 174.4, 160.3, 152.2 (11 ^ = 265.7 Hz), 152.2, 150.9, 149.6, 146.3 ( ⁴ Jcf = 5.8 Hz), 139.5, 135.0 ( ² JCF = 17.3 Hz), 134.5 ( ² JCF = 35.3, 11.9 Hz), 129.2, 127.6, 126.8, 121.7, 120.6 (1JCF = 274.0 Hz, ³ Jcf = 4.8 Hz), 117.4, 113.0, 100.0, 61.4, 60.5, 57, 0, 44.2, 33.4, 28.6, 27.9, 27.2, 24.0; ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -64.54 (d, J = 15.8 Hz, 3F), -129.34 (m, 1F); Analytical values calculated for: C32H33F4N9O5: C, 54.93; H, 4.75; F, 10.86; N, 18.02; found: C, 54.68; H, 4.56; F, 10.94; N, 17.90. LCMS calculated for C ₂₆ H ₂₄ F ₄ N ₉ O (M + H) + for the free base: m / z 554.2; found 554.2.

Reverse phase HPLC determined the chemical purity to be 99.57 area%; The DSC thermogram revealed one major endothermic event with a peak onset at 175.9 °, which is thought to be related to the melting of the compound with a peak at 177.9 ° (see FIG. 1). The DSC was scanned from an initial temperature of 30 ° C to a final temperature of 280 ° C at a heating rate of 10 ° C / min. The TGA thermogram showed a slight 0.29% weight loss observed from 20 to 100 ° C and a significant 62% weight loss that was observed upon further heating from 100 to 600 ° C (see FIG. 2). The TGA thermogram was obtained by heating the sample from 20 to 600 ° C at a heating rate of 20 ° C / min. The XRPD diagram demonstrates the crystalline nature of the adipic acid salt (see FIG. 3). DSC, TGA and XRPD data were consistent with Form I.

DSC parameters: Mettler Toledo Differential Scanning Calorimetry (DSC) instrument, model no. 822; aluminum sample cuvette (40 μl); general conditions: 30-280 ° C at 10 ° C / min.

TGA parameters: TA device, model no. Q500. General initial conditions of the method: change at a rate of 20 ° C / min to 600 ° C.

XRPD conditions: instrument - X-ray powder diffractometer (XRPD) Rigaku MiniFlex; X-ray radiation from copper Cu at 1.054056A with a Kr filter; the sample powder was dispersed on a zero background sample holder; general measurement conditions:

The starting angle is 3.

Stopping angle - 45.

Sampling - 0.02.

Scanning speed - 2.

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Table 1

XRPD Data_______________

2-Theta (°) d (A) Background Height H% Area AND% Full width of the curve on semi-maxi mum level (FWHM) 3.84 22.9919 7 341 24.4 19142 100 0.955 6, 92 12.7638 169 461 33 6796 35, 5 0.25 8.78 10.0638 164 52 3.7 1571 8.2 0.513 9.28 9.5227 161 47 3.4 760 4 0.275 10, 4 8.4994 203 1399 100 15230 79, 6 0, 185 10.981 8.0506 208 135 9, 6 3688 19, 3 0.465 11.74 7,532 179 302 21, 6 6396 33.4 0.36 14.92 5, 933 165 723 51.7 15980 83.5 0.376 15, 4 5.7492 179 377 27 6733 35, 2 0.303 16,859 5.2547 295 123 8.8 843 4.4 0, 117 17.52 5, 058 249 316 22, 6 12179 63, 6 0.655 18, 68 4.7463 238 482 34.4 7294 38.1 0.257 19,861 4.4668 240 361 25, 8 5072 26, 5 0.239 20, 98 4.2309 261 547 39, 1 11823 61.8 0.368 22.12 4.0153 267 273 19, 5 6037 31.5 0.377 22.46 3.9553 280 414 29, 6 8893 46, 5 0.365 23.28 3.8178 300 546 39 10395 54.3 0.324 23, 74 3.7449 254 216 15, 5 9220 48.2 0.725 24.38 3.6481 270 256 18.3 2926 15, 3 0.194 25.062 3.5503 219 54 3, 9 791 4.1 0.249 25.979 3.427 247 212 15, 1 3384 17.7 0.272 26, 901 3.3116 241 60 4,3 1124 5, 9 0.32 27.76 3.2111 213 78 5, 6 1985 10.4 0.431 28,839 3.0933 203 170 12.1 2489 thirteen 0.249 29,841 2.9917 205 98 7 1115 5, 8 0.194 30, 94 2.8879 184 127 9, 1 5062 26, 4 0, 677 31,562 2.8324 184 66 4.7 623 3.3 0.161 32, 92 2.7185 181 125 8.9 3846 20, 1 0.522 35, 14 2.5518 182 147 10.5 4215 22 0.488 35, 62 2.5185 173 83 6 5361 28 1.093 36, 96 2.4302 178 52 3.7 1724 9 0.559 37.359 2.4051 178 89 6, 4 2358 12.3 0.45 38, 86 2.3156 173 72 5, 2 3599 18.8 0.846 39,279 2.2918 177 77 5, 5 2214 11, 6 0.486

Example 4. Pharmaceutical compositions {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-b] pyrimidine-4- yl) -1 N-pyrazol-1-yl] azetidin-3-yl} acetonitrile, adipic acid salt.

The test capsules were produced using a conventional dry mix process. Initial test batches were run on 200 mg mix weight for both 10 mg and 50 mg capsules. The composition of silicated microcrystalline cellulose was selected based on the data of solubility and stability of the composition obtained in experimental batches. {1- {1- [3-Fluoro-2 (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-b] pyrimidin-4-yl) -1H-pyrazole1 -yl] azetidin-3-yl} acetonitrile, adipic acid salt (adipic acid salt) can be obtained as shown in example 358. The composition of the experimental capsule of silicated microcrystalline cellulose is listed in table. A and B below.

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Table A

Components and composition of 10 mg capsule

Component Composition in mg / capsule Adipic acid salt 12.64 * Silicated microcrystalline cellulose * * 187.36 Capsule size 2 (white opaque) - Total 200, 0

* - salt conversion factor is 0.7911;

** - consists of 98% microcrystalline cellulose NF and 2% colloidal silicon dioxide NF.

Table B

Components and composition of a 50 mg capsule

Component Composition in mg / capsule Adipic acid salt 63.20 * Silicated Microcrystalline Cellulose ** 136.80 Capsule size 2 (white opaque) - Total 200, 0

* - salt conversion factor is 0.7911;

* * - consists of 98% microcrystalline cellulose NF and 2% colloidal silicon dioxide NF.

Mixing formula for 10 and 50 mg capsules is presented in table. C and D. Capsules are prepared in the following steps:

1. Pre-mix the required amount of adipic acid salt and an approximately equivalent amount of silicated microcrystalline cellulose (SMCC).

2. Pass the 1 step mixture through an appropriate sieve (eg 40 mesh).

3. The remaining SMCC is sieved through the same sieve used in step 2.

4. The screened 3-stage SMCC is mixed with the 2-stage mixture in a suitable mixer (eg Turbula mixer) for about 5 minutes.

5. The capsules are filled with the mixture to the specified fill weight.

Table C

Mixing formula for 225 g mix for 10 mg capsules

Component g / batch Adipic acid salt 15, 80 Silicated Microcrystalline Cellulose 209.20 Capsule size 2 (white opaque) - Total 225, 0

Table D

Mixing formula for 936 g mix for 50 mg capsules

Component g / batch Adipic acid salt 328.66 Silicated microcrystalline cellulose** 607.34 Capsule size 2 (white opaque) - Total 963, 0

Example A. In vitro assay of JAK kinase.

The compounds described herein were assayed for the inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and were purified. The catalytic activity of JAK1, JAK2 or JAK3 was

- 17 036970 analyzed by measuring the phosphorylation of biotinylated peptide. The phosphorylated peptide was detected by homogeneous time resolved fluorescence (HTRF). IC ₅₀ of the compounds were measured for each kinase in 40 microliter reactions containing enzyme, ATP and 500 nM peptide in 50 mM Tris buffer (pH 7.8) with 100 mM NaCl, 5 mM DTT, and 0.1 mg / ml (0 , 01%) BSA. For IC ₅₀ measurements, 1 mM ATP concentration in the reactions was 1 mM. Reactions were performed at room temperature for 1 h and then stopped by the addition of 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the europium-labeled antibody occurred in 40 min and the HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, MA). Table I presents data related to the compounds of the present invention.

Table I

IC ₅₀ data for JAK enzyme assay (measured at 1 mM ATP)

Example JAK1 1C ₅₀ (nM) ¹ JAK2 1C ₅₀ (nM) ² Ratio 1C ₅₀ JAK2 / JAK1 one + + + + + 24.5 2 + + 13.9 3 + + + + 49, 2

¹ - for JAK1: 5 nM or less (+);

² - for JAK2: 10 nM or less (+); > 50 nM to 100 nM (+++) and> 100 nM (++++).

Various modifications of the present invention, in addition to those described herein, will be apparent to a person skilled in the art from the previous descriptions. Such modifications are also intended to be within the scope of the appended claims. Every reference, including all patents, patent applications and publications cited in this application, is incorporated into this application by reference in its entirety.

Claims (8)

1. A method of treating a disease selected from allograft rejection and graft versus host disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound that is {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] piperidine -4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof. 2. The method according to claim 1, wherein the salt is an adipic acid salt {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile. 3. The method according to claim 1 or 2, wherein the disease is allograft rejection. 4. The method of claim 1 or 2, wherein the disease is a graft versus host disease. 5. Application of a compound representing {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidine-4 -yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a disease selected from allograft rejection and graft versus host disease. 6. Use according to claim 5, wherein the salt is an adipic acid salt {1- {1- [3-fluoro-2 (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1H-pyrazol1-yl] azetidin-3-yl} acetonitrile. 7. Use according to claim 5 or 6, wherein the disease is allograft rejection. 8. Use according to claim 5 or 6, wherein the disease is a graft versus host disease.