EA041836B1 - METHODS AND INTERMEDIATES IN THE PRODUCTION OF A JAK INHIBITOR - Google Patents

Description

Technical field

This invention relates to processes and intermediates for the preparation 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, 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 (PCs) 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 wide range of human diseases, including cancer. Cytokines, low molecular weight polypeptides or glycoproteins, regulate a variety of 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, 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, as well as signal transducers and transcriptional activators (STAT). There are four known mammalian JAKs: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as leukocyte Janus kinase; JAKL and L-JAK) and TYK2 (protein tyrosine kinase 2).

Cytokine-stimulated immune and inflammatory responses contribute to the pathogenesis of diseases: pathologies such as severe combined immunodeficiency (SCID) that result from suppression of the immune system, 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., Cheng, T. et al. (2000), Arthritis Res., 2(1):16-32).

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

The JAK/STAT metabolic pathway, and in particular all four JAKs, are 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. Many 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 implicated in inflammatory eye diseases/conditions and chronic allergic reactions.

Activation of JAK/STAT in cancers can occur by stimulating cytokines (eg, IL-6 or GM-CSF) or by reducing endogenous suppressors of JAK signaling, such as SOCS (suppressor or cytokine signaling) or PIAS (a protein-based inhibitor of activated STAT) (Boudny, V., 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 in cancer patients by further enhancing potential antitumor activity.

JAK2 tyrosine kinase may be useful in patients with myeloproliferative diseases, eg, polycythemia vera (PV), idiopathic thrombocythemia (IT), myeloid metaplasia with MMM myelofibrosis (Levin et al., Cancer Cell, vol. 7, 2005, 387-397). Inhibition of JAK2V617F kinase 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 skin 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, 113:1664-1675), 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 are elevated in rheumatoid arthritis, a disease in which this is thought to be detrimental (Fonesca, J.E. et al., Autoimmunity Reviews, 8:538-42, 2009). Since IL-6 signals at least in part through JAK1, counteracting IL-6 directly or indirectly through inhibition of JAK1 is expected to provide a beneficial clinical effect (Guschin, D.N. et al., Embo J., 14:1421, 1995; Smolen, J.S. et al., Lancet, 371:987, 2008). In addition, in some types of cancer, JAK1 has mutated as a result, leading to undesired growth of tumor cells and their survivability.

- 1 041836 (Mullighan C.G., Proc. Natl. Acad. Sci., USA, 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 have 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 can be effective as long as it is possible to prevent the unnecessary and potentially unwanted effects of inhibiting other JAK kinases.

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 (Thro) (Parganas E. et al., Cell, 93:385-95, 1998 ). Yero 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). Thro is another example of a growth factor dependent JAK2 that plays a central role in controlling the proliferation and maturation of megakaryocytes, the cells from which platelets are made (Kaushansky K., NEJM, 354:2034-45, 2006). Thus, a decrease in Thro signaling will decrease the number of megakaryocytes (megakaryocytopenia) and decrease the number of circulating platelets (thrombocytopenia). This can lead to unwanted and/or uncontrollable bleeding. Decreased inhibition of other JAKs, such as JAK3 and Tyk2, may also be desirable, as 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, 25:745-55, 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 the preparation of JAK inhibitors. The present invention addresses this and other needs.

The essence of the invention

The present invention provides, in particular, compounds of formula VII

or its salts, where R ¹ and R ² each independently represents H or C _1-6 -alkyl; or

R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1, 2, 3 or 4 C _1-4 -alkyl groups.

The present invention also relates to processes for the preparation of a compound of formula VII, which comprises contacting a compound of formula VIII

with compound of formula IX

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in the presence of a coupling agent to obtain a compound according to claim 1, where R ¹ and R ² each independently represents H or C _1-6 -alkyl; or

R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1, 2, 3 or 4 C _1-4 -alkyl groups.

The present invention further provides a compound of formula VIII

or its salt.

The present invention further provides a process for the preparation of a compound of formula VIII, comprising contacting a compound of formula VI

with a compound of formula X

or its salt in the presence of a reducing agent.

Detailed Description of the Invention

At various places in this description, the substituents of the compounds of the invention are described in groups or in ranges. In particular, this implies that the present invention includes every single subcombination of the indicated members of such groups and ranges. For example, the term C _1-6 alkyl is specifically intended to describe individually methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C 6 alkyl.

This further implies that certain features of the invention, which for clarity are described in the context of separate embodiments of the invention, can also be presented in combination in one embodiment of the invention. Conversely, various features of the invention, which for brevity are described in the context of one embodiment of the invention, may also be presented separately or in any suitable subcombination.

As used herein, the phrase optionally substituted means unsubstituted or substituted. As used herein, the term substituted means that a hydrogen atom has been removed and replaced by a substituent. It is clear that the substitution at any given atom is limited by valency.

As used herein, the term alkyl, alone or in combination with other terms, refers to a saturated hydrocarbon group, which may be straight or branched chain. In some embodiments, the alkyl group contains 1 to 12, 1 to 8, or 1 to 6 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, n-heptyl, n-octyl and the like. In some embodiments, the alkyl moiety is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, or 2,4,4-trimethylpentyl . In some embodiments of the invention, the alkyl moiety is methyl.

As used herein, heterocycloalkyl refers to a non-aromatic monocyclic ring, including cyclized alkyl or alkenyl groups, wherein one or more of the carbon atoms forming the ring is replaced by a heteroatom such as O, N, S, or a B atom.

The methods described herein may 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 ¹³ C), infrared spectroscopy, or spectrophotometry (eg UV-visible); or using chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) or other related methods.

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 manner as to allow them to interact at the molecular level to achieve a chemical or physical conversion. In some embodiments of the invention, bringing into contact includes two reactants, and one or more equivalents of the second reactant are used in relation to the first reactant. The contacting steps of the methods described herein may be carried out for a period of time and under conditions suitable to obtain the identified product.

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

The reactions of the methods described herein can be carried out in suitable solvents, which can be easily selected by a person skilled in the art of organic synthesis. Suitable solvents may be substantially non-reactive with the starting materials (reagents), intermediates at the temperatures at which the reaction takes place, 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 may be carried out in the absence of a solvent, such as 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, trichloroethylene, 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, etc.

Suitable ethereal solvents include dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, t-butyl methyl ether, mixtures thereof, and the like.

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, 2-propanol, 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-dimethyl-3,4,5,6tetrahydro-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 easily determined by a person skilled in the art. The reaction temperature will depend on, for example, the melting and boiling points of the reactants and the solvent,

- 4 041836 if present; the thermodynamics of the reaction (eg, highly exothermic reactions may be required at lower temperatures); and reaction kinetics (eg, elevated temperatures may be required to overcome a high activation energy barrier). Elevated temperature refers to a temperature 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 reactants or products that are substantially reactive with air can be carried out using air-sensitive synthetic techniques that are well known to those skilled in the art.

In some embodiments of the invention, the preparation of compounds may include the addition of acids or bases to carry out, for example, catalysis of the desired reaction or the formation of salt forms, such as acid addition salts.

For example, acids can be inorganic and organic. Inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid. Organic acids include formic acid, acetic acid, propionic acid, butanoic acid, benzoic acid, 4-nitrobenzoic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, tartaric acid, trifluoroacetic acid, propiolic acid, butyric acid, 2-butyric acid , vinylacetic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid and decanoic acid.

Examples of bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides, and aromatic amines, the alkoxides including lithium, sodium, and potassium salts of methyl, ethyl, and tert-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include the sodium and potassium salts of methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, trimethylsilyl, and cyclohexyl substituted amides.

Intermediates and products may also include salts of the compounds described herein. As used herein, the term salt refers to a salt formed by adding an acceptable acid or base to a compound described herein. In some embodiments, the salts are pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable refers to a substance that is toxicologically acceptable for use in the pharmaceutical industry and does not interact adversely with the active ingredient. Pharmaceutically acceptable salts, including mono- and bi-salts, include, but are not limited to, salts derived from organic and inorganic acids such as, but not limited to, acetic, lactic, citric, cinnamon, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, oxalic, propionic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, glycolic, pyruvic, methanesulfonic, ethanesulfonic, toluenesulfonic, salicylic, benzoic and similar known acceptable acids. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; and Journal Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein in its entirety by reference.

In preparing compounds according to the methods described herein, conventional isolation and purification steps such as concentration, filtration, extraction, solid phase extraction, recrystallization, chromatography, and the like can be used to isolate the desired product.

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 found. 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 compounds and their salts are common in the art.

- 5 041836

Methods and connections.

Accordingly, the present invention relates to a compound of formula VII

R ¹ O' ^B OR ²

VII or its salts, where R ¹ and R ² each independently represents H or C^b-alkyl; or

R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1,2,3 or 4 C1. ₄ -alkyl groups.

In some embodiments, the compound is a compound having the formula Vila

Vila or its salt.

The present invention relates to a process for the preparation of a compound of formula VII, which comprises contacting a compound of formula VIII

with compound of formula IX

N-NH

R ¹ O' OR ²

IX in the presence of a coupling agent, where R ¹ and R ² are each independently H or C^-alkyl; or

R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1, 2, 3 or 4 C1. ₄ -alkyl groups.

In some embodiments, the binding agent is

-6041836

1,8-diazabicyclo[5.4.0]undecene.

In some embodiments of the invention, from 1.05 to 1.2 equivalents of a coupling agent is used based on the compound of formula VIII.

In some embodiments of the invention, contacting a compound of formula VIII with a compound of formula IX (a) is carried out in a solvent component that contains acetonitrile;

(b) is carried out in a solvent component that contains acetonitrile at a temperature of 40 to 60°C.

In some embodiments, the reaction conditions employ 1-1.2 equivalents of a compound of formula IX based on a compound of formula VIII.

In some embodiments, the reaction conditions include contacting a compound of Formula VIII with r^N yCF ₃

IQ ^F Ν Υ

VIII with a compound of formula IXa N-NH

V o o

IXa in the presence of a coupling agent to give a compound of formula VIIa

Villa.

In some embodiments, the coupling agent is 1,8-diazabicyclo[5.4.0]undecene.

In some embodiments of the invention, the reaction conditions apply from 1.05 to 1.2 equivalents of a coupling agent based on the compound of formula VIII.

In some embodiments, the reaction conditions include contacting a compound of formula VIII with a compound of formula IXa (a) in a solvent component that contains acetonitrile; or (b) is carried out in a solvent component that contains acetonitrile at a temperature of 40 to 60°C.

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

- 7 041836

The present invention also relates to a compound of formula VIII

VIII or its salts.

The present invention also relates to a process for the preparation of a compound of formula VIII

or a salt thereof, comprising bringing into contact a compound of formula VI

with a compound of formula X

or its salt in the presence of a reducing agent.

In some embodiments, the compound of formula X, or a salt thereof, is 2-(azetidine-3-ylidene)acetonitrile hydrochloride.

In some embodiments, the reducing agent is sodium cyanoborohydride or sodium triacetoxyborohydride.

In some embodiments of the invention, from 1.5 to 2.5 equivalents of the reducing agent is used based on the compound of formula X or its salt; or use 2 equivalents of a reducing agent based on a compound of formula X or a salt thereof.

In some embodiments, the reaction conditions include contacting a compound of formula VI and a compound of formula X, or a salt thereof, in a solvent component that contains dichloromethane.

Examples

The present invention will be described in more detail using specific examples. The following examples are presented for the purpose of illustration and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to lead to essentially the same results.

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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).

scheme I 1-carboxylate (3).

Isopropanol (IPA, 200 ml), 1,8-diazabicyclo[5.4.0]undecene (DBU, 9.8 g, 64.4 mmol , 0.125 eq.), 4-(4,4,5,5-Tetraαmethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrαzol (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 a slurry reaction mixture. The resulting reaction mixture was heated at reflux for 30 min to obtain a homogeneous solution and the mixture was kept at reflux for an additional 2-3 h. After the reaction was complete, monitoring was carried out by HPLC, n-heptane (400 ml) was gradually added to the reaction mixture over 45 minutes while maintaining the mixture at reflux. The dry residue was precipitated during the addition of n-heptane. After the addition of n-heptane was complete, the mixture was gradually cooled to room temperature and stirred at ambient temperature for an additional 1 h. with nitrogen to constant weight to give tert-butyl-3-(cyanomethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 H-pyrazole -1-yl)-azetidine 1-carboxylate (3.181 g, 199.9 g theoretical, 90.5%) as a white to pale yellow solid.

For 3: 1H NMR (400 MHz, DMSO^) 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-de) 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;

C ₁₉ H ₂₉ BN ₄ O ₄ (mol. wt. 388.27), VZhMS (EI) m/e 389 (M ++ H).

tert-Butyl-3 - (4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1 H-pyrazol-1 -yl) -3 - (cyanomethyl) -azetidine-1 carboxylate (5) .

To a 1 L flask equipped with a nitrogen inlet, a thermocouple and a mechanical stirrer was added 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)azetigine-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 eq.) at room temperature. The resulting reaction mixture was degassed and filled with nitrogen 3 times before being heated to reflux and kept at reflux under nitrogen for 20-24 hours. When HPLC data indicated that the reaction was complete, the reaction mixture was cooled to 45-55° From 30 min, separation into two phases occurred, the aqueous phase was discarded. n-heptane (125 ml) was added to the organic phase over 30 min at 45-55°C. The resulting mixture was slowly cooled to ambient temperature over 1 h and stirred at ambient temperature for an additional 2 h. The solid was collected by filtration, washed with n-heptane (100 ml) and dried under vacuum at 50° C. equilibrating with nitrogen to constant weight to give tert-butyl-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-9 041836 (cyanomethyl)-azetidine -1-carboxylate (5.96.8 g, 97.7 g theoretical, 99%) as a pale yellow solid.

For 5: 1H NMR (400 MHz, DMSO^) δ 8.89 (s, 1H), 8.68 (s, 1H), 8.44 (s, 1H), 7.60 (d, J=3.5 Hz , 1Н),

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-d6) 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 (br.), 56.80, 27.99, 26.83 ppm;

C ₁₉ H ₂₁ N ₇ O ₂ (mol. wt 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).

To a 0.5 L flask equipped with a nitrogen inlet, a thermocouple, an additional funnel, and a mechanical stirrer were added tert-butyl-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H -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 (HC1) in isopropanol (55 ml, 275 mmol, 7.0 eq.) was added to the suspension over 5 minutes. The resulting reaction mixture was heated at 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 slurry. The mixture was gradually cooled to room temperature and stirred for another 1 h. The solid was collected by filtration, washed with tert-butyl methyl ether (TBME, 45 ml) and dried under vacuum at 50° C. equilibrating with nitrogen to constant weight to give the dihydrochloride salt 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3yl)acetonitrile (6, 13.6 g, 13.9 g theoretical, 98%) as an almost white to light yellow solid.

For 6: ¹ H NMR (400 MHz, D2O) 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, D ₂ O) 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;

C14H ₁₅ Cl ₂ N ₇ (C ₁₄ H ₁₃ N ₇ for free base, mol. wt 279.30), HPMS (EI) m/e 280 (M ⁺ +H).

2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1 H-pyrazol-1-yl)-1-(1-(3-fluoro-2(trifluoromethyl) isonicotinoyl)pipyridin-4-yl)azetidin-3-yl)acetonitrile (8, free base).

To a 0.5 L flask equipped with a nitrogen inlet, a thermocouple, an additional funnel, and a mechanical stirrer were added 2-(3-(4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)- 1H-pyrazol-1-yl)azetidin-3-yl)acetonitrile (6, 20 g, 56.78 mmol), dichloromethane (200 ml) and triethylamine (TEA, 16.62 ml, 119.2 mmol, 2, 1 eq.) at room temperature. The mixture was stirred at ambient temperature for 30 minutes until 1-(3-fluoro-2-(trifluoromethyl)isoisonicotinoyl)piperidin-4-one (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 minutes at ambient temperature (below 26°C). The resulting reaction mixture was stirred at ambient temperature for 2 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 the change of 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 )-1H-pyrazol-1-yl)-1-(1-(3-fluoro2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile (8 free base) as off-white solid .

For 8: 1H NMR (400 MHz, DMSO^) 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-d6) 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;

C26H23F4N9O (mol. wt 553.51), HPMS (EI) m/e 554.1 (M ⁺ +H).

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 adipate (9).

A solution of crude 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl) -1H- 10 041836 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. Then the reaction mixture was heated under reflux to obtain a solution. n-Heptane (220 ml) was gradually added to the reaction mixture at 40-50°C over 1 h. The resulting mixture was gradually cooled to room temperature over 1 h and stirred at ambient temperature for an additional 16 h. The solid was collected by filtration , washed with n-heptane (2x60 ml) and dried under vacuum at 50°C equilibrating with nitrogen to constant weight to give 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidine-4- yl)-1Hpyrazol-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.

For 9: ¹ H NMR (400 MHz, DMSO^) δ 12.16 (s, 1H), 12.05 (br. s, 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-d 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. wt. 699.66; C ₂₆ H ₂₃ F ₄ N ₉ O for free base, mol. wt. 553.51), HPMS (EI) m / e 554.0 (M++H).

Example 2 Alternative 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.

Scheme II

2-(Azetidin-3-ylidene)acetonitrile hydrochloride (2a).

To a 0.5 L flask equipped with a nitrogen inlet, a thermocouple and a mechanical stirrer were added tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate (2.30 g, 154.46 mmol) and methylene chloride (300 ml) at ambient temperature. The solution was then treated with a solution of 5 M hydrogen chloride (HCl) in a solution of isopropanol (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, which was determined by 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 ambient temperature over 3 hours to give 2-(azetidine3-ylidene)acetonitrile hydrochloride (2a, 13.7 g, 20.2 g theoretical, 67.8%) as a white solid.

- 11 041836

For 2a: ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.99 (s, 2H), 5.94 (n, J=2.5 Hz, 1H), 4.85-4.80 (m , 2H), 4.77-4.71 (m, 2H) ppm;

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 155.65, 114.54, 94.78, 55.26, 54.63 ppm;

C ₅ H ₇ ClN ₂ (mol. wt 130.58; C ₅ H ₆ N ₂ for free base, mol. wt 94.11), HPMS (EI) m/e (M++H).

2-(1-(1-(3-Fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidine-3-ylidene)acetonitrile (10).

To a 0.25 L flask equipped with a nitrogen inlet, a thermocouple and a magnetic stirrer were added 2-(azetidine-3-ylidene)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 eq) at ambient temperature. The reaction mixture was stirred at ambient temperature for 2 hours before quenching with saturated sodium bicarbonate (NaHCO ₃ ) 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 hexane under gradient elution) to give 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4yl)azetidin-3-ylidene)acetonitrile (10, 9.5 g, 12.7 g theoretical, 74 .8%) as a white solid.

For 10: ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.57 (d, J=4.7 Hz, 1H), 7.54 (t, J=4.6 Hz, 1H), 5.29 ( n, 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)m. d.;

¹³ C NMR (101 MHz, CDCl3) 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 (q.d., 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. wt 368.33), VZhMS (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) -1 H-pyrazol-1 -yl)azetidin-3-yl)acetonitrile (11).

To a 25 ml flask equipped with a nitrogen inlet, a thermocouple and a magnetic stirrer were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1, 210 mg, 1.08 mmol, 1.08 eq.), 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 1,8 diazabicyclo[5.4.0]undecene (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 onto a silica gel column for chromatographic purification (0-2.5% MeOH in ethyl acetate gradient elution) to give 2-(1-(1-(3-fluoro-2 -(trifluoromethyl)isonicotinoyl)piperidin-4-yl)-3-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)azetidine -3-yl)acetonitrile (11.263 mg, 562.4 mg theoretical, 46.7%) as a white solid.

For 11: ¹ H NMR (400 MHz, DMSO-d _b ) 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 (br.), 83.10, 61.40, 60.53 (2С), 56.49, 44.17, 38.99, 28.55, 27.82, 27.02, 24.63 ppm;

C26H31BF4N6O3 (mol. wt 562.37), HPMS (EI) m/e 563 (M ⁺ +H).

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).

To a 25 ml flask equipped with a nitrogen inlet, a thermocouple and a magnetic stirrer were added 2-(1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)-3-(4 -(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrazol-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 filled with nitrogen 3 times before heating to 85°C. The reaction mixture was stirred at 85° C. under nitrogen overnight. When the reaction was complete, as determined by HPLC, the reaction mixture was concentrated to dryness under reduced pressure and the desired product, 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H -pyrazol1-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

- 12 041836 substances by direct purification on silica gel (SiO ₂ ) column chromatography (0-10% ethyl acetate in hexane gradient elution) of the dried reaction mixture. The compound obtained by this synthetic method is identical in all comparable aspects to compound 8 obtained using the synthetic method described in Example 1 above.

Example 3 Synthesis of (3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8-azaspiro[4,5]decan-8yl)methanone.

Scheme III (3-fluoro-2-(trifluoromethyl)pyridin-4-yl) (1,4-dioxa-8-azaspiro[4,5]decan-8-yl)methanone (14).

A 30 L reactor equipped with a mechanical stirrer, addition 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-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 minutes before being saturated with solid sodium chloride (1.3 kg) and extracted with 2-methyl-tetrahydrofuran (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 mm Hg) at 50°C after removal of the drying agent, sodium sulfate (Na ₂ SO ₄ ), filtering. The yellow oil thus obtained was distilled under reduced pressure (80 mmHg, bp 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, addition funnel, thermometer and vacuum outlet was charged with 3-fluoro-2-(trifluoromethyl)isonicotinic acid (13.3.0 kg, 14.35 mol), benzotriazole-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 eq.) and N,N-dimethylformamide (DMF, 18 L) at room temperature. The resulting solution was stirred at ambient temperature for 20 min before cooling from 5 to 10°C. Then triethylamine (Et ₃ N, 4 L, 28.67 mol, 2.0 eq.) was added to the reaction mixture over 1 h 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 ) 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 min at approximately 50°C. The brown solution was withdrawn before being gradually cooled to ambient temperature (about 20°C) over 3 h with stirring and stirred at ambient temperature for 12 h. The 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 h to obtain (3-fluoro-2-(trifluoromethyl)pyridine-4- yl) (1,4-dioxa8-azaspiro[4,5]decan-8-yl)methanone (14, 3.98 kg, 4.797 kg theoretical, 83.0%) as a white solid.

For 14: ¹ H NMR (300 MHz, DMSO-d6) 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 (br. s, 2H, one NCH2 proton in the piperidine ring, one proton of the other NCH ₂ in piperidine ring, both in axial position), 3.26 (t, ³ J _HH = 5.86 Hz, 2H, one proton NCH2 in piperidine ring, one proton

- 13 041836 of another NCH2 in piperidine ring, both in equatorial position), 1.67 (d, ³ J _HH = 5.86 Hz, 2H, one proton of NCCH2 in piperidine ring, one proton of another NCCH2 in piperidine ring, both in equatorial position ), 1.58 (broad s, 2H, one NCCH2 proton in the piperidine ring, one proton of another NCCH2 in the piperidine ring, both in axial position) ppm;

¹³ C NMR (75 MHz, DMSO-d6) δ 161.03 (N-C=O), 151.16 (d, J0=266.03 Hz, CF), 146.85 (d, ⁴ Jcf=4 .32 Hz, NCH in pyridine), 135.24 (d, ² JCF=11.51 Hz, C-C=O), 135.02 (quartet, ² Jcf=34.57 Hz, NcCf ₃ ), 128, 24 (d, ⁴ Jcf=7.48 Hz, NCCH in pyridine), 119.43 (dχquartet, 1JCF=274.38 Hz, ³ Jcf=4.89 Hz, CF ₃ ), 106.74 (RSD), 64 .60 (OCCO), 45.34 (NC in piperidine ring), 39.62 (NC in piperidine ring), 34.79 (N(NCC in piperidine ring), 34.10 (NCC in piperidine ring) ppm .;

19F NMR (282 MHz, DMSO-d6) δ -64.69 (d, ⁴ Jff=15.85 Hz, F ₃ C), -129.26 (dχquartet, ⁴ Jff=15.85 Hz, ⁴ Jfh=3 .96 Hz, FC) ppm;

C14H14F4N2O3 (mol. wt. 334.27), HPMS (EI) m/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-necked round bottom flask equipped with a mechanical stirrer, thermocouple, addition funnel and nitrogen inlet was charged with (3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1,4-dioxa-8- azaspiro[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 a temperature below 10°C before treatment with 6.0N. an aqueous solution of hydrochloric acid (HCl) (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. an aqueous solution of hydrochloric acid (HCl) (1050 ml, 6.30 mol, 21.0 eq.) was slowly introduced into the reaction mixture at ambient temperature over 8 h through an addition funnel. When the reaction was complete, as monitored by HPLC, the reaction mixture was then cooled to 0°C before being treated with 30% aqueous sodium hydroxide (NaOH, 860 mL, 8.57 mmol, 28.6 eq.) in while the internal temperature was kept below 10°C. The resulting reaction mixture was warmed to room temperature before solid sodium bicarbonate (NaHCO ₃ , 85.0 g, 1.01 mol, 3.37 eq.) was added over 1 hour. The mixture was then extracted with ethyl acetate (2x1.2 L) and combined organic the phase was washed with 16% aqueous sodium chloride solution (2x800 ml) and concentrated to approximately 1.0 l using 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 was 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 dried on the filter under nitrogen under traction vacuum to give (3-fluoro-2-(trifluoromethyl)pyridine-4- yl) (1,4-dioxa8-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-d ₆ ) 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 (br. s, 2H, one NCH2 proton in piperidine ring, one proton of another NCH2 in piperidine ring, both in axial position), 3.54 ( t, ³ JHH=6.15 Hz, 2H, one proton of NCH2 in piperidine ring, one proton of another NCH2 in piperidine ring, both in 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-d6) 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 (dxquartet, 1J _CF =274.38 Hz, ^3J _cf =4.89 Hz, CF ₃ ), 45.10 (NC in piperidine ring) ppm, one carbon (NCC in the piperidine ring) is missing due to overlap with (CD ₃ ) ₂ SO;

¹⁹ F NMR (282 MHz, DMSO-d6) δ -64.58 (d, ⁴ Jff=15.85 Hz, F ₃ C), -128.90 (dxquartet, ⁴ Jff=15.85 Hz, ⁴ Jfh= 4.05 Hz, FC) ppm;

C ₁₂ H ₁₀ F4N ₂ O ₂ (mol. wt., 290.21), VZhMS (EI) m/e 291.1 (M ⁺ + H).

- 14 041836

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 an addition funnel at ambient temperature. During the initial addition, a slight endotherm was observed. The resulting reaction mixture was stirred at ambient temperature for 3 days before being heated to reflux for an additional 3 days. When TLC showed 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 give the first crop of crude title product (1516 g). The filtrate was concentrated under reduced pressure and the resulting semi-solid product was diluted with acetone (1 L). This solid was then collected by filtration to give a second crop of crude desired product (221 g). The crude product, 1-benzhydrylazetidin-3-ol hydrochloride (1737 g, 3998.7 g theoretical yield 43.4%), was pure enough to be used without further purification in the subsequent reaction.

1H NMR (300 MHz, DMSO-d ₆ ) 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 (mol. wt. 275.77; C ₁₆ H ₁₇ NO for free base, mol. wt. 239.31), HPMS (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 10% aqueous sodium carbonate (Na ₂ CO ₃ , 5 L) and dichloromethane (CH ₂ Cl ₂ , 5 L) was stirred at ambient temperature until complete dissolution of all solids. The two layers were separated and the aqueous layer was extracted with dichloromethane (CH ₂ Cl ₂ , 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 psig 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 psig) at ambient temperature for 18 hours. When HPLC showed that the reaction was complete (no more hydrogen was taken up), the reaction mixture was filtered through a pad of celite and the pad of celite 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 minimum 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, t-butyl-3hydroxyazetidine-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, 90.8%) as a colorless oil which solidified on standing at ambient temperature under vacuum.

1H NMR (300 MHz, CDCl ₃ ), 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. Saturated aqueous sodium bicarbonate solution (NaHCO ₃ , 450 ml) and aqueous sodium hypochlorite solution (NaClO, 10-13% available chlorine, 450 ml) were added while maintaining the reaction temperature between 0-5°C. After the sodium hypochlorite solution was added, the color of the reaction mixture instantly changed.

- 15 041836

As more 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 (Na2SO4). The solvent was then removed under reduced pressure to give the crude product, tert-butyl 3-oxoazetidine-1-carboxylate (48 g, 49.47 g theoretical, 97% yield), which was sufficiently pure to be used directly in the subsequent reaction without additional cleaning.

1H NMR (CDCl ₃ , 300 MHz) δ 4.65 (s, 4H), 1.42 (s, 9H) ppm.

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

Diethylcyanomethyl phosphate (745 g, 4.20 mol, 1.20 eq.) and anhydrous tetrahydrofuran (THF, 9 L) were added to a 4-necked flask equipped with thermowell, addition funnel and nitrogen protective 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 within 20 min, maintaining the reaction temperature below -5°C. The resulting reaction mixture was stirred for 3 h at −10° C. and a solution of 1-tert-butoxycarbonyl-3-azetidinone (600 g, 3.50 mol) in anhydrous tetrahydrofuran (THF, 2 L) was added over 2 h 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 solution (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 (Na2SO4). The solvent was removed under reduced pressure and the residue was diluted with dichloromethane (CH2Cl2, 4 L) until 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: ¹ H 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. wt., 194.23), VZhMS (EI) m/e 217 (M ⁺ +Na).

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

20 21 1

C _; H _t N ₂ CjHgINj CgHj.INjSi

Mol, 6006 Mo|. wt; 193.97 Mo I. Wt: 266.15 Mol Wt.: 194 0*

Example V

4-iodopyrazole (20).

A flask equipped with a nitrogen inlet, addition funnel, thermowell, and mechanical stirrer was charged with pyrazole (1.450 g, 6.62 mol) and tetrahydrofuran (THF, 5 L) at ambient temperature. The mixture was then cooled to 10°C and N-iodosuccinimide (NIS, 1490 g, 6.62 mol, 1.0 eq.) was added to the mixture in portions as a solid at approximately 10°C. The resulting reaction mixture was then stirred at ambient temperature for 1 hour (longer reaction time may be necessary depending on ambient temperature). The mixture was then filtered and the THF was removed under reduced pressure. The residue was suspended in ethyl acetate (6 L) and the insolubles were filtered off. The dark filtrate was washed successively with saturated aqueous sodium thiosulfate (2x3 L) (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 around 30°C during the night.

1H NMR (400 MHz, DMSO-dg) δ 13.17 (br. s, 111), 7.93 (br. s, 11), 7.55 (br. s, 1H) ppm;

C3H3IN2 (Mol. wt., 193.97) HPMS (EI) m/e 195 (M ⁺ + H).

1-Trimethylsilyl-4-iodopyrazole (21). A flask equipped with a reflux condenser, nitrogen inlet, mechanical stirrer, and thermowell was charged with 4-iodopyrazole (200 g, 1.03 mol) and THF (2 L) at room temperature. To this solution was added triethylamine (TEA, 158 ml, 1.13 mol, 1.1 eq.) 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

- 16 041836 was removed and the reaction mixture was warmed to room temperature. The progress of the reaction was monitored by GC and was found to be complete after about 1 h (reaction sampling must be done in the absence of air and diluted with dry solvent to prevent hydrolysis of TMS). The reaction mixture was dissolved in n-heptane (2 L) before being filtered under nitrogen. The solvent was removed from the filtrate under reduced pressure with a vented rotary evaporator under a nitrogen atmosphere. The residual oil was diluted with n-heptane (1 L) and re-concentrated. If solids formed upon addition of n-heptane, then a second filtration was necessary. The residue was then distilled under reduced pressure (70-90° C. about 0.5 Torr) using a Kugelroff distiller to afford 1-trimethylsilyl-4-iodine pyrazole (263 g, 274.1 g theoretical, 96%) as a colorless oils. This material must be stored under nitrogen at all times as the TMS group hydrolyzes rapidly. Subsequently, it was found that 1-trimethylsilyl-4-iodopyrazole can be obtained by heating iodopyrazole with 2 equivalents of hexamethyldisilazane for 1 hour.

4-(4,4,5,5-T etramethyl-1,3,2-dioxaborolan-2-yl)-1 H-pyrazole (1).

A flask equipped with a mechanical stirrer, nitrogen inlet, addition 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-cold salt bath before adding a solution of isopropylmagnesium chloride in tetrahydrofuran (2M 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 degree of metal/halogen exchange was monitored by GC and the reaction was found to be complete after about 10 minutes. Then 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2dioxaborolane (isopropylpinacolborate, 347 ml, 1.7 mol, 2.0 eq.) was added to the orange-brown solution slowly at first, keeping the temperature below 0° C, and then faster after about half of the 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 min before warming to room temperature for 1 h and stirring at ambient temperature for an additional 1 h. The reaction mixture was cooled to approximately 6°C and saturated aqueous ammonium chloride (NH4Cl, 2.2 L) was added with the temperature raised to 25°C. The mixture was stirred for 5 minutes, after which it was diluted with toluene (10 L). The layers were separated (large amount of solid present in the aqueous layer) and the organic layer was washed successively with water (6x2.2 L) and brine (2x2.2 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. Residual toluene was co-evaporated with n-heptane to give 4-(4,4,5,5-tetramethyl-1,3,2-dioxαborolan-2-yl)-1H-pyrazole (1.90.3 g, 164.9 g theoretical, 54.8%) as a white solid.

For 1: 1H NMR (400 MHz, DMSO-d ₆ ) δ 13.8 (br. s, 1H), 7.94 (s, 1H), 7.62 (s, 1H), 1.23 (s, 12H) ppm;

C ₉ H ₁₅ BN ₂ O ₂ (mol. wt., 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.

Scheme VI

4-Bromopyrazole (22).

Pyrazole (19, 34.0 g, 0.5 mol) and NBS (89.0 g, 0.5 mol, 1.0 eq.) were suspended in water (625 ml) at ambient temperature. The resulting suspension was stirred at ambient temperature overnight. Then the reaction mixture was 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 used directly in the subsequent reaction without further purification.

- 17 041836

4-Bromo-1 -(ethoxyethyl)-1 H-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 equiv.) 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 separated. The organic layer was washed with water, dried over Na ₂ SO ₄ and concentrated under reduced pressure to dryness to give 4-bromo-1-(ethoxyethyl)-III-pyrazole (113 g, 104.3 g theoretical, 97% yield) as an oil ( GC purity: 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 100 ml of a solution of iPrMgCl.LiCl (50 mmol, 1.8 eq.) 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 h, then cooled to -20°C. Methoxypinacoluorate (10.6 g, 67 mmol, 2.4 eq) was added to the reaction mixture at -20°C. The resulting mixture was stirred at 0-10°C for 1 h. 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 to yellowish solid (GC purity: 99%).

For 24: Ή NMR (DMSO-d^ 400 MHz) 8.09 (s, 1H), 8.58 (s, 1H), 7.62 (s, 1H), 5.55 (q, 1H, J= 6.1 Hz), 3.37 (br.q., 1H, J=7.1, 9.6 Hz), 3.12 (br.q., 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;

C13H ₂ 3BN ₂ O3 (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-2,3-diol (25.0 kg, 211.6 mol) and 1-(1-ethoxyethyl)-4-(4,4,5,5-tetramethyl1,3,2- dioxaborolan-2-yl)-1H-pyrazole (24, 55.0 kg, 206.7 mol) in 1,2-dichloroethane (750 kg) was slowly added a solution of HCl in MTBE (25.0 kg, 20-30% HCl) at 0-5°C. The resulting reaction mixture was stirred at 10-20°C for 3-5 hours. After the selective deprotection was completed, which was monitored by HPLC (1: below 1%), the reaction mixture was degassed and filled with nitrogen, then cooled to -15° WITH. Triethylamine (TEA, 30.0 kg, 296.5 mol) was then added to the cooled reaction mixture, adjusting to pH 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 give the crude desired product (1, 33 .75 kg, theoretical 40.11 kg, 84.1%). Then the crude target product was suspended in 1,2-dichloroethane (30 kg) and the resulting mixture was heated under reflux to obtain a clear solution. Petroleum ether (150 kg) was then added to the hot solution 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 under vacuum at 50-60°C to give 4-(4.4 ,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-pyrazole (1.31.0 kg, 40.11 kg theoretical, 77.3%) as an off-white solid which identical in all comparable aspects to the material synthesized using the synthesis method as described in Example 5 above.

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

Scheme VII

- 18 041836

4,6-Dichloropyrimidine-5-carbaldehyde (26).

A 5 L 4-necked flask equipped with a mechanical stirrer, addition funnel, condenser, thermocouple, and N ₂ sweep in an aqueous NaOH wash was charged and cooled in an ice-cold phosphorus oxychloride salt bath (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 about 100 ml of 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 left to re-cool to approximately 2°C. The residual amount of DMF was added within 2.5 h at a temperature below 8°C. The slurry became very thick, making mixing difficult. When the addition of DMF was complete, the mixture was stirred at 3-5°C for 0.5 h. 4,6-Dihydroxypyrimidine (250 g, 2.232 mol) was added in portions as a solid. After adding about one third of 4,6-dihydroxypyrimidine, the reaction mixture became thinner and a slow exotherm was observed with an increase in the reaction temperature to approximately 12°C over 0.5 h. The remaining amount of 4,6-dihydroxypyrimidine was added in portions over 0.25 h with 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 moderate exotherm was observed.

The heating grid was removed while the reaction temperature continued to rise to about 63°C and remained at that temperature for several minutes before dropping. Heating of the mixture was resumed until a low boil (about 100° C.) was reached. At about 95°C there was a constant fairly rapid evolution of HCl gas and the reaction mixture gradually became thinner and darker. After about 0.5 h, a clear brown solution was obtained when the heating temperature was slowly raised 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 POCl3 (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 separating funnel, adding ice as needed to keep the aqueous mixture near 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), saturated NaHCO ₃ aqueous solution (1 L), brine (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 at 90-100°C, 225 mTorr) to give 15.3 g of pure 4,6-dichloropyrimidine-5-carbaldehyde as a white solid which turned yellow. when stored at ambient temperature.

Ή 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 formed. Light cooling was used to keep the temperature of the reaction mixture below 26°C. The slurry was stirred at ambient temperature for 3.5 hours before filtering off the solids. The solids were 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-amino-6-chloropyrimidine-5-carbaldehyde as a yellow solid. The starting solid was filtered from the reaction mixture which contained additional 4-amino-6-chloropyrimidine-5-carbaldehyde. The product was extracted from the filtered solid by stirring in EtOAc (1.25 L) for 1.5 h, filtered, then stirred in THF (750 ml) for 1 h and filtered again. Both the EtOAc and the THF filtrate 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 give an additional 44.1 g of 4-amino-6- chloropyrimidine-5-carbaldehyde as a yellow solid. The overall 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;

C ₅ H ₄ ClN ₃ O (mol. wt., 157.56), VZhMS (EI) m/e 158 (M++H).

6-Chloro-5-(2-methoxyvinyl)pyrimidin-4-ylamine (28).

Suspension of (methoxymethyl)triphenylphosphonium chloride (276.0 g, 0.807 mol, 1.1 eq.) in THF (1.5 L)

- 19 041836 was cooled in an ice salt bath to -2°C and 1 M potassium tert-butoxide (KO'Bu) in THF (807 ml, 0.807 mol, 1.1 eq.) was added over 1.5 h at -2 down to -3°С. The dark red-orange mixture was stirred at -2 to -3°C for 1 h. 4-Amino-6-chloropyrimidine-5-carbaldehyde (115.2 g, 0.7338 mol, 1.0 eq.) to the reaction mixture as a solid using THF (200 ml) to wash the container and separating funnel. During the 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 NH ₄ Cl (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. HCl (4x250 ml) then 3N. HCl (2x250 ml). The combined HCl extracts were re-extracted with ethyl acetate (500 ml) then filtered through celite to remove insoluble material.

The filtrate was cooled in an ice-cold salt bath, adjusted to pH 8 with 6N hydrochloric acid. 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 charcoal (10 g) and silica gel (10 g) for 1 h. The mixture was filtered through celite, washing the celite with ethyl acetate (1 L) . The filtrate was concentrated by co-evaporating 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 crude product sample (2.3 g) was purified by silica gel column chromatography eluting with 0-35% EtOAc/nheptane to give 1.7 g of pure 6-chloro-5-(2-methoxyvinylyl)pyrimidin-4-ylamine in as a white solid which was a mixture of 1 to 2 E/Z isomers.

¹ H NMR (300 MHz, DMSO-d ₆ ) for the 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;

C7H ₈ ClN ₃ O (mol. wt. 185.61), HPMS (EI) m/e 186/188 (M++H).

4-Chloro-7Н-[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 under reflux. refrigerator for 7.5 hours. Upon heating, a thin suspension was formed, which gradually redissolved. When the reaction was complete, as confirmed by HPLC control, 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 h. 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 give crude 4-chloro-7H-[pyrrolo[2,3-d]pyrimidine (4.58.1 g, 58.1 g theoretical, 100%) in form of a yellow-brown solid. This crude target product was dissolved in EtOAc (1 L) at 50-55° C. and treated with activated charcoal (3 g). The mixture was filtered through celite with 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 slurry 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-d6) 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).

Example A In vitro analysis of JAK kinase.

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. Catalytic domains of human JAK1 (a.a. 837-1142) and JAK2 (a.a. 828-1132) with N-terminal tags were expressed with baculovirus in insect cells and purified. The catalytic activity of JAK1 and JAK2 was analyzed by measuring the phosphorylation of the biotinylated peptide. The phosphorylated peptide was detected by homogenous time-separated fluorescence (HTRF). IC ₅₀ compounds were measured for each kinase in 40 µl reactions that contained the 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 concentration of ATP in the reactions was 1 mM. Provo reactions

- 20 041836 Dil at ambient temperature for 1 hour and then stopped with 20 μl of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Massachusetts). Binding to the europium-labeled antibody occurred within 40 min and the HTRF signal was measured on a Fusion plate counter (Perkin Elmer, Boston, Massachusetts). 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 >10 (measured at 1 mM ATP).

EXAMPLE B Cell studies.

Cancer cell lines dependent on cytokines and hence 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, MA) quantitation. Potential off-target effects of compounds are measured in parallel using non-JAKs, which are cell line driven with the same scoring assay. All experiments were usually performed in duplicate.

The cell lines mentioned above can also be used to investigate the effects of compounds on the phosphorylation of JAK kinases or potential substrates downstream of the biochemical pathway such as the STAT, Akt, Shp2 or Erk proteins. These experiments can be performed overnight on cytokine starvation followed by a short pre-incubation with compound (2 hours or less) and cytokine stimulation of about 1 hour or less. The 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. In these experiments, normal or cancer cells can be used to investigate the effects of compounds on tumor cell survival biology or inflammatory disease mediators. be used to stimulate JAK activation leading to phosphorylation of STAT protein(s) and potentially to transcriptional profiles (as assessed by array or qPCR technology) or 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 widely known to those skilled in the art.

The compounds of this invention may further be tested in cell models designed to evaluate their efficacy and activity against JAK mutants, for example with the JAK2V617F mutation found in myeloid proliferative disorders. Such experiments often use cytokine-dependent cells of a hematologic cell line (e.g., BaF/3) that ectopically express wild-type or mutant JAK kinases (James, C. et al., Nature, 434:1144-1148; Staerk, J. et al., JBC, 280:41893-41899). Endpoints include the effects of the compounds on survival, cell proliferation, and phosphorylated JAK, STAT, Akt, or Erk proteins.

Some of the compounds of this invention can be evaluated for their activity in suppressing T cell proliferation. As such an assay, a second cytokine (ie, JAK) triggered proliferation assay, as well as a simplified immunosuppression or inhibition of immune activation assay, can be considered. The following is a brief 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 calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin) at a density of 2x106 cells/ml at 37°C for up to 2 days. To analyze IL-2 stimulated cell proliferation, T cells are first treated with phytohemagglutinin (PHA) at a final concentration of 10 μg/mL for 72 h. concentrations in culture medium in the presence of 100 U/ml human IL-2 (ProSpec-Tany TechnoGene; 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 immunocompromised mouse human tumor xenograft models. For example, a carcinogenic variant of the INA-6 plasmacytoma cell line can be used for subcutaneous inoculation in SCID mice (Burger, R. et al., Hematol. J., 2:42-53, 2001). Subsequently, tumor-bearing animals may be randomized into drug or vehicle treatment groups, and various doses of compounds may be administered by any number of conventional routes, including oral, intraperitoneal, or continuous

- 21 041836 infusion using implantable pumps. Tumor growth is monitored over time with a caliper. Subsequently, tumor samples can be taken for analysis at any time after the start of treatment, as described above (example B), in order to evaluate 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 triggered by other known kinases (eg Bcr-Abl), such as the K562 tumor model.

Example D Delayed Type Contact Hypersensitivity Skin Reaction Test in Mice.

The compounds of this invention can further be tested for their efficacy (inhibition of JAK targets) in a test model of T cell-driven delayed hypersensitivity in mice. The cutaneous delayed-type contact hypersensitivity (DTH) reaction in mice is considered a valid model for clinical contact dermatitis and other T cell-mediated immune skin disorders such as psoriasis (Immunol Today., 1998 Jan, 19(1):37-44). Numerous characteristics unite HRT in mice with psoriasis, including immune infiltrate, concomitant elevation of 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(1-2):116-21).

On days 0 and 1, Balb/c mice were sensitized by topical application of 2,4-dinitro-fluorobenzene (DNFB) antigen to the shaved belly. On the 5th day, the thickness of the ears was measured using an engineering micrometer. The measurement results were recorded and used as a baseline. Then, both ears of the animals were loaded with topical application of DNFB at a total dose of 20 μl (10 μl on the inner part and 10 μl on the outer part of the auricle) at a concentration of 0.2%. Ears were measured again 24-72 hours after exercise. Treatment with test compounds was administered during the sensitization and exercise phases (day -1 to day 7) or before and during the exercise phase (typically from day 4 pm to day 7). Treatment with test compounds (at various concentrations) was administered systemically or topically (topical application of the drug to the ears). The efficacy of the test compounds was indicated by a reduction 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) results in the formation 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 investigated and quantified. Formalin-fixed and paraffin-embedded ear sections (sampled after the loading phase in the DTH model) are 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 an HRT model for comparison. Test compounds and dexamethasone can produce similar transcriptional changes both qualitatively and quantitatively, and test compounds and dexamethasone can reduce the number of infiltrating cells. Systemic and local application of the studied 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 prophylactically or therapeutically. These models include, without limitation, 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, airway sensitization (asthma), lupus, or colitis, can be used to evaluate the therapeutic potential of compounds. according to this invention. These models are well understood in the research community and 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 eye, 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 rabbit lacrimal gland model using concanavalin A (ConA), the scopolamine model (subcutaneous or transdermal) in

- 22 041836 mice, mouse lacrimal gland model using botulinum toxin, or any of a variety of spontaneous autoimmune rodent models that lead to ocular gland dysfunction (e.g. 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 in its entirety by reference). Endpoints in these models may include histopathology of the ocular glands and the eye (cornea, etc.), as well as the classic Schirmer test or modified versions (Barabino et al.), which measure tear fluid production. Activity is assessed by administration of doses in several ways (eg, systemically or locally), which can begin before or after the onset of a measurable disease.

The agents may be evaluated in one or more preclinical models of uveitis known to those skilled in the art. These include, without limitation, models of experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (IEU). EAU experiments can be performed in rabbit, rat or mouse, and may include passive or active immunization. For example, any of a variety of retinal antigens can be used to sensitize an animal invention to an appropriate immunogenic agent, after which the animal's eyes can be loaded with the same antigen. The IED model is more acute and includes local or systemic administration of lipopolysaccharides at sublethal doses. The endpoints of the EIA and EAU models may include fundoscopy and histopathology, among others. These models are reviewed by Smith et al. (Immunology and Cell Biology, 1998, 76, 497-512, which is incorporated herein in its entirety by reference). Activity is assessed by administration of doses in several ways (eg, systemically or locally), which can begin before or after the onset of a measurable disease. In addition, some of the models listed above may develop scleritis/episcleritis, choroiditis, cyclitis, or inflammation of the iris, and thus are useful for investigating the potential activity of compounds for the therapeutic treatment of these diseases.

Additionally, the agents may be evaluated in one or more preclinical models of conjunctivitis known to those skilled in the art. These include, but are not limited to, guinea pig, rat, or mouse rodent models. Guinea pig models include models using active or passive immunization and/or immune loading protocols with 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 administration of doses in several ways (eg, systemically or locally), which can begin before or after the onset of a 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 act on signs and markers of remodeling and/or bone density (W.S.S. Jee and W. Yao, J. Musculoskel. Nueron. Interact., 2001, 1(3), 193- 207, which is incorporated herein in its entirety by reference). Alternatively, bone density and architecture can be assessed in control or compound-treated rodents in models of treatment-induced (eg, glucocorticoid) 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 effect of compounds on bone resorption and density can be assessed in the rodent arthritis models discussed above (Example E). Endpoints for all of these models may vary, but often include histological and radiological evaluation, as well as immunohistology and relevant biochemical markers of bone remodeling.

A number of embodiments of the invention have been described. However, it should be understood that various modifications can 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 appended claims.

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Claims (18)

CLAIM 1. Compound of formula VII or a salt thereof, wherein R ¹ and R ² are each independently H or C _1-6 alkyl, or R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1, 2, 3 or 4 C _1-4 -alkyl groups. 2. The compound according to claim 1 having the formula VIIa or its salt. 3. A process for the preparation of a compound of formula VII according to claim 1, which comprises contacting a compound of formula VIII with a compound of formula IX in the presence of a coupling agent to obtain a compound according to claim 1, where R ¹ and R ² each independently represents H or C _1-6 -alkyl, or R ¹ and R ² together with the two oxygen atoms to which they are attached and the boron atom to which the oxygen atoms are attached form a 5- or 6-membered heterocycloalkyl ring which is optionally substituted with 1, 2, 3 or 4 C _1-4 -alkyl groups. 4. The method according to claim 3, characterized in that the binding agent is 1,8-diazabicyclo[5.4.0]undecene. 5. The method according to any one of claims 3, 4, characterized in that from 1.05 to 1.2 equivalents of agent are used - 24 041836 that binding based on the compounds of formula VIII. 6. The method according to any one of claims 3 to 5, characterized in that bringing into contact a compound of the formula VIII with a compound of formula IX (a) is carried out in a solvent component which contains acetonitrile; (b) is carried out in a solvent component that contains acetonitrile at a temperature of 40 to 60°C. 7. Method according to any one of claims 3 to 6, characterized in that 1 to 1.2 equivalents of a compound of formula IX are used, starting from a compound of formula VIII. 8. The method according to claim 3, characterized in that it comprises bringing into contact a compound of formula VIII with a compound of formula IXa in the presence of a binding agent to obtain a compound of formula Vila 9. The method according to claim 8, characterized in that the binding agent is 1,8-diazabicyclo[5.4.0]undecene. 10. The method according to claim 8 or 9, characterized in that from 1.05 to 1.2 equivalents of the binding agent based on the compound of formula VIII are used. 11. The method according to any one of claims 8 to 10, characterized in that the contact of the compound of formula VIII with the compound of formula IXa (a) is carried out in a solvent component that contains acetonitrile; or (b) is carried out in a solvent component that contains acetonitrile at a temperature of 40 to 60°C. 12. A method according to any one of claims 8 to 11, characterized in that 1 to 1.2 equivalents of a compound of formula IXa are used starting from a compound of formula VIII. 13. Compound of formula VIII - 25 041836 VIII or its salt. 14. A process for preparing a compound according to claim 13, or a salt thereof, comprising contacting a compound of formula VI with a compound of formula X X or its salt in the presence of a reducing agent. 15. The method according to claim 14, wherein the compound of formula X or a salt thereof is 2-(azetidine-3-ylidene)acetonitrile hydrochloride. 16. The method according to claim 14 or 15, characterized in that the reducing agent is sodium cyanoborohydride or sodium triacetoxyborohydride. 17. The method according to any one of claims 14 to 16, characterized in that (a) using from 1.5 to 2.5 equivalents of a reducing agent based on a compound of formula X or a salt thereof; or (b) using 2 equivalents of a reducing agent based on a compound of formula X or a salt thereof. 18. The method according to any one of paragraphs. 14-17, characterized in that the contacting of a compound of formula VI and a compound of formula X or a salt thereof is carried out in a solvent component that contains dichloromethane. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2