WO2009048474A1 - 2,7,9-substituted purinone derivatives for immunosuppression - Google Patents

Abstract

The present invention provides novel purinone and related derivatives useful for the prevention and treatment of autoimmune diseases, inflammatory disease, mast cell mediated disease and transplant rejection. The compounds are of the general formula (I).

Description

2,7,9-SUBSTITUTED PURINONE DERIVATIVES FOR IMMUNOSUPPRESSION

FIELD OF THE INVENTION [0001] The invention relates to purinone derivatives useful as immunosuppressants.

BACKGROUND OF THE INVENTION

[0002] Immunosuppression is an important clinical approach in treating autoimmune disease and in preventing organ and tissue rejection. The clinically available immunosuppressants, including azathioprine, cyclosporine and tacrolimus, although effective, often cause undesirable side effects including nephrotoxicity, hypertension, gastrointestinal disturbances and gum inflammation. Inhibitors of the tyrosine kinase Jak3 are known to be useful as immunosuppressants (see US patent 6,313,129).

[0003] The members of the Janus kinase (Jak) family of non-receptor intracellular tyrosine kinases are components of cytokine signal transduction. Four family members have been identified to date: Jakl, Jak2, Jak3 and Tyk2. The Jaks play a key role in the intracellular signaling mediated through cytokine receptors. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for other signaling molecules, in particular members of the signal transducer and activator of transcription (STAT) family. While expression of Jakl, Jak2 and Tyk2 is relatively ubiquitous, Jak3 expression is temporally and spatially regulated. Jak3 is predominantly expressed in cells of hematopoietic lineage; it is constitutively expressed in natural killer (NK) cells and thymocytes and is inducible in T cells, B cells and myeloid cells (reviewed in Ortmann, et al., 1999 and Yamaoka, et al., 2004). Jak3 is also is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcεRI cross-linking (Malaviya and Uckun, 1999).

[0004] A specific, orally active Jak3 inhibitor, CP-690,550, has been shown to act as an effective immunosuppressant and prolong animal survival in a murine model of heart transplantation and a primate model of kidney transplantation (Changelian, et al., 2003).

[0005] Furthermore, aberrant Jak3 activity has been linked to a leukemic form of cutaneous T-cell lymphoma (Sezary's syndrome) and acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. The identification of Jak3 inhibitors has provided the basis for new clinical approaches in treating leukemias and lymphomas (reviewed in Uckun, et al, 2005). Two dimethoxyquinazoline derivatives, WHI-P131 (JANEX-I) and WHI-P154 (JANEX-2), have been reported to be selective inhibitors of Jak3 in leukemia cells (Sudbeck et al., 1999).

[0006] Jak3 has also been shown to play a role in mast cell-mediated allergic reactions and inflammatory diseases and serves as a target in indications such as asthma and anaphylaxis.

[0007] Therefore, compounds that inhibit Jak3 are useful for indications such as leukemias and lymphomas, organ and bone marrow transplant rejection, mast cell- mediated allergic reactions and inflammatory diseases and disorders.

SUMMARY OF THE INVENTION

[0008] It has now been found that compounds of general formula I are potent and selective inhibitors of Jak3:

In these compounds,

X is selected from the group consisting of hydrogen, halogen and cyano;

Ri is selected from the group consisting of substituted (C₁-C₄)alkyl, heteroarylalkyl, and (C₁-C₈)hydrocarbon; and

R₂ and R₃ are selected independently for each occurrence from the group consisting of hydrogen, fluorine and chlorine.

[0009] The members of these genera are useful in inhibiting Jak3 activity and as such are useful in indications where clinical immunosuppression is desired and in the treatment of hematological cancers. The compounds are more selective for Jak3 than for Aurora A kinases than are the corresponding compounds in which Ri is hydrogen.

[0010] In another aspect, the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of general formula I, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0011] In another aspect, the invention relates to a method for treating a disease by altering a response mediated by Jak3 tyrosine kinase. The method comprises bringing into contact with Jak3 at least one compound of general formula I.

[0012] In yet another aspect the present invention relates to a method of suppressing the immune system in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one compound of general formula I.

[0013] In a further aspect of the present invention relates to a method for treating a disease or disorder selected from an autoimmune disease, an inflammatory disease, a mast cell mediated disease, hematological malignancy and organ transplant rejection in a subject in need thereof comprising administering to a subject a therapeutically effective amount of at least one compound of general formula I.

[0014] Suppression of immune system activity is desirable for preventing or treating tissue or organ rejection following transplant surgery and for preventing and treating diseases and disorders arising from aberrant activity of the immune system, in particular autoimmune disorders and diseases. Exemplary autoimmune disorders include graft versus host disease (GVHD), insulin-dependent diabetes (Type I), Hashimoto's thyroiditis and Graves' disease, pernicious anemia, Addison's disease, chronic active hepatitis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis, scleroderma and myasthenia gravis.

[0015] The compounds of the present invention are useful in preventing and treating diseases and disorders related to mast cell-mediated allergic reactions and inflammation, such as keratoconjuctivitis sicca.

[0016] Other indications in which the Jak3 inhibitors are useful include leukemias and lymphomas.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Throughout this specification the substituents are defined when introduced and retain their definitions.

[0018] In a first aspect the invention relates to purinones and imidazopyridinones having general formula I:

[0019] In certain embodiments, X is selected from hydrogen, halogen and cyano. In other embodiments Ri is selected from substituted (Ci-C₃)alkyl, heteroarylalkyl, and (Ci-GOhydrocarbon. In yet other embodiments, R₂ and R₃ are independently selected from the group containing hydrogen, fluorine and chlorine.

[0020] Some examples of the aforementioned substituted (Ci-C₄)alkyl groups include but are not limited to:

[0021] Some examples of the aforementioned (C_t-Cg)hydrocarbons include but are not limited to:

[0022] All of the compounds falling within the foregoing parent genera and their subgenera are useful as Jak3 inhibitors. It may be found upon examination that species and genera not presently excluded are not patentable to the inventors in this application. In this case, the exclusion of species and genera in applicants' claims are to be considered artifacts of patent prosecution and not reflective of the inventors' concept or description of their invention. The invention, in a composition aspect, is all compounds of formula I except those that are in the public's possession.

Definitions

[0023] For convenience and clarity certain terms employed in the specification, examples and claims are described herein. [0024] Alkyl is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof. Lower alkyl refers to alkyl groups of from 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, (both n- propyl and isopropyl), butyl (including s-and t-butyl) and the like. Preferred alkyl groups are those of C₂₀ or below; more preferred are (Ci-C₄)alkyl. Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl and the like.

[0025] (Ci to Cs)hydrocarbon includes alkyl, cycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include phenethyl, cyclohexylmethyl, camphoryl and naphthylethyl.

[0026] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to groups containing one to four carbons. The term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the American Chemical Society, 1J196, but without the restriction of |127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups.

[0027] Acyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. One or more carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers to groups containing one to four carbons.

[0028] Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9- or 10- membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S. The aromatic 6- to 14-membered carbocyclic rings include, e.g., benzene and naphthalene, and for the purposes of the present invention, fused moieties such as tetrahydronaphthalene (tetralin), indane and fluorine, in which one or more rings are aromatic, but not all need be. The 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.

[0029] Arylalkyl refers to a substituent in which an aryl residue is attached to the parent structure through alkyl. Examples are benzyl, phenethyl and the like. Heteroarylalkyl refers to a substituent in which a heteroaryl residue is attached to the parent structure through alkyl. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl and the like.

[0030] Heterocycle means a cycloalkyl in which from one to three carbons is replaced by a heteroatom selected from the group consisting of N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized. In some contexts (other than the present) the term heterocycle may be interpreted to include heteroaryl; for the purpose of this application, heterocycle is a saturated heterocycle and does not include heteroaryl. When heteroaryl is intended, it is expressly named. Examples of heterocycles include pyrrolidine, morpholine, dioxane, tetrahydrofuran, and the like. Examples of heterocyclyl residues additionally include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxo-pyrrolidinyl, 4-piperidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, quinuclidinyl, tetrahydrofuryl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinylsulfoxide, and thiamorpholinylsulfone. A nitrogenous heterocycle is a heterocycle containing at least one nitrogen in the ring; it may contain additional nitrogens, as well as other heteroatoms.

[0031] Substituted alkyl, aryl, cycloalkyl, heterocyclyl, heteroaryl etc. refer to alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl wherein up to three H atoms in each residue are replaced with halogen, haloalkyl, hydroxy, loweralkoxy, hydroxyloweralkyl, carboxy, carboalkoxy (also referred to as alkoxycarbonyl), carboxamido (also referred to as alkylaminocarbonyl), heterocyclylcarbonyl, cyano, carbonyl, nitro, amino, alkylamino, dialkylamino, loweralkoxyamino, arylaminocarbonyl, mercapto, alkylthio, sulfoxide, sulfoxide amino, sulfone, acylamino, amidino, alkenyl, cycloalkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, heteroaryl, phenoxy, benzenesulfonyl, benzyloxy, or heteroaryloxy. When the parent is a heterocycle that allows such substitution, the term also includes oxides, for example pyridine-N-oxide, thiopyran sulfoxide and thiopyran-S,S-dioxide. As mentioned above, two hydrogens on a single carbon may be replaced by a carbonyl to form an oxo derivative. Noteworthy oxo-substituted aryl residues include tetralone (3,4-dihydronaphthalen-l(2H)-one) and indanone (2,3- dihy droinden- 1 -one) .

[0032] The terms "halogen" and "halo" refer to fluorine, chlorine, bromine or iodine.

[0033] Some of the compounds described herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present invention is meant to include all such possible isomers, as well as mixtures thereof, including racemic and optically pure forms. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be Z, E or a mixture of the two in any proportion.

[0034] The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are taken from Maehr J. Chem. Ed. 62, 114-120 (1985): solid and broken wedges are used to denote the absolute configuration of a chiral element; wavy lines indicate disavowal of any stereochemical implication which the bond it represents could generate; solid and broken bold lines are geometric descriptors indicating the relative configuration shown but denoting racemic character; and wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate absolute configuration. For

example, the graphic representation

indicates either, or both, of

the two trans enantiomers

[0035] It will be recognized that the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain an unnatural ratio of one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include ³H, ¹⁴C, ³⁵S, ¹⁸F, ³⁶Cl and ^12SI, respectively. Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e. ³H, and carbon- 14, i.e., ¹⁴C, radioisotopes are particularly preferred for their ease in preparation and detectability. Radiolabeled compounds of this invention can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent. Because of the high affinity for the JAK3 enzyme active site, radiolabeled compounds of the invention are useful for JAK3 assays. Chemical Synthesis

[0036] Terminology related to "protecting", "deprotecting" and "protected" functionalities occurs throughout this application. Such terminology is well understood by persons of skill in the art and is used in the context of processes that involve sequential treatment with a series of reagents. In that context, a protecting group refers to a group which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable. The protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or "deprotection" occurs after the completion of the reaction or reactions in which the functionality would interfere. Thus, when a sequence of reagents is specified, as it is in the processes of the invention, the person of ordinary skill can readily envision those groups that would be suitable as "protecting groups". Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T.W. Greene [John Wiley & Sons, New York, 1991], which is incorporated herein by reference.

[0037] A comprehensive list of abbreviations utilized by organic chemists appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled "Standard List of Abbreviations", is incorporated herein by reference.

[0038] In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants that are in themselves known, but are not mentioned here. The starting materials, for example in the case of suitably substituted benzimidazole ring compounds, are either commercially available, synthesized as described in the examples or may be obtained by the methods well known to persons of skill in the art

[0039] The present invention further provides pharmaceutical compositions comprising as active agents, the compounds described herein. [0040] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the compounds described herein, or physiologically acceptable salts or solvents thereof, with other chemical components such as physiologically suitable carriers and excipients.

[0041] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0042] Compounds that inhibit Jak-3 can be formulated as pharmaceutical compositions and administered to a mammalian subject, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical, transdermal or subcutaneous routes.

[0043] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate. [0044] In addition, enteric coating may be useful as it is may be desirable to prevent exposure of the compounds of the invention to the gastric environment.

[0045] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.

[0046] In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0047] For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's or Ringer's solution or physiological saline buffer. For transmucosal and transdermal administration, penetrants appropriate to the barrier to be permeated may be used in the composition. Such penetrants, including for example DMSO or polyethylene glycol, are known in the art.

[0048] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e. g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e. g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0049] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions.

[0050] The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0051] Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on many factors including the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician. The compounds of the invention may be administered orally or via injection at a dose from 0.001 to 2500 mg/kg per day. The dose range for adult humans is generally from 0.005 mg to 10 g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5 mg to 500 mg, usually around 10 mg to 200 mg. The precise amount of compound administered to a patient will be the responsibility of the attendant physician. However, the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also, the route of administration may vary depending on the condition and its severity.

[0052] As used herein, and as would be understood by the person of skill in the art, the recitation of "a compound" is intended to include salts, solvates and inclusion complexes of that compound. The term "solvate" refers to a compound of Formula I or II in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19th Ed. (1995) volume 1, page 176-177, which is incorporated herein by reference. The most commonly employed inclusion complexes are those with cyclodextrins, and all cyclodextrin complexes, natural and synthetic, are specifically encompassed within the claims.

[0053] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, benzenesulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.

[0054] The term "preventing" as used herein refers to administering a medicament beforehand to forestall or obtund an attack. The person of ordinary skill in the medical art (to which the present method claims are directed) recognizes that the term "prevent" is not an absolute term. In the medical art it is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or seriousness of a condition, and this is the sense intended herein. [0055] It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0056] The compositions may be presented in a packaging device or dispenser, which may contain one or more unit dosage forms containing the active ingredient. Examples of a packaging device include metal or plastic foil, such as a blister pack and a nebulizer for inhalation. The packaging device or dispenser may be accompanied by instructions for administration. Compositions comprising a compound of the present invention formulated in a compatible pharmaceutical carrier may also be placed in an appropriate container and labeled for treatment of an indicated condition.

Indications

[0057] The compounds of the present invention are useful in inhibiting the activity if Jak3 or in inhibiting Jak3 mediated activity and are useful as immunosuppressive agents for tissue and organ transplants, including bone marrow transplant and in the treatment of autoimmune and inflammatory diseases and of complications arising therefrom.

[0058] Hyperacute, acute and chronic organ transplant rejection may be treated. Hyperacute rejection occurs within minutes of transplantation. Acute rejection generally occurs within six to twelve months of the transplant. Hyperacute and acute rejections are typically reversible where treated with immunosuppressant agents. Chronic rejection, characterized by gradual loss of organ function, is an ongoing concern for transplant recipients because it can occur anytime after transplantation.

[0059] There are about 75 different autoimmune disorders known that may be classified into two types, organ-specific (directed mainly at one organ) and non- organ-specific (affecting multiple organs).

[0060] Examples of organ-specific autoimmune disorders are insulin-dependent diabetes (Type I) which affects the pancreas, Hashimoto's thyroiditis and Graves' disease which affect the thyroid gland, pernicious anemia which affects the stomach, Cushing's disease and Addison's disease which affect the adrenal glands, chronic active hepatitis which affects the liver; polycystic ovary syndrome (PCOS), celiac disease, psoriasis, inflammatory bowel disease (IBD) and ankylosing spondylitis.

[0061] Examples of non-organ-specific autoimmune disorders are rheumatoid arthritis, multiple sclerosis, systemic lupus and myasthenia gravis.

[0062] Type I diabetes ensues from the selective aggression of autoreactive T-cells against insulin secreting β cells of the islets of Langerhans. Targeting Jak3 in this disease is based on the observation that multiple cytokines that signal through the Jak pathway are known to participate in the T-cell mediated autoimmune destruction of β cells. Indeed, a Jak3 inhibitor, JANEX-I was shown to prevent spontaneous autoimmune diabetes development in the NOD mouse model of type I diabetes.

[0063] Graft-versus-host disease (GVHD) is a donor T-cell initiated pathological condition that frequently follows allogeneic bone marrow transplantation (BMT). Substantial experimental and clinical research have demonstrated that donor T-cells are the principal mediators and effectors of GVHD. Jak3 plays a key role in the induction of GVHD and treatment with a Jak3 inhibitor, JANEX-I, was shown to attenuate the severity of GVHD (reviewed in Cetkovic-Cvrlje and Ucken, 2004).

[0064] Mast cells express Jak3 and Jak3 is a key regulator of the IgE mediated mast cell responses including the release of inflammatory mediators. Jak3 was shown to be a valid target in the treatment of mast cell mediated allergic reaction.

[0065] Allergic disorders associated with mast cell activation include Type I immediate hypersensitivity reactions such as allergic rhinitis (hay fever), allergic urticaria (hives), angioedema, allergic asthma and anaphylaxis, i.e., "anaphylatic shock." These disorders are treated or prevented by inhibition of Jak3 activity, for example, by administration of a Jak3 inhibitor according to the present invention.

[0066] According to the present invention, the Jak3 inhibitors may be administered prophylactically, i.e., prior to onset of acute allergic reaction, or they may be administered after onset of the reaction, or at both times. [0067] Inflammation of tissues and organs occurs in a wide range of disorders and diseases and in certain variations, results from activation of the cytokine family of receptors. Exemplary inflammatory disorders associated with activation of Jak3 include, in a non-limiting manner, skin inflammation due radiation exposure, asthma, allergic inflammation and chronic inflammation.

[0068] The Jak3 inhibitors of the present invention are also useful in treating certain malignancies, including skin cancer and hematological malignancy such as lymphomas and leukemias.

[0069] The following examples will further describe the invention, and are used for the purposes of illustration only, and should not be considered as limiting the invention being disclosed.

EXAMPLES

[0070] The following abbreviations and terms have the indicated meaning throughout:

[0071] Examples 1-25 describe syntheses of certain precursors and intermediates of the invention.

Unsubstituted system: preparation of (lR,4R)-4-(tert-butyldiphenylsilyloxy)- 1,2,3,4-tetrahydronaphthalen-l-amine.

[0072] Example 1: (R)-2,2,2-Trifluoro-N-(l,2,3,4-tetrahydronaphthalen-l- yl)acetamide. A 250 ml round bottom flask was charged with of (R)- 1,2,3, 4- tetrahydronaphthalen-1 -amine (5 g, 34 mM), then purged with argon when DCM (50 ml) was added, and the solution was cooled to 0 ⁰C. Triethylamine (9.5 mL) was added by syringe followed be trifluoroacetic anhydride (7.1 mL) of was added over 5 min by syringe. The solution was then stirred overnight, slowly warming to room temp. The mixture was diluted with DCM and washed with water, 1 N HCl, and brine. The organic layer was dried over MgSO4 and concentrated in vacuo, and purified by column chromatography (eluting with 1:1 DCM/Hex), to give 6.6g (80%) of the titled compound. IH-NMR (300 MHz, CDC13) δ 7.2 (m, 3H), 7.1 (t,lH), 6.5 (broad s, IH), 5.2 (q, IH), 2.8 (q, 2H), 2.1 (m IH), 1.9 (m, 3H) ppm.

[0073] Example 2: (R)-2,2,2-Trifluoro-N-(4-oxo-l,2,3,4-tetrahydronaphthalen- l-yl)acetamide. To a solution of (R)-2,2,2-trifluoro-N-( 1,2,3, 4-tetrahydronaphthalen- l-yl)acetamide (1 g) in acetone (30 mL) at 0 °C was added a solution of MgSO4.7H2O (2.0 g) in water (15 mL). After 5 min of stirring, KMnO4 (2 g) was added in small portions over 1 hr. The mixture was then stirred overnight, slowly warming to room temp. Celite was added and the mixture was filtered. The filtrate was treated with saturated sodium metabisulfite and filtered. The filtrate was extracted with DCM several times. The combined organic extracts were washed with distilled water and brine, dried over MgSO4 and concentrated in vacuo to afford 0.96 g (91%) of the title compound, whose purity was good enough to use crude. IH- NMR (300 MHz, CDC13) δ 8.2 (d, IH), 7.7 (t,lH), 7.6 (t, IH), 7.5 (d, IH), 6.8 (broad s, IH), 5.5 (m, IH), 2.9 (m, 2H)₅ 2.6 (m IH), 2.4 (m, IH) ppm.

[0074] Example 3 : 2,2,2-Trifluoro-N-((lR,4R)-4-hydroxy-l ,2,3,4- tetrahydronaphthalen-l-yl)acetamide. (R)-2,2,2-Trifluoro-N-(4-oxo-l,2,3,4- tetrahydronaphthalen-l-yl)acetamide (0.5 g), RuCl[(R,R)-Tsdpen(p-cymene)] and DMF (25mL) were added to a flask that was purged with argon (catalyst prepared using procedure from Org Syn, VoI 82, pg 10-17, note 5). Triethylamine and HCOOH were added and the reaction mixture was stirred at 50 ⁰C overnight. Further addition of catalyst (0.06g), TEA (0.4 mL) and HCOOH (0.12 mL) were required to drive the reaction to completion after continued heating at 50oC for an additional 6-8 hr. The reaction mixture was cooled to room temp, diluted with 150 mL EtOAc, and washed with 20 mL distilled water. The organic phase was dried over MgSO4 and concentrated in vacuo, and purified by column chromatography (e luting with 1% MeOH/DCM), to give 0.417g (83%) of the titled compound. IH-NMR (300 MHz, CDC13) δ 7.5 (d, IH), 7.3 (m, 2H), 7.2 (d, IH), 6.4 (broad s, IH), 5.3 (q, IH), 4.8 (d, IH), 2.4 (m, IH), 2.2 (m IH), 1.8 (m, 2H) ppm.

[0075] Example 4: N-((lR,4R)-4-(tert-butyldiphenyIsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-2,2,2-trifluoroacetamide. tert- Butylchlorodiphenylsilane (4.4 mL) was added to a stirred solution of 2,2,2-trifluoro- N-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)acetamide (2.2 g) and imidazole (1.7 g) in DMF (35 mL). The reaction mixture was stirred at room temp overnight. The reaction mixture was diluted with EtOAc (150 mL), and washed with distilled water (20 mL). The organic phase was dried over MgSO4 and concentrated in vacuo and purified by column chromatography (eluting with 1 :1 DCM/Hex), to give 4.0 g (95%) of the titled compound. IH-NMR (300 MHz, CDC13) δ 7.7 (d, 2H), 7.6 (d,2H), 7.4 (m, 6H), 7.2 (m, 4H), 6.3 (broad s, IH), 5.2 (q, IH), 4.8 (t, IH), 2.4 (m, IH), 1.8 (m, 2H), 1.6 (m, IH), 1.0 (s, 9H) ppm.

[0076] Example 5: (lR,4R)-4-(tert-Butyldiphenylsilyloxy)-l, 2,3,4- tetrahydroiiapkthalen-1-amine. Potassium carbonate (8.3 g) in water (16 mL) was added to a solution of N-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-2,2,2-trifluoroacetamide (4 g) in MeOH (160 mL). The reaction mixture was stirred at room temp overnight then diluted with EtOAc (150 mL), and after separation, the aqueous phase was extracted several times with EtOAc. The organic phase was dried over MgSO4 and concentrated in vacuo to afford 3.1 g of the title compound, whose purity was good enough to use crude. IH- NMR (300 MHz, CDC13) δ 7.7 (d, 2H), 7.6 (d,2H), 7.4 (m, 7H), 7.2 (m, 3H), 4.8 (t, IH), 4.2 (t, IH), 3.8 (broad s, 2H), 2.4 (m, IH), 1.8 (m, 2H), 1.6 (m, IH), 1.0 (s, 9H) ppm.

Substituted systems: preparation of (lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fiuoro-

1,2,3,4-tetrahydronaphthalen-l-amine

[0077] Example 6: (S)-7-Fluoro-l,2,3,4-tetrahydronaphthaIen-l-ol. To ice bath- cooled triehtylamine (37.6 mL, 270.5 mmol, 7.4 equiv.) was added dropwise formic acid (10.4 mL, 270.5 mmol, 7.4 equiv). The reaction solution was stirred at RT for 20 min. 7-Fluoro-3, 4-dihydronaphthalen-l(2H)-one (6.0 g, 36.6 mmol, 1 equiv.) and Noyori(S,S) catalyst (560 mg, 2.5 mol%, prepared according to lit.l) were added. Most was dissolved. DMF (10 mL) was added to make the reaction homogeneous. The reaction turned to dark red and was stirred at 40 o C overnight. TLC was checked and showed no more starting material. The solvents were removed by evaporation. The residue was mixed with water (100 mL) and stirred for 30 min to result a red solid, which was collected on funnel and washed with water and small amount of cold EtOH, then was dried in vacuo overnight. A red solid product (10.1 g, 100%) was obtained. IH NMR (400 MHz, DMSO-d6) δ 7.16(dd, IH), 7.08(t, IH), 6.86-6.97(m, IH), 5.25(d, IH), 4.53(q, IH), 2.58-2.67(m, 2H), 1.82-1.90(m, 2H), 1.59-1.70(m, 2H).

[0078] Example 7: (R)-l-azido-7-fluoro-l,2,3,4-tetrahydronaphthaIene. DBU

(27.1 mL, 180.5 mmol, 3 equiv.) was added to a solution of (S)-7-Fluoro-l, 2,3,4- tetrahydronaphthalen-1-ol (10 g, 60.2 mmol, 1 equiv.) in toluene (300 mL). The resulted dark red solution was cooled in ice bath. Diphenylphosphoryl azide (38.6 mL, 180.6 mmol, 3 equiv) was added dropwise within 30 min. The reaction solution was slowly brought to RT and stirred overnight. TLC was checked and showed no more SM. The reaction was diluted with methylene chloride (200 mL). The methylene chloride layer was washed with saturated NaHCO3, brine, water, saturated NH4C1, and brine. The organic layer was separated and dried (Na2SO4), evaporated under reduced pressure to give a dark-red syrup which was purified on ISCO (80 g column, 0-2% EtOAc-hexane) to give a colorless oil product (7.9 g, 70%). IH NMR(400MHz, CDC13) δ 7.07-7.1 l(m, IH), 7.02(dd, IH), 6.91-6.96(m, IH), 4.51(t, IH), 2.69- 2.86(m, 2H), 1.76-2.05(m, 4H).

[0079] Example 8: (R)-7-fluoro-l,2,3,4-tetrahydronaphthaIen-l-amine. (R)-I- azido-7-fluoro-l,2,3,4-tetrahydronaphthalene (4.85 g) was dissolved in methanol (70 mL) and mixed with Pd/C(10%, wet, 485 mg), applied to Parr apparatus under H2(35 psi) overnight. The catalyst was removed by filtration and washed twice with MeOH. The filtrate and washings were combined and evaporated under reduced pressure to give a yellow oil product (3.82 g, 91%). IH NMR (400 MHz, DMSO-d6) δ 7.28(dd, IH), 7.06(t, IH), 6.91(m, IH), 3.74-3.76(m, IH), 2.60-2.72(m, 2H), 2.10(broad, 2H), 1.80-1.90(m, 2H), 1.45-1.67(m, 2H). 19F NMR(400 MHz, DMSO-d6) δ -117.6.

[0080] Example 9: (R)-2,2,2-trifluoro-N-(7-fluoro-l,2,3,4- tetrahydronaphthaleii-l-yl)acetamide. (R)-7-fluoro- 1,2,3, 4-tetrahydronaphthalen- 1-amine (6.13 g, 37.1 mmol, 1 equiv) was dissolved in methylene chloride (80 niL) containing triethyamine(10.3 niL, 2 equiv) and cooled in ice bath. Trifluoroacetic anhydride (7.6 mL, 55.7 mmol, 1.5 equiv) was added dropwise within 20 min. The reaction solution was stirred at RT under argon for 16 h. The reaction was diluted with dichloromethane (20 mL) and washed with saturated NaHCO3, brine and dried (Na2SO4). Solvents were removed by evaporation. The residue was purified on ISCO ( 0-20% EtOAc-hexane) to give a white solid product (7.85g, 81%). IH NMR(400 MHz, DMSO-d6) δ 9.83(s, IH), 7.15-7.20(m, IH), 7.04(m, IH), 6.88(dd, IH), 4.99- 5.02(m, IH), 2.69-2.79(m, 2H), 1.70-1.99(m, 4H).

[0081] Example 10: (R)-2,2,2-trifIuoro-N-(7-fluoro-4-oxo-l,2,3,4- tetrahydronaphthalen-l-yl)acetamide. To a solution of (R)-2,2,2-trifiuoro-N-(7- fluoro-l,2,3,4-tetrahydronaphthalen-l-yl)acetamide (3.51 g, 13.4 mmol, 1 equiv) in acetone (217 mL) was added magnesium sulfate heptahydrate (9.92 g, 40.3 mmol, 3 equiv) with water(167 mL). The mixture was stirred in ice bath. Potassium permangnate (6.37 g, 40.3 mmol, 3 equiv) was added in portions within 1 hour period. After addition, the reaction mixture was stirred at RT for 16 h. The resulting black solid was filtered off and washed with acetone. The combined filtrate and washings were diluted with CH2C12 (200 mL). The aqueous phase was separated and washed with CH2C12. The CH2C12 solution was washed with saturated Na2S2O7 solution (150 mL). The resulting black solid was filtered off. The filtrate was evaporated to remove organic solvents. The concentrate was diluted with CH2C12(400 mL). The aqueous phase was separated and washed with CH2C12. The combined CH2C12 phase was concentrated to give a white solid product (3.54 g, 96%). IH NMR (400 MHz, DMSO-d6) δ 10.00(S, IH), 8.00(q, IH), 7.30-7.36(m, IH), 7.15(dd, IH), 5.32-5.38(m, IH), 2.60-2.85(m, 2H), 2.16-2.30(m, 2H).

[0082] Example 11: 2,2,2-Trifluoro-N-((lR,4R)-7-fluoro-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl) acetamide. To ice bath- cooled triehtylamine (13.3 mL, 95.2 mmol, 7.4 equiv.) was added dropwise formic acid (3.65 mL, 95.2 mmol, 7.4 equiv.). The reaction solution was stirred at RT for 20 min. A solution of (R)-2,2,2- trifluoro-N-(7-fluoro-4-oxo-l,2,3,4-tetrahydronaphthalen-l-yl)acetamide (3.54 g, 12.9 mmol, 1 equiv) in DMF (10 ml) and Noyori catalyst(R,R form) (204.3 mg, 2.5mol%) were added and stirred at RT overnight. TLC was checked and found only small amount of product formed. The reaction solution was stirred at 40 ⁰C for 16 h. TLC showed reaction completed. The solvents were evaporated off. The residue was treated with CH2C12 to result a white solid precipitate, which was collected on funnel and washed with small amount of CH2C12 (2.15 g). The filtrate gave additional 0.58 g of product after chromatography (ISCO, 30-40% EtOAc-hexane). Total: 2.73 g (77%). IH NMR (400 MHz, DMSO-d6) δ 9.83(s, IH), 7.52(q, IH), 7.13(m, IH), 6.85(dd, IH), 5.36(d, IH), 5.05(m, IH), 4.55-4.62(m, IH), 2.06-2.14(m, 2H), 1.64- 1.80(m, 2H).

[0083] Example 12: N-((lR,4R)-4-(tert-butyIdiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthaIen-l-yl)-2,2,2-trifluoroacetamide. 2,2,2-Trifiuoro-N-((lR,4R)- 7-fluoro-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl) acetamide (2.71 g, 9.78 mmol, 1 equiv) and imidazole(1.33g, 19.56 mmol, 2 equiv) were dissolved in anhydrous DMF(40 niL) and cooled in ice bath. Tert-Butyldiphenylsilyl chloride (3.25 mL, 12.71, 1.3 equiv) was added dropwise. The reaction solution was stirred at RT for 16 h. The DMF was removed by evaporation. The residue was dissolved in EtOAc(200 mL) and washed with water, brine and dried(Na2SO4). The organic phase was evaporated to dryness to give an oil residue, which was purified by ISCO (4Og column, EtOAc-hexane 10-50%) to give a white solid product (2.16g, TLC: pure) and a mixture (3.0 g).

IH NMR (400 MHz, DMSO-d6) δ 9.1 l(s, IH), 7.26-7.30(q, IH), 7.09(m, IH), 6.98(dd, IH), 5.07-5.14(m, IH), 4.77-4.80(m, IH), 2.06-2.15(m, IH), 1.74-1.92(m, 2H), 1.56-1.669m, IH), 1.03(s, 9H).

[0084] Example 13: (lR,4R)-4-(tert-butyIdiphenylsiIyIoxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen-1-amine. A mixture of N-((lR,4R)-4-(tert- buty ldiphenylsilyloxy)-7-fluoro- 1 ,2,3,4-tetrahydronaphthalen- 1 -yl)-2,2,2- trifluoroacetamide (2.16g, 4.19 mmol, 1 equiv) and K2CO3 (4.06g, 29.3 mmol, 7 equiv) in MeOH (166 mL) plus water (16.7 mL) was stirred at 50 o C for 3 days. TLC showed no more SM. The solvents were evaporated off. The residue was dissolved in EtOAc (100 mL) and washed with water, brine and dried (Na2SO4). Evaporation of EtOAc solution gave an oil crude which was purified by ISCO (4Og column, MeOH- DCM 0-20%) to give a colorless oil product (1.5 g). A mixture from (8) was treated by the same method as above to give a colorless oil product (2.3 g). Total: 3.8 g (yield from 7 to 9: 91%). MS(ESI): 420.4; IH NMR (400 MHz, DMSO-d6) δ 7.60-7.85(m, 4H), 7.42-7.45(m, 6H), 7.27-7.33(dd, IH), 7.17-7.22(m, IH), 6.92-6.97(t, IH), 4.75(m, IH), 3.77- 3.84(m, IH), 3.33(broad, IH), 2.00-2.09(m, IH), 1.82-1.90(m, IH), 1.63-1.73(m, IH), 1.27-1.39(m, IH), 1.02(s, 9H); 19F NMR(400 MHz, DMSO-d6) δ -115.3.

[0085] Example 14: (lR,4R)-4-(tert-butyIdiphenyIsilyIoxy)-5,7-difluoro-l,2,3,4- tetrahydronaphthalen-1-amine. The title compound was synthesized in the same manner as (lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen-1 -amine. IH-NMR (300 MHz, CD3OD ) δ 7.7 (d, 2H), 7.5 (d, 2H), 7.2-7.4 (m, 6H), 6.8 (d, IH), 6.5 (t, IH), 4.9 (m, IH), 4.1 (m, IH), 2.5 (m, IH), 1.8 (m, 2H), 1.6 (m, IH), 1.0 (s, 9H) ppm.

It will be apparent that the above method is generally applicable to stereocontrolled synthesis of protected 4-hydroxytetrahydronapthalen-l -amines:

OTBDPS

Where Xl and X2 may be substituents such as hydrogen, lower alkyl, halogen, nitrile, trifluorormethyl, lower alkoxy, carboxy, carboxamido and the like.

[0086] Example 15: l-(5-nitro-4-thiocyanatopyrimidin-2-yI)benzimidazoIe. A mixture of 2-chloro-5-nitro-4-thiocyanatopyrimidine (5.0 g, 23 mmol, WO 2003 /032994) and benzimidazole (2.72 g, 23 mmol) in 200 mL of DMF was cooled to 0 C and Et3N (2.33 g, 23 mmol) was added. The ice-bath was removed and the contents were stirred for 3h at RT. The reaction mixture was diluted with water and filtered. The solid was washed with water, followed by ice-cold methanol and dried in vacuo to provide 3.1 g of l-(5-nitro-4-thiocyanatopyrimidin-2-yl)benzimidazole. IH NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1 H), 9.15 (s, 1 H), 8.90-8.80 (m, 1 H), 7.95-7.80 (m, 1 H), 7.55-7.35 (m, 2H).

Preparation of 6-fluoro- 1 -(5 -nitro-4-thiocyanatopyrimidin-2-yl)- 1 H- benzo [d] imidazole

Synthesis of tert-butyl 2-amino-4-fluorophenylcarbamate

[0087] Example 16: 4-Fluoro-2-nitro-phenyl di-tert-butyl imidodicarbonate. A catalytic amount of DMAP was added to a mixture of 4-fluoro-2-nitrobenzenamine (0.78 g) and di-tert-butyl dicarbonate (2.18 g) in DCM (20 mL) and stirred at room temperature for 15 hr. The mixture was diluted with H2O and twice extracted with DCM, the combined organics were dried, filtered and evaporated to yield the bis-BOC material (quant), IH-NMR (300 MHz, CDC13) δ 7.8 (dd, IH), 7.3 (m, 2H), 1.4 (s, 18H); 19F δ -109 ppm that was used as such in the next step.

[0088] Example 17: tert-Butyl 4-fluoro-2-nitrophenylcarbamate. (procedure: Connell, R. D.; Rein, T.; Akermark, B.; Helquist, P. J. J. Org. Chem. 1988, 53, 3845) To a stirred solution of the Bis-BOC material in DCM (20 niL) was added TFA (0.58 mL). After 3 hr the reaction was quenched with aq. NaHCO3 (5 mL), brine was added, the mixture separated and extracted with additional DCM. The combined organics were evaporated, purified via column chromatography (eluted with 7.5% EtOAc/Hex) to give the titled product (1.12 g), IH-NMR (300 MHz, CDC13) δ 9.5 (br IH), 8.5 (dd, IH), 7.9 (dd, IH), 7.3 (m, IH), 1.5 (s, 9H); 19F δ -119 ppm.

[0089] Example 18: tert-Butyl 2-amino-4-fluorophenylcarbamate. To a solution of tert-butyl 4-fluoro-2-nitrophenylcarbamate (0.34 g) in THF (30 mL) was added a premixed solution of sodium hydrosulfite (2 g) and sodium bicarbonate (Ig) in water (50 mL). MeOH (10 mL) was also added to aid solution of the mixture, which was stirred at room temperature for 30 min, when sodium chloride was added to saturate the solution. The resultant mixture was extracted with EtOAc (2x). The combined organics were dried, filtered and evaporated to yield the titled compound (quant) that was used as such for the next step. IH-NMR (300 MHz, CDC13) δ 7.5 (dd, IH), 6.6 (dd, IH), 6.5 (m, IH), 6.4 (br IH), 4.7 (br 2H), 1.5 (s, 9H); 19F δ -119 ppm; MH+ = 227 (minor) 127 (-BOC), 171 (-tBu).

Synthesis of 6-fluoro-l-(5-nitro-4-thiocyanatopyrimidin-2-yl)-lH-benzo[d]imidazole

[0090] Example 19: 2-Chloro-5-nitro-4-thiocyanatopyrimidine. (compound known, e.g. WO 2003/032994) Potassium thiocyanate (0.97 g, 10 mM) was added to a solution of 2,4-dichloro-5-nitropyrimidine (1.94 g 10, mM) in EtOH (40 niL) cooled to 0 ⁰C via an ice bath. The solution was stirred at 0⁰C for 30 min, then the bath was removed and the resulting suspension allowed to come to RT over 60 min, when water (100 mL) was added. The precipitate was collected via filtration, washed with ice cold water, dissolved with DCM, dried (MgSO4), filtered and evaporated to yield the titled compound (1.7 g). NMR CDC13 IH δ 9.4 (s, IH) ppm.

[0091] Example 20: tert-Butyl 4-fluoro-2-(5-nitro-4-thiocyanatopyrimidin-2- ylamino)phenylcarbamate. Potassium carbonate (207 mg) was added to a stirred solution of 2-chloro-5-nitro-4-thiocyanatopyrimidine (108 mg) and tert-butyl 4- fluoro-2-nitrophenylcarbamate (113 mg) in ACN (5 mL) and stirred for 15 hr. The solution was diluted with brine and extracted with EtOAc (2x). The combined organics were evaporated and purified via column chromatography, elution with 30 % EtOAc/Hex gave the titled compound (144 mg, 71 % yield) IH-NMR (300 MHz, DMSO-d6) δ 10.5 (br s, IH), 9.3 (br s, IH), 8.9 (br s, IH), 7.7-7.4 (m, 2H), 7.1 (br s, IH), 1.5 (s, 9H), 1.5 (s, 9H) ppm; MH+ = 407, 307 (-BOC), 351 (-tBu).

[0092] Example 21: 6-FIuoro-l-(5-nitro-4-thiocyanatopyrimidin-2-yl)-lH- benzo[d]imidazole. tert-Butyl 4-fluoro-2-(5-nitro-4-thiocyanatopyrimidin-2- ylamino)phenylcarbamate (4.06g) was dissolved in 30% TFA/DCM (5OmL) and stirred until no starting material remained (90 min). The reaction solvents were removed, to yield crude 5-fluoro-Nl-(5-nitro-4-thiocyanatopyrimidin-2-yl)benzene- 1,2-diamine (MH+ = 306) as a TFA salt that was used immediately as such in the next step.

Trimethyl ortho formate (15 mL) and MeOH (100 mL) were added to the above diamine and the solution was stirred for 16 hrs. The resulting orange ppt was collected via filtration, washed with MeOH and dried under reduced pressure to yield the titled compound (2.62 g), IH-NMR (400 MHz, d6-DMSO) δ 9.2 (s, IH), 9.1 (s, IH), 8.5 (dd, IH), 7.8 (dd, IH), 7.3 (dd, IH) ppm; MH+ = 317. Some similar thiocyanatopyrimidines, such as tert-butyl 4-chloro-2-(5-nitro-4- thiocyanatopyrimidin-2-ylamino)phenylcarbamate, tert-butyl 2-(5 -nitro-4- thiocyanatopyrimidin-2-ylamino)-4-(trifluoromethoxy)phenylcarbamate, and tert- butyl 2-(5 -nitro-4-thiocy anatopyrimidin-2-ylamino)-4-

(trifluoromethyl)phenylcarbamate, were prepared via procedures described above for the synthesis of tert-butyl 4-fluoro-2-(5-nitro-4-thiocyanatopyrimidin-2- ylamino)phenylcarbamate, from corresponding 4-chloro-2-nitrobenzenamine, 2-nitro- 4-(trifluoromethyl)benzenamine and 2-nitro-4-(trifluoromethoxy)benzenamine.

Preparation of 3-(5-nitro-4-thiocyanatopyrimidin-2-yl)-3H-benzo[d]imidazole-5- carbonitrile:

[0093] Example 22: 4-(2,4-Dimethoxybenzylamino)-3-nitrobenzonitrile. A solution of 4-fluoro-3-nitrobenzonitrile (5.0 g) in THF (100 mL) was treated with DIEA (6.3 mL) and 2,4-dimethoxybenzylamine (5.0 mL), and then stirred for 24 h. The solvent was evaporated and the crude mixture was dissolved in EtOAc (100 mL). The solution was washed once with 1 M HCl and twice with saturated aqueous NaCl (100 mL each). The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuo. Column chromatography (20% EtOAc / DCM) provided 9.25 g of the title compound.

[0094] Example 23: 4-(2,4-DimethoxybenzyIamino)-3-aminobenzonitrile. A solution of 4-(2,4-dimethoxybenzylamino)-3-nitrobenzonitrile (4.54 g) in THF (400 mL) was treated with a solution of sodium hydrosulfite (20 g) and sodium bicarbonate (10 g) in distilled water (350 mL). Enough methanol was immediately added (50 mL) to maintain a homogeneous solution. After 15 minutes, EtOAc (500 mL) and saturated aqueous NaCl (500 mL) were added and the organic layer was separated. The aqueous layer was extracted again with 400 mL EtOAc. The combined organic layers were washed with saturated aqueous NaCl (500 mL) and separated. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to provide 4.33 g of the title compound.

[0095] Example 24: 4-(2,4-DimethoxybenzyIamino)-3-(5-nitro-4- thiocyanatopyrimidin-2-ylammo)benzonitrile. A solution of 4-(2,4- dimethoxybenzylamino)-3-aminobenzonitrile (3.9 g) in acetonitrile (100 mL) was cooled to 0 ⁰C and treated with potassium carbonate (6.3 g) followed by a solution containing 3 g of 2-chloro-5-nitro-4-thiocyanatopyrimidine (WO 2003/032994) in acetonitrile (50 mL). The mixture was stirred for 30 minutes at 0 ⁰C and 30 minutes at room temperature resulting in the formation of a precipitate. The mixture was quenched at 0 ⁰C by the addition of 4% acetic acid (150 mL) and filtered. The precipitate was swirled in 100 mL acetonitrile and filtered again. The precipitate was washed with acetonitrile, which resulted in the slow dissolution of product into the filtrate. After air-drying, 1.5 g of the title compound remained as the precipitate cake. The filtrate was extracted with EtOAc, dried over Na2SO4, filtered, and concentrated in vacuo. Column chromatography (0->20% EtOAc / DCM) and recrystallization from acetonitrile provided 0.415 g of additional title compound.

[0096] Example 25: 3-(5-Nitro-4-thiocyanatopyrimidin-2-yI)-3H- benzo[d]imidazole-5-carbonitrile. To a solution of 0.58 g (1.25 mmol) 4-(2,4- dimethoxybenzylamino)-3-(5-nitro-4-thiocyanatopyrimidin-2-ylamino)benzonitrile in 10 niL 30% (v/v) TFA in DCM was added 0.01 mL of triethylsilane. The mixture was stirred for 0.5 hr, LCMS indicated the completion of de-protection. A red residue was obtained after removing the volatiles on rotary evaporator, and was suspended (majority dissolved) in 10 mL 1:1 trimethyl orthoformate: MeOH. The resulting mixture was stirred for 2 hrs at room temperature. Yellow solids were precipitated. Desired product (0.38 g, -95% overall yield) was obtained after suction filtration and washing with cold MeOH twice. IH-NMR (300 MHz, CDC13 + 10% CD3OD) δ 9.5 (s, IH), 9.2 (s, IH), 9.1 (s, IH), 7.9 (d, IH), 7.7 (d, IH) ppm; MS (ESI), m/z 324 ([M₊H]₊).

Preparation of 2-(6-fluoro- 1 H-benzo[d]imidazol- 1 -yl)-9-(( lR,4R)-4-hydroxy- 1 ,2,3 ,4- tetrahydronaphthalen-l-yl)-7-methyl-7H-purin-8(9H)-one.

[0097] Example 26: N-((lR,4R)-4-(tert-Butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-5- nitropyrimidin-4-amine. A solution of (lR,4R)-4-(tert-butyldiphenylsilyloxy)- 1, 2,3 ,4-tetrahydronaphthalen-l -amine (1.8 g) and DIEA (1.4 mL) in anhydrous THF (10 mL) were added via syringe over 1 min to 6-fluoro-l-(5-nitro-4- thiocyanatopyrimidin-2-yl)-lH-benzo[d]imidazole (1.27 g, 4 mM) in THF at 0 ⁰C. The resulting solution was allowed to warm slowly to RT overnight, then partitioned between water and DCM. Column purification (ISCO, DCM -> 2% MeOH/DCM) gave the titled product (2.5 g).

[0098] Example 27: 9-((lR,4R)-4-(tert-ButyldiphenyIsiIyloxy)-l,2,3,4- tetrahydronaphthaIen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-7H-purin- 8(9H)-one. Under a flush of Ar, a catalytic amount of a Raney Ni solution in water (« 1 mL) was added to a solution of N-((lR,4R)-4-(tert-butyldiphenylsilyloxy)- 1 ,2,3 ,4-tetrahydronaphthalen- 1 -yl)-2-(6-fluoro- 1 H-benzo[d]imidazol- 1 -yl)-5- nitropyrimidin-4-amine (450 mg) in THF (30 mL). The mixture was capped with a septum stopper, briefly evacuated under low vacuum and then hydrogen was added (balloon). The resulting suspension was stirred at RT for 15 hr, when the H2 balloon was removed, mixture evacuated and filtered through a plug of celite, that was thoroughly rinsed with THF, DCM and MeOH. Evaporation of the solvents under vacuum afforded N4-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen- 1 -y l)-2-(6-fluoro- 1 H-benzo [d] imidazol- 1 -yl)pyrimidine-4, 5 - diamine (MH+ = 629) that was used as such.

The above diamine was dissolved in anhydrous THF and treated with an excess of l,l'-carbonyldiimidazole (3+ eq.) and stirred under an Ar atmosphere for 15 hr. Upon completion of the reaction the mixture was partition between water and DCM, separated and solvents removed under reduced pressure. Purification via column chromatography (elution with 2.5 and 4 % MeOH/DCM) yielded the titled product (0.36 g), MH+ = 655.

[0099] Example 28: 9-((lR,4R)-4-(tert-butyldiphenyIsilyIoxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazoI-l-yI)-7-methyI-7H- purin-8(9H)-one. A mixture of 9-((lR,4R)-4-(tert-Butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen- 1 -yl)-2-(6-fluoro- 1 H-benzo[d]imidazol- 1 -yl)-7H-purin-8(9H)- one (0.19 g), 2-tert-butylimino-2-diemylamino-l,3-dimethyl-perhydro-l,3,2- diazaphosphorine on polystyrene (0.5 g, 2.2 mM/g) and iodomethane (0.5 mL) in ACN (3 mL) was slowly stirred for 15 hr at RT, then filtered. The solid residue was thoroughly rinsed with DCM and MeOH. Evaporation of the solvents and purification through a short column (elution with 2.5 % MeOH/DCM) gave the titled compound (0.12 g), MH+ = 669.

[00100] Example 29: 2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-7H-purin-8(9H)-one. A mixture of the above compound and CsF (xs, 90 mg) in DMF were stirred and heated at 55 ⁰C for 15 hr at which time LC analysis indicated no SM remained. The cooled mixture was treated with water and extracted twice with DCM. The combined organics were concentrated and purified via column chromatography (elution with 2.5 and 4 % MeOH/DCM) to yield the titled product (55 mg), IH-NMR (400 MHz, CDC13) δ 8.9 (s, IH), 8.2(s, IH), 7.8 (d, IH), 7.7 (dd, IH), 7.5 (d, IH), 7.3 (t, IH), 7.1 (m, 2H), 6.9 (d, IH), 5.9 (dd, IH), 5.2 (dd, IH), 3.6 (s, 3H), 2.6-2.3 (m, 3H), 1.9 (m, lH) ppm; MH+ = 430. Preparation of 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl- 8-oxo-8,9-dihydro-7H-purin-2-yl)-3H-benzo[d]imidazole-5-carbonitrile.

[00101] Example 30: 3-(4-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-ylamino)-5-nitropyrimidin-2-yl)-3H-benzo[d]imidazole- 5-carbonitrile. A solution of (lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-1 -amine (4.0 g) and DIEA (5.3 mL) in anhydrous THF (20 mL) were added via syringe over 1 min to 6-cyano-l-(5-nitro-4-thiocyanatopyrimidin-2- yl)-lH-benzo[d]imidazole (3.43 g, 10.601 mM) in THF at 0 ⁰C. The resulting solution was allowed to warm slowly to RT overnight, then partitioned between water and DCM. Column purification (1% MeOH/DCM) gave the titled product (5.21 g), MH+ = 666.

[00102] Example 31: 3-(9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitriIe. Under a flush of Ar, 0.52 g of 5% Pt/C (sulfided) was added to a solution of 3-(4-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-ylamino)-5-nitropyrimidin-2-yl)-3H-benzo[d]imidazole-5- carbonitrile (5.2 g) in 3:1 EtOAc/MeOH (30 mL), in a Parr hydrogenation vessel. The mixture was then evacuated under vacuum / filled with hydrogen 3 times. The resulting suspension was stirred at RT for 15 hr under 40 psi of hydrogen. The mixture was then evacuated, air was slowly let in, and then it was filtered through a plug of ce lite, that was thoroughly rinsed with EtOAc and MeOH. Evaporation of the solvents under vacuum afforded 3-(5-amino-4-((lR,4R)-4-(tert- butyldiphenylsilyloxy)-l,2,3,4-tetrahydronaphthalen-l-ylamino)pyrimidin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile (MH+ = 636) that was used as such.

The above diamine was dissolved in anhydrous THF and treated with an excess of 1 , 1 '-carbonyldiimidazole (3+ eq.) and stirred under an Ar atmosphere for 15 hr. Upon completion of the reaction the mixture was partition between water and DCM, separated and solvents removed under reduced pressure. Purification via column chromatography (elution with 1 % MeOH/DCM) yielded the titled product (4.43 g), MH+ = 662.

[00103] Example 32: 3-(9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yI)-3H- benzo [d] imidazole-5-carbonitrile. A mixture of 3 -(9-(( 1 R,4R)-4-(tert- butyldiphenylsilyloxy)-l,2,3,4-tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H- purin-2-yl)-3H-benzo[d]imidazole-5-carbonitrile (3.85 g), 2-tert-butylimino-2- diethylamino-l,3-dimethyl-perhydro-l,3,2-diazaphosphorine on polystyrene (9.6 g, 2.2 mM/g) and iodomethane (3.6 mL) in ACN (20 mL) was stirred for 15 hr at RT, then filtered. The solid residue was thoroughly rinsed with DCM and MeOH. Evaporation of the solvents and purification through a short column (elution with 1 % MeOH/DCM) gave the titled compound (3.90 g), MH+ = 676.

[00104] Example 33: 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthaIen-l- yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H-benzo[d]imidazole-5- carbonitrile. A mixture of 3-(9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-l,2,3,4- tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile (0.18 g) and CsF (xs, 60 mg) in DMF were stirred and heated at 55 ⁰C for 15 hr at which time LC analysis indicated no SM remained. The cooled mixture was treated with water and extracted twice with DCM. The combined organics were concentrated and purified via column chromatography (elution with land 2 % MeOH/DCM) to yield the titled product (100 mg), IH-NMR (300 MHz, CDC13) δ 9.1 (s, IH), 8.4 (s, IH), 8.3 (s, IH), 8.0 (d, IH), 7.9 (d, IH), 7.5 (d, IH), 7.4 (t, IH), 7.2 (t, IH), 7.0 (d, IH), 6.0 (dd, IH), 5.4 (dd, IH), 3.7 (s, 3H), 2.8-2.4 (m, 3H), 2.1 (q, IH), 1.8 (br s, IH) ppm; MH+ = 438.

[00105] Example 34: 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l- yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H-benzo[d]imidazole-5-carbonitrile.

Prepared in a similar fashion to 3-(9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile, but without the penultimate alkylation step before Cesium Fluoride deprotection.. IH-NMR (300 MHz, CDC13) δ 9.1 (m, 2H), 8.4 (d, 2H), 8.1 (d, IH), 8.0 (d, IH), 7.7 (d, IH), 7.4 (t, IH), 7.2 (t, IH), 7.0 (d, IH), 6.1 (dd, IH), 5.5 (dd, IH), 2.8-2.4 (m, 4H), 2.2 (q, IH), 1.5 (bs, IH) ppm; MH+ = 424.

[00106] Example 35: 3-(7-allyl-9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with Allyliodide used to alkylate instead of Iodomethane. IH-NMR (300 MHz, CDC13) δ 8.9 (s, IH), 8.2 (s, IH), 8.1 (s, IH), 7.8 (d, IH), 7.7 (d, IH), 7.5 (d, IH), 7.3 (t, IH), 7.0 (t, IH), 6.8 (d, IH), 5.9 (m, 2H), 5.3 (m, 3H), 4.6 (d, 2H), 2.6-2.3 (m, 3H), 2.0 (q, IH), 1.5 (br s, IH) ppm; MH+ = 464.

[00107] Example 36: Methyl 2-(2-(6-cyano-lH-benzo[d]imidazol-l-yl)-9- ((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydropurin- 7-yl)acetate. Prepared in a similar fashion to 3-(9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphmalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile, but with Methyl bromoacetate used to alkylate instead of Iodomethane. IH-NMR (300 MHz, CDC13) δ 8.9 (s, IH), 8.2 (s, IH), 8.1 (s, IH), 7.8 (d, IH), 7.7 (d, IH), 7.5 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 6.8 (d, IH), 5.9 (dd, IH), 5.3 (m, IH), 4.7 (s, 2H), 3.8 (s, 3H), 2.6-2.3 (m, 3H), 2.0 (q, IH), 1.5 (br s, IH) ppm; MH+ = 496.

[00108] Example 37: 3-(7-ethyI-9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yI)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazoIe-S-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with Iodoethane used to alkylate instead of Iodomethane. IH-NMR (300 MHz, CDC13) δ 9.0 (s, IH), 8.3 (s, IH), 8.2 (s, IH), 7.9 (d, IH), 7.8 (d, IH), 7.6 (d, IH), 7.3 (t, IH), 7.2 (t, IH), 6.9 (d, IH), 5.9 (dd, IH), 5.3 (dd, IH), 4.2 (q, 2H), 2.6-2.3 (m, 3H), 2.0 (q, IH), 1.5 (q, 4H) ppm; MH+ = 452.

[00109] Example 38: 3-(7-(cyclopropylmethyl)-9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthaIen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazoIe-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with (Bromomethyl)cyclopropane used to alkylate instead of Iodomethane. IH-NMR (300 MHz, CDC13) δ 9.0 (s, IH), 8.3 (d, 2H), 7.9 (d, IH), 7.8 (d, IH), 7.6 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 6.9 (d, IH), 5.9 (dd, IH), 5.3 (dd, IH), 3.9 (d, 2H), 2.7-2.3 (m, 4H), 2.0 (q, IH) 1.6 (bs, IH), 0.7 (d, 2H), 0.5 (d, 2H) ppm; MH+ = 478.

[00110] Example 39: 3-(7-(cyanomethyl)-9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. A mixture of 3-(9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purm-2-yl)-3H-benzo[d]imidazole- 5-carbonitrile (0.08 g), 2-tert-butylimino-2-diethylamino-l,3-dimethyl-perhydro- 1,3,2-diazaphosphorine on polystyrene (0.09 g, 2.2 niM/g) and Bromoacetonitrile (0.1 mL) in ACN (10 niL) was stirred for 15 hr at RT, then filtered. The solid residue was thoroughly rinsed with DCM and MeOH. Evaporation of the solvents and purification through a short column (elution with 1 % MeOH/DCM) gave the titled compound (0.045 g), IH-NMR (300 MHz, CDC13) δ 9.1 (s, IH), 8.5 (s, IH), 8.3 (s, IH), 8.1 (d, IH), 8.0 (d, IH), 7.7 (d, IH), 7.5 (t, IH), 7.2 (t, IH), 7.0 (d, IH), 6.0 (dd, IH), 5.4 (m, IH), 5.1 (s, 2H), 2.8-2.4 (m, 3H), 2.1 (q, IH) 1.6 (bs, IH) ppm; MH+ = 463.

[00111] Example 40: 2-(2-(6-cyano-lH-benzo[d]imidazoI-l-yl)-9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydropurin-7- yl)acetamide. Prepared in a similar fashion to 3-(9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile, but with 2-Bromoacetamide used to alkylate instead of Iodomethane. ¹H-NMR (300 MHz₃ CDCl₃) δ 9.0 (s, IH), 8.9 (s, IH), 8.1(s, IH), 7.8 (d, IH), 7.5 (m, 2H), 7.2 (m, 3H), 5.5 (t, IH), 4.7 (t, IH), 4.1 (s, 2H), 2.3 (m, IH), 2.2 (m, IH), 1.9 (q, 2H) ppm; MH⁺ = 481.

[00112] Example 41: 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l- yl)-7-(3-methoxypropyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with l-Bromo-3-methoxypropane used to alkylate instead of Iodomethane. ¹H-NMR (300 MHz, CDCl₃) δ 8.9(s, IH), 8.2 (m, 2H), 7.9 (d, IH), 7.8 (d, IH), 7.5 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 6.8 (d, IH), 5.9 (dd, IH), 5.3 (dd, IH), 4.1 (t, 2H), 3.4 (t, 2H), 3.3 (s, 3H), 2.6-2.2 (m, 3H), 2.1-1.8 (m, 3H) 1.6 (bs, IH) ppm; MH⁺ = 496.

RT

17

Synthesis of (18a) is used to demonstrate the synthetic method of the series of compounds.

[00113] Example 42: N-((lR,4R)-4-(tert-butyldiphenyIsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-5- Ditropyrimidin-4-amine. 6-Fluoro-l-(5-nitro-4-thiocyanatopyrimidin-2-yl)-lH- benzo[d]imidazole (498 mg, 1.57 mraol, 1.1 equiv) was added to a solution of (lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4-tetrahydronaphthalen-l- amine (600 mg, 1.43 nimol, 1 equiv) in THF (20 mL) containing DIEA (1.5 mL), followed by adding 2 mL of DMSO. The resulted red solution was stirred at RT for 16 h. THF was evaporated off. The residue was dissolved in EtOAc, washed with water and brine, dried (Na2SO4). The organic layer was evaporated to dryness. The residue was purified on ISCO (12 g column, MeOH-CH2C12 0-5%) to give a yellow solid product (633 mg, 65%). MS (ESI): 677.3.

[00114] Example 43: N4-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro- l,2,3,4-tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l- yl)pyrimidine-4,5-diamine. N-(( 1 R,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro- l,2,3,4-tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-5- nitropyrimidin-4-amine (633 mg, 0.975 mmol) was dissolved in EtOAc (25 mL) and MeOH (25 mL), and mixed with platium on carbon, sulfided (10%, wet) (127 mg, 20%w/w). The reaction mixture was applied to Parr apparatus under H2 (40psi) for 5 h. TLC showed the reaction completed. The catalyst was filtered off and washed with MeOH. The filtrate and washings were combined and evaporated to dryness to give a white solid product (498 mg, 82%). MS (ESI): 647.5; IH NMR (400 MHz, DMSO- d6) δ 8.95(s, IH), 8.18(dd, IH), 7.63-7.76(m, 6H), 7.41-7.53(m, 6H), 7.25-7.30(m, IH), 7.02-7.22(m, 3H), 5.57-5.65(m, IH), 4.92(broad, 2H), 4.87-4.91(m, IH), 2.28- 2.38(m, IH), 1.86-1.98(m, 2H), 1.72-1.82(m, IH), 1.02(s, 9H).

[00115] Example 44: 9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthaIen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-7H-purin- 8(9H)-one. N4-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen- 1 -y l)-2-(6-fluoro 1 H-benzo [d]imidazol- 1 -y l)pyrimidine-4, 5 - diamine (490 mg, 0.751 mmol, 1 equiv) was dissolved in anhydrous THF (15 mL) and CDI (737 mg, 4.54 mmol, 7 equiv) was added. The reaction was stirred at RT under argon overnight. The solvent was removed by evaporation. The residue was dissolved in EtOAc (30 mL) and washed with saturated NaHCO3, water and brine, dried (Na2SO4). The organic layer was evaporated to dryness. The residue was applied to ISCO (12 g column, MeOH-CH2C12 0-2%) to give a white solid product (420 mg, 82%). MS (ESI): 673.3.

[00116] Example 45: 9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-7-methyl-7H- purin-8(9H)-one. 9-((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4- tetrahydronaphthalen- 1 -yl)-2-(6-fluoro- lH-benzo[d]imidazol-l -yl)-7H-purin-8(9H)- one (260 mg, 0.386 mmol, 1 equiv) was dissolved in anhydrous acetonitrile (10 niL) and mixed with 2-tert-butylimino-2-diethylamino-l,3-dimethyl-perhydro-l,3,2- diazaphosphorine on polystyrene (701 mg, 4 equiv). Iodomethane (72 uL, 1.159 mmol, 3 equiv) was added via syringe. The reaction mixture was stirred at RT under argon for 16 h. The resin was filtered off and washed with acetonitrile. The combined filtrate and washings were evaporated to dryness to give a yellow semisolid product which was used into the next step without further purification. MS (ESI): 687.1.

[00117] Example 46: 2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-9-((lR,4R)-7-fluoro- 4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-7H-purin-8(9H)-one. 9-

((lR,4R)-4-(tert-butyldiphenylsilyloxy)-7-fluoro-l,2,3,4-tetrahydronaphthalen-l-yl)- 2-

(6-fluoro-lH-benzo[d]imidazol-l-yl)-7-methyl-7H-purin-8(9H)-one (248 mg, 0.361 mmol, 1 equiv) and cesium fluoride (218 mg, 1.444 mmol, 4 equiv) were added to DMF (5 niL). The reaction mixture was stirred at 50 o C for 20 h. DMF was removed by evaporation. The residue was treated with 3 mL MeOH. The solid was filtered off and washed with MeOH. The combined fitrate and washing were applied to HPLC (Varian ProStar, Sunfire C18 column, 19x100 mm, ACN-H2O gradient elution) to give a white solid product (85 mg, 52%). IH NMR (400 MHz, DMSO-d6) δ 8.91(s, IH), 8.55(s, IH), 7.71-7.75(m, 2H), 7.48(dd, IH), 7.07-7.18(m, 2H), 6.88(dd, IH), 5.70-5.76(m, IH), 5.60(d, IH), 4.84- 4.91(m, IH), 3.48(s, 3H), 2.43-2.45(m, IH), 2.23-2.31(m, IH), 2.12-2.20(m, IH), 1.80-1.90(m, IH); 19F NMR (400 MHz, DMSO-d6) δ -115.8, 117.0; LC-MS: M+H ^: 449.1, RT = 5.52 min.

[00118] Example 47: 2-(6-Chloro-lH-benzo[d]imidazol-l-yl)-9-((lR,4R)-7- fluoro-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-7H-purin-8(9H)- one. IH NMR (400 MHz, MeOD): δ 8.91(s, IH), 8.41(s, IH), 8.03(d, IH), 7.79(q, IH), 7.64(d, IH), 7.31(dd, IH), 7.03(t, IH), 6.70(dd, IH), 5.84(q, IH), 5.19(q, IH), 3.57(s, 3H), 2.60-2.72(m, IH), 2.42-2.50(m, IH), 2.24-2.34(m, IH), 1.88-2.00(m, IH); 19F NMR (MeOD): -116.8; LC-MS: 465.2, 467.2, RT = 5.82 min.

[00119] Example 48: 3-(9-((lR,4R)-7-Fluoro-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile.

IH NMR (400 MHz, MeOD): δ 9.06(s, IH), 8.44(s, IH), 8.26(s, IH), 7.89(q, IH), 7.82(d, IH), 7.63(dd, IH), 7.06(t, IH), 6.74(dd, IH), 5.82-5.88(m, IH), 5.11-5.16(m, IH), 3.58(s, 3H), 2.54-2.62(m, IH), 2.43-2.50(m, IH), 2.27-2.34(m, IH), 1.89- 2.00(m, IH); 19F NMR (MeOD): δ -115.8; LC-MS: M+H = 456.3, RT = 5.55 min.

By routes exactly analogous to the above, using (lR,4R)-4-(tert- butyldiphenylsilyloxy)-5,7-difluoro-l,2,3,4-tetrahydronaphthalen-l-amine the compounds below have been prepared:

[00120] Example 49: 2-(lH-benzo[d]imidazol-l-yI)-9-((lR,4R)-7-fluoro-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-7H-purin-8(9H)-one. IH NMR (400 MHz, DMSO-dό): δ 8.89(s, IH), 8.53(s, IH), 7.80(q, IH), 7.71(d, IH), 7.59(d, IH), 7.29(t, IH), 7.21(t, IH), 7.14(t, IH), 6.92(dd, IH), 5.70-5.76(m, IH), 5.659d, IH), 4.79-4.84(m, IH), 3.48(s, 3H), 2.34-2.47(m, IH), 2.20-2.29(m, IH), 2.12-2.20(m, IH), 1.79-1.91(m, IH); 19F NMR (DMSO-d6): δ -115.7; LC-MS: M+H = 417.3, RT = 4.60 min.

[00121] Example 50: 3-(9-((lR,4R)-5,7-difluoro-4-hydroxy-l,2,3,4- tetrahydronaphthaIen-l-yI)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. IH-NMR (300 MHz, CD3OD ) δ 8.8 (s, IH), 8.6 (s, IH), 8.2 (s, IH), 7.8 (d, IH), 7.6 (d, IH), 6.8 (t, IH), 6.5 (d, IH), 5.8 (m, IH), 5.5 (m, IH), 3.6 (s, 3H), 2.3-2.6 (m, 3H), 2.1 (m, IH) ppm, MS (ESI), m/z 474.1 ([M+H]+).

[00122] Example 51: 9-((lR,4R)-5,7-difluoro-4-hydroxy-l,2,3,4- tetrahydronaphthaIen-l-yl)-2-(6-fluoro-lH-benzo[d]imidazol-l-yl)-7-methyl-7H- purin-8(9H)-one. IH-NMR (300 MHz, CD3OD) δ 8.7 (s, IH), 8.2 (s, IH), 7.7(m, IH), 7.6 (d, IH), 7.0 (m, IH), 6.8 (m, IH), 6.5 (d, IH), 5.8 (m, IH), 5.4 (m, IH), 3.6 (s, 3H), 2.3-2.6 (m, 3H), 2.0 (m, IH) ppm, 19F-NMR (300 MHz, CD3OD ) δ -109.9, -112.4, -117.0 ppm, MS (ESI), m/z 467.1 ([M+H]+).

[00123] Example 52: 2-(lH-benzo[d]imidazol-l-yl)-9-((lR,4R)-5,7-difluoro-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yI)-7-methyl-7H-purin-8(9H)-one. IH-

NMR (300 MHz, CD3OD) δ 8.8 (s, IH), 8.3 (s, IH), 7.8 (m, IH), 7.6 (m, IH), 7.3 (m, IH), 7.0 (t, IH), 6.8 (d, IH), 5.8 (m, IH), 5.3 (m, IH), 3.5 (s, 3H), 2.2-2.6 (m, 3H), 2.0 (m, IH) ppm, 19F-NMR (300 MHz, CD3OD ) δ -112.5, -114.1 ppm, MS (ESI), m/z 449.3 ([M+H]+).

Possible synthetic route to inhibitors containing Azabenimidazole(3H-imidazo[4,5- c]pyridin-3-yl) moiety:

[00124] Preparation of 9-(( 1 R,4R)-4-hydroxy- 1 ,2,3 ,4-tetrahydronaphthalen- 1 -yl)-2- (3H-imidazo[4,5-c]pyridin-3-yl)-7-methyl-7H-purin-8(9H)-one. A suspension of 9- (( lR,4R)-4-(tert-butyldiphenylsilyloxy)- 1 ,2,3,4-tetrahydronaphthalen- 1 -yl)-2-(3H- imidazo[4,5-c]pyridin-3-yl)-7H-purin-8(9H)-one 2-ter/-butylimino-2-diethylamino- l,3-dimethyl-perhydro-l,3,2-diazaphosphorine on polystyrene (3 - 5 mass equiv.) and iodomethane (xs) in ACN would be slowly stirred at RT for 2 to 15 hr. Upon completion of the reaction the product would be purified via a short plug of silica to yield 9-((lJ?,4i?)-4-(tert-butyldiphenylsilyloxy)- 1 ,2,3,4-tetrahydronaphthalen- 1 -yl)-2- (3H-imidazo[4,5-c]pyridin-3-yl)-7-methyl-7H-purin-8(9H)-one that would yield the above titled compound followed the removal of the silicon protecting group as described above.

[00125] Example 53: 2-(2-(6-Fluoro-lH-benzo[rf]imidazol-l-yl)-9-((li?,4J?)-4- hydroxy-l,2,3,4-tetrahydronaphthaIen-l-yl)-8-oxo-8,9-dihydropurin-7- yl)acetonitrile. Prepared in the same manner as 3-(7-(cyanomethyl)-9-((li?,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. ¹H-NMR (400 MHz, CDCl₃ + 1% CD₃OD)) δ 8.8 (s, IH), 8.4 (s, IH), 7.8 (d, IH), 7.7 (dd, IH), 7.5 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 7.0 (td, IH), 6.9 (d, IH), 5.9 (m, IH), 5.1 (m, IH), 5.0 (s, 2H), 2.5-2.3 (m, 3H), 2.2 (br s, IH) 1.9 (m, IH); ¹⁹F 5 -115 PPm₅ MH⁺ = 456. ^■

[00126] Example 54: 3-(7-(Cyanomethyl)-9-((lR,4R)-7-fluoro-4-hydroxy-l,2,3,4- tetrahydronaphthaIen-l-yI)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d] imidazole-5-carbonitriIe. 3 -(9-(( lR,4R)-7-Fluoro-4-hydroxy- 1 ,2,3 ,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purm-2-yl)-3H-benzo[d]imidazole- 5-carbonitrile (58 mg, 0.126 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (10 mL) and mixed with BEMP (174 mg). Bromoacetonitrile (28 μL, 0.378 mmol, 3 equiv.) was added via syringe. The reaction mixture was stirred at RT for 16 h. TLC and LC-MS showed reaction completed. The volatile was removed by evaporation. The residue was purified by HPLC (Varian ProStar, Sunfire C18 column, 19x100 mm, ACN-H₂O gradient elution) to give a white solid product (28 mg, 44%). ¹H NMR (400 MHz, MeOD) δ 9.07(s, IH), 8.60(s, IH), 8.25(s, IH), 7.89(q, IH), 7.82(d, IH)₅ 7.62(dd, IH), 7.06(t, IH), 6.81(d, IH), 5.85-5.89(m, IH), 5.19(d, 2H), 5.11- 5.14(m, IH), 2.58-2.61(m, IH), 2.42-2.50(m, IH), 2.30-2.38(m, IH), 1.93-1.96(m, IH); ¹⁹F NMR (400 MHz, MeOD) δ -116.5; LC-MS: M+H= 481.3, RT= 5.61 min.

[00127] Example 55: 3-(7-(but-2-ynyl)-9-((lR,4R)-4-hydroxy-l,2,3,4- tetrahydronaphthalen-l-yl)-8-oxo-8,9-dihydro-7H-purin-2-yI)-3H- benzo[d]imidazole-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with l-Bromo-2-butyne used to alkylate instead of Iodomethane. ¹H-NMR (300 MHz, CDCl₃) δ 9.0 (s, IH), 8.4 (s, IH), 8.3(s, IH), 7.9 (d, IH), 7.8 (d, IH), 7.6 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 6.9 (d, IH), 5.9 (dd, IH), 5.5 (dd, IH), 4.8 (s, 2H), 2.7-2.3 (m, 3H), 2.0, (q, IH), 1.9 (s, 3H) ppm; MH⁺ = 476.

[00128] Example 56: 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l- yl)-8-oxo-7-(pyridin-4-ylmethyl)-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy-l,2,3,4-tetrahydronaphthalen-l-yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with 4-(Bromomethyl)pyridine (HBr salt), used to alkylate instead of Iodomethane. ¹H-NMR (300 MHz, CDCl₃) δ 8.9(s, IH), 8.7, (d, 2H), 8.2 (s, IH), 8.0 (s, IH), 7.9 (d, IH), 7.8 (d, IH), 7.6 (d, IH), 7.3 (t, IH), 7.2 (d, 2H), 7.1 (t, IH), 6.8 (d, IH), 6.0 (dd, IH), 5.3 (dd, IH), 5.2 (s, 2H), 2.7-2.4 (m, 3H), 2.0 (q, IH) 1.6 (bs, IH) ppm; MH⁺ = 515.

[00129] Example 57: 3-(9-((lR,4R)-4-hydroxy-l,2,3,4-tetrahydronaphthalen-l- yI)-7-(3-hydroxypropyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)-3H- benzo[d]imidazole-5-carbonitrile. Prepared in a similar fashion to 3-(9-((lR,4R)-4- hydroxy- 1 ,2,3 ,4-tetrahydronaphthalen- 1 -yl)-7-methyl-8-oxo-8,9-dihydro-7H-purin-2- yl)-3H-benzo[d]imidazole-5-carbonitrile, but with 2-(3-

Bromopropoxy)tetrahydropyran used to alkylate instead of Iodomethane to give 3-(9- (( lR,4R)-4-hydroxy- 1 ,2,3,4-tetrahydronaphthalen- 1 -yl)-8-oxo-7-(3 -(tetrahydro-2H- pyran-2-yloxy)propyl)-8,9-dihydro-7H-purin-2-yl)-3H-benzo[d]imidazole-5- carbonitrile, MH⁺ = 566. Removal of THP was carried out by refluxing material with catalytic PPTS in 20 ml of 9: 1 EtOH/water, to give title compound. ¹H-NMR (300 MHz, CDCl₃) δ 9.0 (s, IH), 8.3 (d, 2H), 7.9 (d, IH), 7.8 (d, IH), 7.6 (d, IH), 7.3 (t, IH), 7.1 (t, IH), 6.8 (d, IH), 5.9 (dd, IH), 5.3 (dd, IH), 4.2 (t, 2H), 3.7 (d, 2H), 3.0 (d, IH), 2.7-2.4 (m, 3H), 2.0, (m, 2H), 1.6 (bs, 2H) ppm; MH⁺ = 482.

Generic Alkylation:

[00130] It will be apparent from the above examples that a wide variety of groups may be added to the 7 position of the by the use of alkylating agents. The alkylating agents may themselves be functionalized (where said functionality is suitably protected), with for example amine, alcohol acid, nitrile, carboxamide, sulsonamide, ether and the like. The method also allows for the introduction of alkyl substituted with aryl and heteroaryl groups such benzyl, substituted benzyl, pyrazin-2-ylmethyl and the like. Alkylating conditions may include the use of alkyl halides or alkyl sulfonates (with suitable protection where necessary) and an appropriate base, for example BEMP, hunigs base, potassium carbonate in a polar aprotic solvent. Alternatively alkylations may be carried out using alcohols under Mitsunobou conditions:

Jak3 kinase assay

[00131] Human Jak3 cDNA was amplified by PCR. A fragment encoding the catalytic domain of Jak3 (508aa to 1124aa) was ligated with GST at 5' end. This fused GST- Jak3 DNA fragment was cloned into the EcoRI site of the donor plasmid pFastBac 1 (Life Technologies #10359-016). The transformation, transposition, and transfection of insect cells (Sf9) were performed according to the manufacture's instructions. The cell lysate containing recombinant GST- Jak3 was used in the kinase assay. Anti-GST antibody (10 μg/ml, Sigma #G1417) was coated onto a 384-well plate at 4⁰C overnight. Cell lysate containing GST- Jak3 (1 :100 dilution) was added to the anti-GST coated plates, and GST- Jak3 was captured by immobilized anti-GST antibody. Testing compounds and substrate mix (50 mM HEPES, pH 7, 0.5 mM Na₃VO₄, 25 mM MgCl₂, 1 mM DTT, 0.005% BSA, 1 μM ATP, and 4.5 μg/ml biotinyl poly-Glu,Ala,Tyr) were added to the plate to initiate the reaction. After a 60- min incubation, the reaction was stopped by 4 mM EDTA, and phosphorylation of biotinyl poly-Glu,Ala,Tyr was detected using 17 μg/ml Cy5-streptavidin (Amersham, #PA92005) and 2.7 μg/ml Europium-conjugated anti-phosphotyrosine antibody (PerkinElmer #AD0069) using homogeneous time-resolved fluorescence (HTRF) technology. Jak3 cellular assay

[00132] The mouse F7 pre-B lymphocyte cell line was used for the cellular Jak3 assay. Human IL-2Rβc cDNA is stably expressed in F7 cells (Kawahara et al., 1995). F7 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum plus IL-3. Cells (30,000 cells/well) in serum-free medium were seeded in 96- well plates for the cell proliferation assay. Testing compounds were added to cells, followed by the addition of IL-2 (final 20 ng/ml). After a 24-h incubation, the number of viable cells was determined by the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, #G7573) according to the manufacturer's instructions.

Aurora A kinase assay

[00133] Aurora A kinase assay was performed using a fluorescence polarization format. A 100 nM solution of fluorescien-labeled FAM PKAtide (Molecular Devices), the substrate for Aurora A (Upstate Biotechnology), was incubated with Aurora A (80 ng/ml) and 30 mM ATP at room temperature for 1 hour in the presence of an appropriate concentration of test inhibitor. The reaction was terminated by adding IMAP Progressive Binding Reagent mix according to the manufacturer's instructions (Molecular Devices). The polarization signal was detected using Aquest (Molecular Devices).

[00134] The results of testing of representative species are shown below. The compounds in Table 1 exhibited IC₅₀ less than 2OnM in the Jak3 Kinase assay.

IL-2-induced IFN-γ production in the mouse

[00135] Administration of IL-2 leads to an increase in serum IFN-γ in the mouse due to NK secretion of the cytokine (Thornton S, Kuhn KA, Finkelman FD and Hirsch R. NK cells secrete high levels of IFN-γ in response to in vivo administration of IL-2. Eur J Immunol 2001 31:3355-3360). The experiment was carried out essentially according to the protocol in Thornton et al. and the test compounds were administered in order to determine the level of inhibition attained. In summary, female BALB/c mice were fasted for 12-18 hours before a study but had free access to water at all times. Test compounds were administered by oral gavage one hour before intraperitoneal injection of IL-2 and capture antibody. At termination of the studies, the mice were sacrificed by carbon dioxide inhalation, terminal blood samples were collected by cardiac puncture and serum was generated. Serum was stored frozen until it was assayed for IFN-γ, as described by the kit manufacturer (BD Pharmingen™, San Diego, CA).

[00136] A reference compound, CP690550, exhibited an ED50 of 1.8 mg/kg on oral dose. Example 30 inhibits with an ED50 = 0.4 mg/kg on oral dose.

[00137] The 7-substituted purinones exhibit increased selectivity for Jak3 compared to their 7-unsubstituted congeners. The compounds are more selective for Jak3 than for Aurora A kinases than are the corresponding compounds in which Ri is hydrogen.

[00138] Some comparative examples are shown below. All of the IC₅₀ ⁵S for Jak3 are below 20 nM.

Aurora Selectivity Modulated by N7 Alkylation

[00139] Similarly, replacing a chroman with tetralin at the N9 position increases the potency of the corresponding compound more than ten-fold. Some comparative examples are shown below.

results in a 14-fold improvement in Jak3 IC50 values.

results in a 16.7-fold improvement in these values.

[00140] Although the foregoing invention has been described in some detail for purposes of illustration, it will be readily apparent to one skilled in the art that changes and modifications may be made without departing from the scope of the invention described herein.

Claims

1. A compound of formula I

wherein

X is selected from the group consisting of hydrogen, halogen and cyano;

Ri is selected from the group consisting of substituted (Ci-C₄)alkyl, heteroarylalkyl and (Ci-Cg)hydrocarbon; and

R₂ and R₃ are selected independently for each occurrence from the group consisting of hydrogen, fluorine and chlorine.

2. A compound according to claim 1 selected from the group consisting of

3. A compound according to claim 1 or 2 wherein the chiral carbons in the tetralin are both of the (R)- configuration.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound according to claim 1, 2 or 3.

5. The use of a compound of claim 1, 2 or 3 for the manufacture of a medicament for the treatment of a disorder responsive to inhibition of Janus kinase 3.

6. The use of a compound of claim 1, 2 or 3 for the manufacture of a medicament for the treatment of a disorder selected from an autoimmune disease, an inflammatory disease, a mast cell mediated disease, cancer, hematological malignancy, and organ transplant rejection.

7. The use according to claim 6 wherein said disorder is bone marrow transplant rejection.

8. The use according to claim 6 wherein said hematological malignancy is selected from leukemia and lymphoma.

9. The use according to claim 6 wherein said disorder is asthma.

10. The use according to claim 6 wherein said autoimmune disease is selected from an organ specific and a non-organ specific autoimmune disease.

11. The use according to claim 6 wherein said disorder is keratoconjuctivitis sicca.

12. The use according to claim 6 wherein said hematological malignancy is chronic myelogenous leukemia.

13. The use according to claim 6 wherein said disorder is selected from a leukemic form of cutaneous T-cell form lymphoma and acute lymphoblastic leukemia.