Substituted heterocyclic compounds and methods of use

This application is a divisional patent application, and claims the benefit, of U.S. patent application Ser. No. 11/034,042 filed Jan. 11, 2005, which claims the benefit of U.S. Provisional Application No. 60/536,617 filed Jan. 14, 2004, both which are hereby incorporated herein by reference.

T cells play a key role in the regulation of immune responses and are important for establishing immunity to pathogens. In addition, T cells are often activated during inflammatory autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, type I diabetes, multiple sclerosis, Sjogren\'s disease, myasthenia gravis, psoriasis, and lupus. T cell activation is also an important component of transplant rejection, allergic reactions, and asthma.

T cells are activated by specific antigens through the T cell receptor (TCR) which is expressed on the cell surface. This activation triggers a series of intracellular signaling cascades mediated by enzymes expressed within the cell (Kane, L P et al. Current Opinion in Immunol. 200, 12, 242). These cascades lead to gene regulation events that result in the production of cytokines, like interleukin-2 (IL-2). IL-2 is a critical cytokine in T cell activation, leading to proliferation and amplification of specific immune responses.

One class of enzymes shown to be important in signal transduction is the kinase proteins. PKC enzymes are members of a distinct family of serine/threonine protein kinases that contain nine members (isotype α, β, γ, δ, ε, ζ, η, θ, ι) (reviewed in Nishizuka Y., Science 1992; 258:607-614), some of which are expressed at particular high levels in T cells (including α, δ, ε, η, θ) (reviewed in Baier, G., Immunological Reviews 2003 192:64-79). Gene disruption studies suggest that inhibition of some members of the PKC family of kinases would potentially lead to therapeutic benefit. PKCα (−/−) mice and mice deficient in PKCθ both have T cell defects (Baier, G., Immunological Reviews 2003 192:64-79; Pfeifhofer C. et. al, Journal of Experimental Medicine, 197:1525-1535; Sun, Nature 2000, 404:402-407), suggesting that inhibition of either of these kinases would be useful in diseases of T cell mediated inflammation and autoimmunity. PKCθ in particular may be a prime target for novel anti-inflammatory or immuno-suppressive therapies, due to its restricted tissue-expression and its nonredundant critical role in TCR-mediated IL-2 secretion (N. Isakov and A. Ammon Annu. Rev. Immunol. 2002 20:761-94). Small molecule drugs selectively inhibiting PKCθ and/or other certain other PKC isoenzymes such as PKC alpha, beta, epsilon and zeta may manifest improved efficacy and/or improved side-effect profile over drugs targeted against other immune-mediators suc has calcineurin and Akt1/PKBalpha. For example, a dual inhibitor of both PKC theta and PKC alpha may effectively prevent mature T cell activation.

PKC alpha, like PKC theta, is involved in TCR signaling in T cells (Iwamoto 1992 JBC 267:18644-18648; Ohkusu 1997 J. Immunol. 159:2082-2084). PKC family kinases are also important for signaling downstream of other immune cell receptors. PKC beta participates in B cell receptor signaling (Leitges M. et al. 1996 Science 273:788-791), neutrophils (Dekker L V et al. 2000 Biochem. J. 347:285-289), and mast cells (Nechushian H et al. 2000 Blood 95:1752-1757). PKC zeta also plays a role in B cell signaling and function (Martin P. et al. 2002 EMBO J. 15:4049-4057) and PKC epsilon is required for macrophage activation (Castrillo A. et al. 2001 J. Exp. Med. 194:1231-1242). These findings suggest that PKC family kinase inhibitors may be useful in treating inflammatory, autoimmune and allergic diseases and asthma.

In addition to its essential function in mature T cell activation and IL-2 secretion, PKC theta provides a survival signal that protects leukemic T cells from Fas-ligand induced apoptosis (M. Villalba and A. Altman 2002 Current Cancer Drug Targets 2:125-134). This feature and the constitutive membrane location of PKC theta in some leukemic T cells suggest that it plays a role in the growth and survival of leukemic T-cells. Furthermore, the high-affinity IL2 receptor (IL-2R alpha) is constitutively expressed by some malignant T cell leukemias suggesting that expansion of these cells may be supported by an IL-2 autocrine loop (M. Villalba and A. Altman 2002 Current Cancer Drug Targets 2:125-134). PKC theta may also promote survival of malignant cells by functioning in development of a multidrug resistance (MDR) phenotype. PKC theta expression is positively correlated with the expression of some genes involved in MDR including MDR1 and MRP1 in acute myelogenous leukemia patients (Beck J. et al. 1996 Leukemia 10:426-433) and PKC theta regulates MDR1 promoter activity in human breast carcinoma cells (Gill P. K. et al. 2001 Eur. J. Biochem 268:4151-4157). Therefore, a PKC theta small molecule inhibitor may facilitate elimination of leukemic T cells and other malignant cells that over-express PKC theta. Concomitant overexpression/activation of both PKC alpha and PKC theta has also been implicated in development of multi-drug resistance. Therefore a dual PKC theta and PKC alpha small molecule inhibitor may also facilitate elimination of malignant cells that overexpress both PKC alpha and PKC theta.

Other groups have published on inhibitors of PKC family kinase and the activities of these inhibitors in various in vitro and in vivo biological systems. For example, PCT Publication No. WO 2004067516 discloses 2,4-diaminopyrimidine derivatives useful as inhibitors of PKC-theta. WO 2003082859 discloses indolylmaleimide derivatives as compounds useful in the treatment and/or prevention of diseases or disorders mediated by T-lymphocytes and/or PKC. The protein kinase C beta inhibitor ruboxistaurin (LY-333531), the lead compound from a series of 14-membered macrocycles, is being developed for the potential treatment of diabetic retinopathy, diabetic macular edema and diabetic neuropathy (Investigational Drug database, Dec. 19, 2003, Ruboxistaurin update). By October 2003, this compound was also being investigated as a potential treatment for cardiovascular disease in diabetic patients. It was in phase III trials for both diabetic retinopathy and macular edema by early 2001.

The compounds disclosed in the present invention possess pharmacological activity not only by virtue of an effect on a single biological process, but it is believed that the compounds modulate T cell activation by way of inhibition of one or more of the multiple protein kinases involved in early signal transduction steps leading to T cell activation, for example by way of inhibition of PKC theta kinase.

The compounds of the present invention inhibit serine threonine kinases, especially PKC theta and to a varying degree other PKC isoenzymes, and are thus useful in the treatment, including prevention and therapy, of protein serine/threonine kinase-associated disorders such as immunologic disorders. “Protein serine-threonine kinase-associated disorders” are those disorders which result from aberrant serine-threonine kinase activity, and/or which are alleviated by the inhibition of one or more of these enzymes. For example, PKC theta inhibitors are of value in the treatment of a number of such disorders (for example, the treatment of autoimmune diseases), as PKC theta inhibition blocks T cell activation. The treatment of T cell mediated diseases, including inhibition of T cell activation and proliferation, is a preferred embodiment of the present invention. Compounds of the present invention which selectively block T cell activation and proliferation are preferred. Also, compounds of the present invention which may block the activation of endothelial cell protein serine-threonine kinase by oxidative stress, thereby limiting surface expression of adhesion molecules that induce neutrophil binding, and which can inhibit protein serine-threonine kinase necessary for neutrophil activation would be useful, for example, in the treatment of ischemia and reperfusion injury.

The present invention also provides methods for the treatment of protein serine-threonine kinase-associated disorders, comprising the step of administering to a subject, such as to those in need thereof, at least one compound of the present invention in an amount effective therefore. The compound(s) may be administered in a pharmaceutical formulation, having been formulated with a suitable pharmaceutically acceptable carrier. Other therapeutic agents such as those described below may be employed with the inventive compounds in the present methods. In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with or following the administration of the compound(s) or pharmaceutical composition of the present invention.

Use of the compound(s) of the present invention in treating protein serine-threonine kinase-associated disorders is exemplified by, but is not limited to, treating a range of disorders such as: arthritis (such as rheumatoid arthritis, psoriatic arthritis or osteoarthritis); transplant (such as organ transplant, acute transplant or heterograft or homograft (such as is employed in burn treatment)) rejection; protection from ischemic or reperfusion injury such as ischemic or reperfusion injury incurred during organ transplantation, myocardial infarction, stroke or other causes; transplantation tolerance induction; multiple sclerosis; inflammatory bowel disease, including ulcerative colitis and Crohn\'s disease; lupus (systemic lupus erythematosis); graft vs. host diseases; T-cell mediated hypersensitivity diseases, including contact hypersensitivity, delayed-type hypersensitivity, and gluten-sensitive enteropathy (Celiac disease); Type 1 diabetes; psoriasis; contact dermatitis (including that due to poison ivy); Hashimoto\'s thyroiditis; Sjogren\'s syndrome; Autoimmune Hyperthyroidism, such as Graves\' Disease; Addison\'s disease (autoimmune disease of the adrenal glands); Autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome); autoimmune alopecia; pernicious anemia; vitiligo; autoimmune hypopituatarism; Guillain-Barre syndrome; other autoimmune diseases; cancers where PKC theta or other PKC-family kinases such as PKC alpha are activated or overexpressed, such as T cell leukemia, thymoma, T and B cell lymphoma, colon carcinoma, breast cancer and lung carcinoma, or cancers where PKC-family kinase activity facilitates tumor growth or survival or provides resistance to chemotherapeutic drugs or radiation; glomerulonephritis, serum sickness; uticaria; allergic diseases such as respiratory allergies (asthma, hayfever, allergic rhinitis) or skin allergies; scleracielma; mycosis fungoides; acute inflammatory responses (such as acute respiratory distress syndrome and ishchemia/reperfusion injury); dermatomyositis; alopecia areata; chronic actinic dermatitis; eczema; Behcet\'s disease; Pustulosis palmoplanteris; Pyoderma gangrenum; Sezary\'s syndrome; atopic dermatitis; systemic schlerosis; morphea; type II diabetes; insulin resistance; diabetic retinopathy; diabetic macular edema; diabetic neuropathy; and cardiovascular disease in diabetic patients. The present invention also provides for a method for treating the aforementioned disorders such as atopic dermatitis by administration of a therapeutically effective amount of a compound of the present invention, which is an inhibitor of protein serine-threonine kinase, to a patient in need of such treatment.

Other PKC-family kinases, such as PKC beta and zeta are also important in B cell function (Leitges M. et al. 1996 Science 273:788-791; Martin P. et al. 2002 EMBO J. 15:4049-4057). This activity would result in additional anti-autoimmune activity for the present compounds in addition to their effects on T cells. This activity would be especially of value, for example, in the treatment of autoimmune/inflammatory diseases, such as lupus, arthritis or inflammatory bowel disease. PKC theta may also function in B-cells (Krappmann D. 2001 Molecular & Cellular Biology. 21:6640-6650). PKC theta is also expressed in mast cells and PKC beta and epsilon plays a role in neutrophils/mast cell and macrophage function respectively. The ability to inhibit neutrophil, monocyte and macrophage responses would result in further anti-inflammatory activity for the present compounds in addition to their effects on T cells. The present compounds may also be of value for the treatment of autoimmune glomerulonephritis and other instances of glomerulonephritis induced by deposition of immune complexes in the kidney.

In addition, certain PKC isoenzymes including PKC theta and beta may function in degranulation of mast cells and basophils that plays an important role in asthma, allergic rhinitis, and other allergic disease. The ability to inhibit mast cell and basophil responses may result in additional anti-inflammatory activity for the present compounds beyond their effect on T cells.

The combined activity of the present compounds towards B cells, monocytes, macrophages, T cells, mast cells, endothelial cells, etc. may prove to be a valuable tool in the treatment of any of the aforementioned disorders.

In a particular embodiment, the compounds of the present invention are useful for the treatment of the aforementioned exemplary disorders irrespective of their etiology, for example, for the treatment of rheumatoid arthritis, transplant rejection, multiple sclerosis, inflammatory bowel disease, lupus, graft v. host disease, T cell mediated hypersensitivity disease, psoriasis, Hashimoto\'s thyroiditis, Guillain-Barre syndrome, cancer, contact dermatitis, allergic disease such as allergic rhinitis, asthma, ischemic or reperfusion injury, or atopic dermatitis whether or not associated with serine-threonine kinases.

PKC theta and certain other PKC isoenzymes are abnormally activated in the skeletal muscle of human patients with type II diabetes as well as in skeletal muscle of a rodent model of high fat induced insulin resistance (Gray S. et al. 2003 European Journal of Clinical Investigation 33:983-987 and references therein). Furthermore a small molecule inhibitor of PKC beta is in late stage clinical trials for treatment of diabetic retinopathy, neuropathy and macular degeneration (IDDB: ruboxistaurin LY-333531). The ability of the present compounds to inhibit abnormal PKC theta and other PKC isoenzyme activity associated with development of insulin resistance, type II diabetes and side-effects thereof may result in a therapeutically beneficial reversal of insulin resistance and retinopathy associated with type II diabetes.

Abnormal activation of PKC theta and other PKC isoenzymes such as PKC alpha has been associated with the development of multidrug resistance (Beck J. et al. 1996 Leukemia 10:426-433; Gill P. K. et al. 2001 Eur. J. Biochem 268:4151-4157). The present compounds may be used to increase the potency of other medicines such as for instance chemotherapeutic drugs in cancer patients.

In addition to T cells, mast cells and skeletal muscle, PKC theta is also specifically expressed in platelets (Chang J D et al. 1993 Journal of Biological Chemistry. 268:14208-14214). The present compounds, bye virtue of inhibiting PKC theta may be used therapeutically to prevent or treat adverse thromboembolic events by regulating platelet activation.

Inhibitors of PKC isoenzymes such as PKC beta also inhibit angiogenesis in solid tumor models in rodents in vivo (IDDB: LY-317615 Update Nov. 24, 2003; Teicher B A at al. International Journal of Antimicrobial Agents 2001, 17:Suppl 1 Abs S6.03). The present compounds may have a therapeutic effect in solid tumors such as brain, breast, ovarian, gastric, non small-cell lung cancer, small-cell lung cancer, gastric, hepatocellular, colon and renal cell cancer by decreasing the number of intratumoral vessels.