EA040009B1 - HETEROARYL-SUBSTITUTED PYRROLO[2,3-D]PYRIMIDINES AS JANUS KINASE INHIBITORS - Google Patents

Description

Field of invention

The present invention relates to heteroaryl-substituted pyrrolo[2,3-d]pyrimidines that modulate Janus kinase activity and can be used to treat diseases associated with Janus kinase activity, including, for example, diseases associated with the immune system, skin diseases, myeloid proliferative diseases, cancer and other diseases.

Prerequisites for the creation of the invention

Protein kinases (PKs) are a group of enzymes that regulate various important biological processes, including cell growth, survival and differentiation, organ formation and morphogenesis, neovascularization, tissue repair and regeneration, among others. Protein kinases exert their physiological functions by catalyzing the phosphorylation of proteins (or substrates) and thus modulating the cellular activity of substrates in various biological contexts. In addition to functions in normal tissues/organs, many protein kinases also play a more specialized role in the host in human diseases, including cancer. A suite of protein kinases (also referred to as oncogenic protein kinases), when dysregulated, can induce tumor formation and growth and further contribute to tumor maintenance and progression (Blume-Jensen P. et al., Nature, 2001, 411 (6835):355 -365). So far, oncogenic protein kinases represent the largest and most attractive group of protein targets for intervention in cancer and drug development.

Protein kinases can be divided into receptor and non-receptor type kinases. Receptor tyrosine kinases (RTKs) have an extracellular portion, a transmembrane domain, and an intracellular portion, while non-receptor tyrosine kinases are entirely extracellular. RTK-mediated signal transduction is usually initiated by an extracellular interaction with a particular growth factor (ligand), usually followed by receptor dimerization, stimulation of intrinsic tyrosine kinase protein activity, and receptor transphosphorylation. This creates binding sites for intracellular signal transduction molecules and this leads to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate appropriate cellular response such as division, cell differentiation, metabolic effects and changes in the extracellular microenvironment.

Currently, at least nineteen (19) different RTK subfamilies have been identified. One of the RTK subfamilies, designated HER, includes EGFR, HER2, HER3, and HER4 and binds ligands such as epithelial growth factors (EGF), TGF-α, amphiregulin, HB-EGF, betacellulin, and heregulin. The second RTK family, referred to as the insulin subfamily, includes INS-R, IGF-1R and IR-R. The third family, the PDGF subfamily, includes PDGF α and β receptors, CSFIR, c-kit, and FLK-II. Another RTK subfamily, referred to as the FLK subfamily, encompasses the kinase domain insert receptor, fetal liver kinase 1 (KDR/FLK-1), fetal liver kinase 4 (FLK-4) and fms-like tyrosine kinase (flt-1). Two other RTK subfamilies have been designated the FGF family of receptors (FGFR1, FGFR2, FGFR3 and FGFR4) and the Met subfamily (c-Met, Ron and Sea). For a detailed discussion of protein kinases, see, for example, Blume-Jensen, P. et al., Nature, 2001, 41, 1 (6835):355365; and Manning, G. et al., Science, 2002, 298(5600):1912-1934.

The non-receptor type of tyrosine kinases also consists of many subfamilies including Src, Btk, AbI, FAK and JAK. Each of these subfamilies can be further subdivided into many members that are often associated with oncogenesis. The Src family, for example, is the largest and includes, among others, Src, Fyn, Lck, and Fgr. For a detailed discussion of these kinases, see Bolen, J.B., Nonreceptor tyrosine protein kinases, Oncogene, 1993, 8(8):2025-31.

A significant number of tyrosine kinases (both receptor and non-receptor) are associated with cancer (see Madhusudan, S., Ganesan, T.S., Tyrosine kinase inhibitors in cancer therapy. Clin. Biochem., 2004, 37(7):618-35). Clinical studies suggest that overexpression or dysfunction of tyrosine kinases may also be of predictive value. For example, members of the HER family of the RTK series are associated with poor prognosis in breast cancer, colorectal cancer, head and neck cancer, and lung cancer. The c-Kit tyrosine kinase mutation is associated with reduced survival in gastrointestinal stromal tumors. In acute myelogenous leukemia, the Flt-3 mutation predicts shorter disease-free survival. Expression of VEGFR, which is important for tumor angiogenesis, is associated with a lower survival rate in lung cancer. Tie-1 kinase expression is inversely correlated with gastric cancer survival. Expression of Bcr-Ab1 is an important predictor of response in chronic myelogenous leukemia, and Src tyrosine kinase is an indicator of poor prognosis at all stages of colorectal cancer.

The immune system responds to the damage and threat of pathogens. Cytokines are low molecular weight polypeptides or glycoproteins that stimulate biological responses in virtually all cell types. For example, cytokines regulate a variety of pathways involved in the host's inflammatory response to sepsis. Cytokines affect cell differentiation, proliferation, and activation, and they can modulate both pro-inflammatory and anti-inflammatory responses, enabling the host to respond appropriately to pathogens.

Binding of a cytokine to a receptor on the cell surface initiates intracellular signaling cascades that transduce an intercellular signal into the nucleus, ultimately leading to changes in gene expression. The pathway, which includes the Janus kinase family of protein tyrosine kinases (JAKs) and signal transducers and transcription activators (STATs), is involved in signal transduction for a wide range of cytokines. Typically, cytokine receptors do not have intrinsic tyrosine kinase activity and therefore require receptor-related kinases to propagate the phosphorylation cascade. JAKs perform this function. Cytokines bind to receptors, causing receptor dimerization, and this allows JAK kinases to phosphorylate each other as well as certain tyrosine motifs at cytokine receptors. STATs that recognize these phosphotyrosine motifs are recruited by the receptor and then themselves activated by JAK-dependent tyrosine phosphorylation. Upon activation, STATs dissociate from the receptor, dimerize, and target the nucleus to bind to specific DNA sites and alter transcription (Scott, M.J., Godshall, C.J. et al. (2002), Jaks, STATs, Cytokines, and Sepsis, Clin Diagn Lab Immunol. , 9(6):1153-9).

The JAK family plays a role in the cytokine-dependent regulation of proliferation and function of cells involved in the immune response. There are currently four known members of the mammalian JAK family: JAK1 (also known as Janus kinase-1), JAK2 (also known as Janus kinase-2), JAK3 (also known as leukocyte Janus kinase, JAKL, 1-JAK and Janus kinase-3) and TYK2 (also known as protein tyrosine kinase 2). JAK proteins range in size from 120 to 140 kDa and include a network of conserved JAK homologous domains (JH); one is a functional catalytic kinase domain, and the other is a pseudokinase domain potentially having a regulatory function and/or serving as a docking site for STATs (Scott, Godshall et al., 2002, supra).

While JAK1, JAK2, and TYK2 are ubiquitously expressed, JAK3 has been reported to be preferentially expressed in natural killer (NK) cells rather than resting T cells, suggesting a role in lymphoid activation (Kawamura, M., McVicar, D. W. et al. (1994), Molecular cloning of 1-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes, Proc. Natl. Acad. Sci., USA, 91(14):6374-8 ).

Not only do cytokine-stimulated immune and inflammatory responses actually contribute to host defense, but they also play a role in disease pathogenesis: pathologies such as severe joint immunodeficiency (SCID) arise from an underactive and suppressed immune system, while hyperactivity and abnormal immune/inflammatory response contribute to the pathology of autoimmune diseases such as rheumatoid and psoriatic arthritis, asthma and systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, type I diabetes mellitus, bulbospinal palsy, thyroiditis, immunoglobulin neuropathies, myocarditis, and diseases , such as scleroderma and osteoarthritis (Ortmann, R.A., Cheng, T. et al. (2000), Janus kinases and signal transducers and activators of transcription: their roles in cytokine signaling, development and immunoregulation, Arthritis Res., 2(1) :16-32). In addition, syndromes with a mixed manifestation of autoimmune and immunodeficiency diseases are absolutely common (Candotti, F., Notarangelo, L. et al. (2002), Molecular aspects of primary immunodeficiencies: lessons from cytokine and other signaling pathways, J. Clin. Invest ., 109(10):1261-9). Thus, therapeutic agents generally aim to increase or suppress immune and inflammatory pathways, respectively.

Deficits in expression of JAK family members are associated with disease states. JAK1 -/ mice are small at birth, poorly fed, and die perinatally (Rodig, S.J., Meraz, M.A. et al. (1998), Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses , Cell, 93(3):373-83). Embryos of JAK2 -/- mice are anemic and die approximately 12.5 days after copulation due to the absence of fixed erythropoiesis. JAK2-deficient fibroblasts do not respond to IFN gamma, although IFN alpha/beta and IL-6 responses are unaffected.

JAK2 functions in signal transduction of a specific group of cytokine receptors required for defining erythropoiesis (Neubauer, H., Cumano, A. et al. (1998), Cell, 93(3): 397-409; Parganas, E., Wang, D ., et al (1998), Cell, 93(3):385-95). It turned out that JAK3 plays a role in the normal development and functioning of B and T lymphocytes. JAK3 mutations have been reported to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans (Candotti, F., Oakes, S.A. et al. (1997), Structural and functional basis for JAK3-deficient severe combined immunodeficiency, Blood, 90( 10):3996-4003).

The JAK/STAT pathway, and in particular all four members of the JAK family, is thought to play a role in the pathogenesis of the asthmatic response in chronic obstructive pulmonary disease, bronchitis and other related inflammatory diseases of the lower respiratory tract. For example, the abnormal immune responses that are characteristic of asthma are associated with a set

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CD4+ T helper cells, defined as T helper 2 (Th2) cells. Signaling through the cytokine receptor IL-4 stimulates JAK1 and JAK3 to activate STAT6, and signaling through IL-12 stimulates the activation of JAK2 and TYK2 and subsequent phosphorylation of STAT4. STAT4 and STAT6 control many aspects of CD4+ T helper cell differentiation (Pernis, A.B., Rothman R.B. (2002), JAK-STAT signaling in asthma, J. Clin. Invest., 109(10):1279-83 ). In addition, TYK2-deficient mice have been found to have an increased propensity for Th2 cell-mediated allergic airway inflammation (Seto, Y., Nakajima, H. et al. (2003), Enhanced Th2 cell-mediated allergic inflammation in Tyk2 -deficient mice, J. Immunol., 170(2):1077-83). Moreover, many cytokines that signal through JAK kinases are associated with inflammatory diseases or conditions of the upper respiratory tract, such as those affecting the nose and sinuses (eg, rhinitis, sinusitis), whether they are classic allergic reactions or not. .

The JAK/STAT pathway is also implicated in inflammatory eye diseases/conditions including, but not limited to, iris inflammation, uveitis, scleritis, conjunctivitis, and chronic allergic responses. Therefore, inhibition of JAK kinases may play a beneficial role in the therapeutic treatment of these diseases.

The JAK/STAT pathway, and in particular JAK3, also plays a role in immune system cancer. In adult T-cell leukemia/lymphoma (ATLL), human CD4+ T cells acquire a transformed phenotype, an event that correlates with the acquisition of constitutive JAK and STAT phosphorylation. In addition, an association between JAK3 and STAT1, STAT3 and STAT5 activation and cell cycle progression was demonstrated by both staining with propidium iodide and incorporation of bromodeoxyuridine into the cells of the four ATLL patients tested. These results suggest that JAK/STAT activation is associated with leukemic cell replication and that therapeutic approaches aimed at JAK/STAT inhibition can be considered to arrest neoplastic growth (Takemoto, S., Mulloy, J.C. et al. (1997), Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins, Proc. Natl. Acad. Sci., USA, 94(25):13897-902).

Blocking signal transduction at the level of JAK kinases offers the prospect of developing an approach to the treatment of human cancer. Cytokines of the interleukin 6 (IL-6) family, which activate the gp130 signal transducer, are major survival and growth factors for human multiple myeloma (MM) cells. It is proposed that signal transduction of gp130 involves JAK1, JAK2 and TYK2 and downstream (5'-3') STAT3 effectors and mitogen-activated protein kinase (Mark) pathways. In IL-6 dependent MM cell lines treated with the JAK2 inhibitor tyrphostin AG490, JAK2 kinase activity and ERK2 and STAT3 phosphorylation were inhibited. In addition, cell proliferation was suppressed and apoptosis was induced (De Vos, J., Jourdan, M. et al. (2000), JAK2 tyrosine kinase inhibitor tyrphostin AG490 downregulates the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transccription (STAT) pathways and induces apoptosis in myeloma cells, Br. J. Haematol., 109(4):823-8). However, in some cases, AG4 90 can induce a dormant state of tumor cells to actually protect them from death.

Activation of JAK/STAT in cancer can occur through numerous mechanisms, including stimulation of a cytokine (e.g., IL-6 or GM-CSF) or by reducing endogenous suppressors of JAK signaling such as SOCS (suppressor or cytokine signaling) or PIAS ( protein inhibitor of activated STAT) (Boudny, V., Kovarik, J., Neoplasm., 49:349-355, 2002). Importantly, activation of signaling by STAT, as well as other pathways downstream of JAK (eg, Akt), correlates with poor prognosis for many types of cancer (Bowman, T. et al., Oncogene, 19:2474-2488 , 2000). In addition, elevated levels of circulating cytokines that signal through JAK/STAT may adversely affect the health of the patient, as they are expected to play an episodic role in cachexia and/or chronic fatigue. As such, inhibition of JAK may be of therapeutic value in the treatment of cancer patients for reasons that greatly expand the potential for antitumor activity. Data for cachexia may lead to additional mechanistic support gain from the fact that the saturation factor - leptin - signals through JAK.

Janus kinase 3 (JAK3) as a pharmacological target has been successfully used to control allograft rejection and graft-versus-host disease (GVHD). In addition to its involvement in cytokine receptor signaling, JAK3 is also involved in the CD40 signaling pathway of peripheral blood monocytes. During CD40-induced maturation of myeloid dendritic cells (DCs), JAK3 activity is induced and co-stimulated molecular expression, IL-12 production is increased, and a strong allogeneic stimulatory capacity is observed. The rationally designed JAK3 inhibitor, WHI-P-154, prevents these actions by stopping DCs at an immature level, suggesting that immunosuppressive therapy targeting JAK3 tyrosine kinase may also affect myeloid cell function (Saemann, M.D., Diakos, C. et al (2003), Prevention of CD40-triggered dendritic cell maturation and induction of T-cell hypo

- 3 040009 reactivity by targeting of Janus kinase 3, Am. J. Transplant., 3(11):1341-9). In a mouse model system, JAK3 has also been shown to be an important molecular target for the treatment of autoimmune insulin-dependent diabetes mellitus (type I). A rationally designed JAK3 inhibitor, JANEX-1, exhibited strong immunomodulatory activity and delayed the onset of diabetes in a NOD mouse model of type I autoimmune diabetes (Cetkovic-Cvrlje, M., Dragt, A.L. et al. (2003), Targeting JAK3 with JANEX- 1 for autoimmune type 1 diabetes prevention in NOD mice, Clin. Immunol., 106(3):213-25).

It has been suggested that inhibition of JAK2 tyrosine kinase may be beneficial in patients with myeloproliferative disorders (Levin et al., Cancer Cell, vol. 7, 2005:387-397). Myeloproliferative disorders (MPD) include polycythemia vera (PV), idiopathic thrombocytopenia (ET), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), and systemic mast cell disease ( SMCD). Although it is believed that myeloproliferative disorders are caused by an acquired somatic mutation in hematopoietic progenitors; the genetic basis of these diseases is unknown. However, the hematopoietic cells of the majority of PV patients and a significant number of ET and MMM patients have been reported to have recurrent somatic activating mutations in JAK2 tyrosine kinase. Inhibition of JAK2V617F kinase by small molecule inhibitors has also been reported to result in inhibition of hematopoietic cell proliferation, suggesting that JAK2 tyrosine kinase is a potential target for pharmacological inhibition in patients with PV, ET, and MMM.

Inhibition of JAK kinases is also expected to be of therapeutic benefit to patients suffering from skin immune disorders such as psoriasis and skin allergic reaction. It is generally accepted that in psoriasis vulgaris, the most common form of psoriasis, T lymphocytes are important in maintaining the disease and associated psoriatic plaques (Gottlieb, A. B. et al., Nat. Rev. Drug Disc., 4:19-34). Psoriatic plaques contain a significant immune infiltrate, including leukocytes and monocytes, as well as numerous layers of the epidermis with increased keratinocyte proliferation. While the initial activation of immune cells in psoriasis occurs through a disease-dependent mechanism, maintenance of the disease is thought to depend on a number of inflammatory cytokines, in addition to various chemokines and growth factors (JCI, 113:1664-1675). Many of them, including interleukins -2, -4, -6, -7, -12, -15, -18 and -23, as well as GM-CSF and IFNg, signal through Janus kinases (JAKs) (Adv Pharmacol , 2000, 47:113-74). As such, blocking signal transduction at the level of JAK kinases could lead to a therapeutic benefit in patients suffering from psoriasis or other skin immune disorders.

Some drugs are known to cause immune reactions such as skin rash or diarrhea in some patients. For example, the use of some novel targeted anti-cancer agents such as Iressa, Eritux and Tarceva has induced acne in some patients. Another example relates to the fact that some topical drugs cause skin irritation, skin rash, contact dermatitis or an allergic skin contact reaction. For some patients, these immune responses may be of concern, but for others, immune responses such as rash or diarrhea may make it impossible to continue treatment. Although the driving force behind these immune responses is not fully understood, to date these immune responses are likely to be associated with an immune infiltrate.

An extensive search is being made for inhibitors of Janus kinase or related kinases, and effective classes of compounds have been reported in several publications. For example, some inhibitors are reported in WO 99/65909, US 2004/0198737, WO 2004/099204, WO 2004/099205 and WO 01/42246. Heteroaryl-substituted pyrroles and other compounds are reported in WO 2004/72063 and WO 99/62908.

Thus, there is a continuing need for new or improved agents that inhibit kinases, such as Janus kinases, which act as immunosuppressive agents in organ transplantation, as well as agents for the prevention and treatment of autoimmune diseases (eg, multiple sclerosis, rheumatoid arthritis, asthma). , type I diabetes, inflammatory bowel disease, Crohn's disease, autoimmune thyroid disorders, Alzheimer's disease), diseases that involve an overactive inflammatory response (eg, eczema), allergies, cancer (eg, prostate cancer, leukemia, multiple myeloma) and some immune reactions (eg skin rash or contact dermatitis or diarrhea) caused by other drugs, some of which are mentioned. The compounds, compositions and methods described herein meet this need and other purposes.

Brief summary of the invention

The present invention relates to a medicament for the treatment of atopic dermatitis in a patient, said disease being associated with JAK activity, comprising a therapeutically effective amount of 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4- yl)-1H-pyrazol-1yl]propanenitrile or a pharmaceutically acceptable salt thereof. The compound is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, or its

- 4 040009 a pharmaceutically acceptable salt or (3S)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)1H-pyrazol-1-yl]propanenitrile, or a pharmaceutically acceptable thereof acceptable salt.

The invention also relates to the use of said compound 3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, or a pharmaceutically acceptable salt thereof, for the treatment of atopic dermatitis in a patient, said disease being associated with JAK activity. Said compound is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, or a pharmaceutically acceptable salt thereof, or ( 3S)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, or a pharmaceutically acceptable salt thereof.

Detailed description of the invention

The present invention relates to a medicament for the treatment of atopic dermatitis in a patient, said disease being associated with JAK activity, comprising a therapeutically effective amount of 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4- yl)-1H-pyrazol-1yl]propanenitrile or a pharmaceutically acceptable salt thereof. The compound is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof ; or (3S)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof.

The invention also relates to the use of said compound 3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof for the treatment of atopic dermatitis in a patient, said disease being associated with JAK activity. Said compound is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof; or (3S)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof.

Additionally, it should be understood that some of the features of the invention, which for clarity are described in the context of individual embodiments, can also be provided in combination with a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided individually or in any suitable subcombination.

The compounds described herein may be asymmetric (eg, containing one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are included within the scope of the invention unless otherwise indicated. Compounds of the present invention which contain asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods known in the art for preparing optically active forms from optically active starting materials include such as separation of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, compounds with C=N double bonds, etc. may also be present in the compounds described here, and all such stable isomers are also meant in the present invention. The cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms.

Separation into optical isomers of racemic mixtures of compounds can be carried out using any of the numerous methods known in this field. An exemplary method includes fractional crystallization using a chiral resolving acid, which is an optically active salt-forming organic acid. Suitable resolving agents for fractional crystallization processes are, for example, optically active acids such as D- and L-forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid; or various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other separating agents useful in fractional crystallization processes include stereoisomerically pure forms of α-methylbenzylamine (e.g., S- and R-forms or diastereomerically pure forms), 2-phenylglycine, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and etc.

Separation into optical isomers of racemic mixtures can also be carried out by elution on a column filled with an optically active resolving agent (eg dinitrobenzoylphenylglycine). A suitable solvent composition for elution can be determined by a person skilled in the art.

The compounds of the invention also include tautomeric forms. The tautomeric forms result from the replacement of a single bond with an adjacent double bond, together with concomitant proton migration. Tautomeric forms include prototropic tautomers, which are isomeric protonated states that share the same empirical formula and overall charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactamlactim pairs, amide-imidic acid pairs, enamine-imine pairs, and ring forms where the proton can

- 5 040009 occupy two or more positions in the heterocyclic system, for example 1H- and 3H-imidazole, 1H-, 2Hi 4H-1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. The tautomeric forms may be in equilibrium or they may be sterically fixed in one form by suitable substitution.

Compounds of the invention further include hydrates and solvates, as well as anhydrous and unsolvated forms.

The compounds of the invention also include all isotopes of atoms present in intermediate or final compounds. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

In some embodiments, the compounds of the invention and their salts are substantially isolated. By substantially isolated is meant that the compounds are at least partially or substantially separated from the environment in which they were formed or found. Partial separation may include, for example, a composition enriched with a compound of the invention. A substantive separation may include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97% or at least about 99% by weight of a compound of the invention or a salt thereof. Methods for isolating compounds and salts are common in the art.

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

The term pharmaceutically acceptable is used throughout this specification to refer to those compounds, substances, compositions and/or dosage forms which, within the reasonable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The present invention also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, the term pharmaceutically acceptable salts refers to derivatives of the described compounds in which the parent compounds are modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids and basic residues such as amines; alkali or organic salts of acid residues such as carboxylic acids; and so on. The pharmaceutically acceptable salts of the present invention include the usual non-toxic salts of the parent compounds formed, for example, with non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds that contain a basic or acid moiety using conventional chemical methods. Typically, such salts can be prepared by reacting the free acidic or basic forms of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent, or in a mixture of the two solvents; generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile (MeCN) are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack-Publishing Company, Easton, Pa., 1985, p. 1418; and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

The present invention also includes prodrugs of the compounds described herein. For use herein, the term prodrugs refers to covalently linked carriers that release the active parent drug upon administration to a mammalian subject. Prodrugs can be prepared by modifying the functional groups present on the compounds such that the modified groups are cleaved either by conventional manipulation or in vivo to give the parent compounds. Prodrugs include compounds in which a hydroxyl, amino, sulfhydryl, or carboxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, the acetate, formate, and benzoate derivatives of the alcohol and amine functional groups in the compounds of the invention. The production and use of prodrugs is discussed in Higuchi, T., Stella, V., Pro-drugs as Novel Delivery Systems, vol. 14 of the A.C.S. Symposium Series; and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entirety.

Synthesis.

The compounds of this invention, including their salts, can be obtained using known methods of organic synthesis and can be synthesized in accordance with any of the

- 6 040009 possible synthetic routes. The reactions for preparing the compounds of the invention may be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents may be substantially non-reactive with the starting materials (reagents), intermediates or products at the temperatures at which the reactions are carried out, for example at temperatures that may range from the freezing point of the solvent to the boiling point of the solvent. This reaction can be carried out in a single solvent or a mixture of solvents. Depending on the particular reaction step, suitable solvents for the particular reaction step may be selected by the skilled artisan.

The preparation of a compound of the invention may involve the introduction and deprotection of various chemical groups. The need for the introduction and removal of the protecting group and the choice of an appropriate protecting group can easily be determined by a person skilled in the art. The chemistry of protecting groups can be found in Green T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

Reactions can be controlled by any suitable method known in the art. For example, product formation can be monitored by spectroscopic methods such as nuclear magnetic resonance spectroscopy (for example, 1H or ¹³ C), infrared spectroscopy, spectrophotometry (for example, in the visible and ultraviolet light regions) or mass spectrometry, or using chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

The compounds of the invention can be prepared according to numerous synthetic routes known in the literature. Examples of synthetic methods for preparing the compounds of the invention are shown in the diagrams below.

As shown in Scheme 1, pyrazole-containing central fragments 1-9 and 1-6 can be synthesized starting from pyrrolo[2,3-d]pyridine or pyrrolo[2,3-b]pyrimidine 1-1. Compound 1-1 can be converted to active derivatives such as the N-oxide analog (1-2) using an oxidizing agent such as m-CPBA. The N-oxide 1-2 can be halogenated using a halogenating agent such as a combination of tetramethylammonium bromide and methanesulfonic anhydride to form a 4-halogenated compound 1-3 such as a 4-bromo compound while reducing the N-oxide at the same time. The amine group in compound 1-3 may be protected with an appropriate amine protecting group to give the protected compound 1-7, which is subsequently Suzuki cross-coupled with boric acid 1-8 to give the pyrazole-containing central moieties 1-9a, which may then be subjected to reaction with the reagent L-(Y)nZ (where L is a leaving group), leading to the compounds of the invention 1-9b. Alternatively, the N-oxide 1-2 can be halogenated using a halogenating agent such as MeSO ₂ Cl to form a 4-halogenated compound 1-4, such as a 4-chloro compound, while reducing the N-oxide at the same time. The 4-chloro compound 1-4 can be condensed with the bromo-substituted pyrazole compound 1-5 under suitable conditions, such as heat, to give the pyrazole-containing central moiety 1-6, which may contain certain functional groups, such as bromine or cyano, suitable for subsequent chemical modification.

Similarly, the imidazole core 1-11 can be synthesized by condensation of the 4-halo compound 1-4 with the imidazole derivative 1-10 under suitable conditions such as heat to give the imidazole core 1-11 which may contain some functional groups such as bromine or cyano, suitable for subsequent chemical modification.

- 7 040009

Scheme 1

As shown in Scheme 2, the pyrazole-containing central fragments 2-3, 2-5 and 2-6 can be synthesized starting from the bromo-substituted pyrazole derivative 2-1 (compound 1-6 in Scheme 1, where one of R ⁵ is Br). The bromo-substituted pyrazole derivative 2-1 may be fused with boron-containing aromatic derivatives such as aromatic boronic acids 2-2 using Suzuki cross-coupling, where Ar is aryl or heteroaryl, each of which may be optionally substituted with one or more substituents such as like alkyl, aryl, CN, nitro, alkoxy, etc. Alternatively, an alkene- or alkine-containing compound, such as an alkene-containing compound 2-5, can be prepared by the condensation of a bromo-substituted pyrazole derivative 2-1 with an unsaturated compound, such as an alkene 2-4, in the presence of a metal catalyst, such as bis(triphenylphosphine)palladium chloride (II) where t may be 0, 1, 2, etc.; and R may be a substituent such as alkyl, aryl, CN, nitro, alkoxy, etc. The alkene group in compound 2-5 can be reduced by hydrogenation to form the corresponding compound 2-6.

Scheme 2

- 8 040009

As shown in Scheme 3, imidazole-containing central fragments 3-7 can be synthesized starting from N-protected 4-bromo-pyrrolo[2,3-d]pyridine or N-protected 4-bromopyrrolo[2,3-b]pyrimidine 3- 1, where P is a suitable amine protecting group such as {[2-(trimethylsilyl)ethoxy]methyl} (SEM). Compound 3-1 can be reacted with a Grignard reagent such as isopropyl magnesium chloride to generate an aromatic anion by ion exchange. Subsequent addition to the anion of a chloroacetyl-containing compound, such as 2-chloro-N-methoxy-N-methylacetamide 3-2, will generally result in the chloroacetyl derivative 3-3. Derivative 3-3 may be reacted with an organic acid salt such as the cesium salt R ⁵ CO2Cs to give compound 3-4. In the presence of a suitable source of ammonia, such as ammonium acetate, compound 3-4 may be reacted with ammonia under suitable conditions such as high temperature to form the imidazole ring of compound 3-5. The free amine nitrogen in the imidazole derivative 3-5 can be subjected to a subsequent modification, such as reaction with an X-(Y)nZ compound, where X is a leaving group such as chlorine, bromine, or iodine, such that a compound 3- 6. The protecting group in compound 3-6 can be removed by a suitable method according to the nature of the protecting group, resulting in compound 3-7. It should be noted that if there are functional groups present in the group R, R ⁵ and -(Y)nZ, further modifications can be made. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol, which in turn can be further modified. Suitable subsequent modifications will be apparent to those skilled in the art.

Scheme 3

As shown in Scheme 4, thiazole-containing central fragments 4-3 can be synthesized starting from the N-protected chloroacetyl derivative 4-1, where P is a suitable amine protecting group, such as SEM. Compound 4-1 can be reacted with thioamide 4-2 to form a thiazole ring, followed by deprotection of the amino nitrogen on the pyrrole ring by removing the P group to give compound 4-3. Various thioureas 4-5 (equivalents of compound 4-2 where -(Y) _n -Z is NR'R; and R' and R are H, alkyl, aryl, or the like; or R' and R together with the N atom to which they are attached form heterocycloalkyl), which can be used to obtain thiazole compounds 4-3, can be obtained from secondary amines 4-4. The secondary amine 4-4 can be reacted with 1,1'-thiocarbonyldiimidazole and the resulting intermediate can be further reacted with ammonia to give thiourea 4-5.

Scheme 4

As shown in Scheme 5, thiazole-containing central fragments 5-5 can be synthesized

- 9 040009 based on the thiazole compound 5-1. Compound 5-1 can be reacted with a metal alkyl such as n-butyllithium to generate an aromatic anion in situ via ion exchange. Subsequent addition of boric acid trimethyl ester followed by hydrolysis will generally give boric acid 5-2. Boric acid 5-2 can enter into a Suzuki cross-coupling with N-protected 4-bromo-pyrrolo[2,3-b]pyridine or N-protected 4-bromo-pyrrolo[2,3-d]pyrimidine 5-3, where P is a suitable amine protecting group such as SEM. The protecting group P in the cross-coupling product 5-4 can be removed using a suitable method according to the nature of the protecting group, resulting in the compound of the invention 5-5.

1. nBuLi, hexanes

2. V(OMe) ₃

5-1

^B(OH) ₂

H ₂ O/DMF ^{5 2} heating

Scheme 5

As shown in Scheme 6, the pyrazole-containing compounds 6-1 can be further modified by replacing the pyrazole NH group with suitable reagents. For example, compound 6-1 in which P is a suitable amine protecting group such as SEM can be reacted with L-(Y) _n -Z where L is a leaving group such as halogen, triflate, or the like. n., with the receipt in basic conditions of compound 6-2. If some functional groups are present in the Y and/or Z groups, further modifications may be made. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol. Suitable subsequent modifications will be apparent to those skilled in the art.

In addition, compound 6-1 may be reacted with alkene 6-3 (wherein R' and R may be H, alkyl, cycloalkyl, etc.; and Z' may be an electron withdrawing group such as an ester group or CN) resulting in compound 6-4. In addition, a substitution of the alkene 6-3 in the alpha position (alpha relative to Z') can be carried out to form substituted derivatives of the product 6-4.

Compounds 6-2 and 6-4 may be deprotected using appropriate methods according to the nature of the protecting group used to form corresponding unprotected equivalents.

6-4

Scheme 6

- 10 040009

As shown in Scheme 7, bromopyrazole-containing compounds 7-1 can be further modified by metalation with reagents such as butyllithium and reaction with electrophiles such as aldehydes to give alcohol-containing compounds 7-2, which can be deprotected to give compounds of the invention having the formula 7-3. A person skilled in the art will understand suitable subsequent modifications, where appropriate.

Scheme 7

As shown in Scheme 8, pyrazole-containing compounds 8-4 and 8-5 can be prepared by reacting the N-protected bromide compound 8-1 with hydrazine in a suitable solvent such as N,N-dimethylformamide (DMF) to give the hydrazine intermediate 8 -2. The hydrazine intermediate 8-2 is reacted with a suitably substituted 1,3-bis-aldehyde such as 8-3 to give the pyrazole-containing compound 8-4. If some functional groups are present in the Y and/or Z groups, further modifications may be made. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol. Those skilled in the art will recognize suitable subsequent modifications.

Scheme 8

As shown in Scheme 9, the 1,2,4-oxadiazole compound 9-6 can be prepared from the N-protected bromide compound 9-1 by treating it with zinc cyanide in DMF in the presence of a catalyst such as bis(tributyl)palladium to give N-protected cyano compound 9-2. The N-hydroxycarboximidamide compound 9-3 can be prepared by heating the N-protected cyano compound 9-2 with hydroxylamine hydrochloride in a suitable solvent such as ethanol in the presence of a base such as potassium carbonate at a temperature below the boiling point of the solvent. An N-protected 1,2,4-oxadiazole compound can be prepared by treating the N-hydroxycarboximidamide compound 9-3 with an appropriately substituted acid chloride 9-4 in a solvent such as pyridine at a temperature sufficient to complete ring closure. If some functional groups are present in the Y and/or Z groups, further modifications may be made. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol. A person skilled in the art will understand suitable subsequent modifications, where appropriate.

- 11 040009

Scheme 9

As shown in Scheme 10, 3- and 4-arylpyrazolo compounds 10-9 can be prepared by reacting the corresponding 3-arylpyrazolo compound 10-4 or 4-arylpyrazolo compound 10-7 with a suitably substituted bromide compound 10-8, as described earlier. The 3-arylpyrazole compound 10-4 can be prepared by reacting a suitably substituted halogen-containing aryl group such as bromine or triflate with an N-protected boronic acid or boronic acid ester of the pyrazole compound 10-2 under conditions such as Suzuki conditions known in literature. The N-protecting group at 10-3 can be removed under conditions previously described and known in the literature for removing a group such as SEM.

The 4-arylpyrazole compounds 10-7 can be prepared by reacting a suitably substituted acetophenone compound 10-5 with a DMF acetal at elevated temperature to give the dimethylamino compound 10-6. 4-Arylpyrazolo compounds 10-7 can be prepared by treating dimethylamino compound 10-6 with hydrazine in a solvent such as ethanol.

Scheme 10

As shown in Scheme 11, the substituted pyrazole compound 11-5 can be prepared in a variety of ways, such as deprotection, eg SEM, on compound 11-4 under the previously described conditions. For example, a substituted N-protected pyrazole compound 11-4 can be prepared by reacting an intermediate N-protected pyrazole compound 11-3 with a suitably substituted alkyl halide, benzyl halide, alkyl sulfonates, e.g. mesylate or tosylate, or other suitable leaving groups L, in a suitable a solvent such as MeCN, DMF or tetrahydrofuran (THF) in the presence of a base such as sodium hydride or cesium carbonate. N-arylpyrazole 11-4 (where Y is an aromatic group) can be prepared by reacting the pyrazole intermediate 11-3 with a suitably substituted arylboronic acid in a solvent such as dichloromethane (DCM) in the presence of copper acetate and pyridine. Alternatively, Narylpyrazole 11-4 (where Y is an aromatic group) can be prepared by reacting the pyrazole intermediate 11-3 with a suitably substituted aryl fluoride in a solvent such as DMF at elevated temperature. Or substituted pyrazole compounds 11-4 (where Z is a group such as nitrile or ester and Y has at least two carbons) can be prepared by reacting the pyrazole intermediate 11-3 with a suitably substituted acrylate, acrylonitrile, or other type acceptors Michael in solvent

- 12 040009 such as DMF in the presence of a base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine (TEA) and at a temperature below the boiling point of the solvent. If some functional groups are present in the Y and/or Z groups, further modifications may be made. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol. A person skilled in the art will understand suitable subsequent modifications, where appropriate.

Scheme 11

As shown in Scheme 12, pyrazole 12-1, where P is a suitable amine protecting group such as SEM and can be reacted with an alkyne-containing conjugated acceptor such as 12-2; and where Z is an electron withdrawing group (eg -CN) optionally in the presence of a base (DBU or K2CO3 etc.) in a solvent such as DMF or MeCN for varying lengths of time to produce olefin-containing adducts 12-3. The compounds represented by formula 12-3 may be deprotected using suitable methods according to the nature of the protecting group used to give compounds of invention 12-4.

Scheme 12

As shown in Scheme 13, oxazole- or thiazole-containing compounds 13-6 can be prepared starting from N-protected 4-chloro-pyrrolo[2,3-d]pyrimidine 13-1, where P is a suitable amine protecting group such as S.E.M. Oxazole- or thiazole-containing products of formula 13-2 can be prepared by palladium-catalyzed condensation of 13-1 with oxazole or thiazole. Compound 13-2 can be reacted with a metal alkyl such as n-butyllithium to generate an in situ aromatic anion to which carboxylic acid derivatives 13-3 ( where W=N(Me)(OMe) when X'=S; and W=Cl when X'=O), in the presence of other auxiliary additives such as zinc chloride or copper(I) iodide when X'= O, in a suitable solvent such as THF to form a variety of ketones 13-4. Ketones 13-4 can be reacted with a variety of reagents such as diethyl(cyanomethyl)phosphonate or triethylphosphonoacetate in the presence of a base such as potassium tert-butoxide followed by reduction (including hydrogenation or copper hydride catalyzed reduction) or with reagents such as tosylmethyl isocyanide to give products of formula 13-5 wherein Z is an electron withdrawing group such as ester or -CN. If some functional groups are present in the group R or they are covered by Z, further modifications can be made and such suitable further modifications will be obvious to a person skilled in the art. In compounds 13-5, the protective

- 13 040009 group using suitable methods in accordance with the nature of the protective group used, to obtain the corresponding unprotected equivalents of 13-6.

Scheme 13

As shown in Scheme 14, aminothiazole-containing central fragments 14-5 can be synthesized starting from a thiazole-containing central fragment 14-1, where P is a suitable amine protecting group, such as SEM. Compound 14-1 can be treated with a metal alkyl such as n-butyllithium to generate an in situ aromatic anion to which a suitable electrophilic halogen source such as carbon tetrabromide can be added to give the halogenated derivative 14-2. The protecting group P in 14-2 can be removed using a suitable method according to the nature of the protecting group used to give the product 14-3. Compound 14-3 may be reacted with amines 14-4 at elevated temperature in a suitable solvent such as DMF to give compound 14-5 of the invention.

Scheme 14

As shown in Scheme 15, pyrrole-containing central fragments 15-4 can be synthesized starting from N-protected 4-chloro-pyrrolo[2,3-d]pyrimidine 15-1, where P is a suitable amine protecting group such as DEM ( diethoxymethyl). Compound 15-1 can be reacted with 1-(triisopropylsilyl)pyrrole-3-boronic acid under Suzuki cross-coupling conditions to simultaneously give the deprotected pyrrole central moiety 15-2. Pyrrole-containing compounds 15-2 can be reacted with alkenes 15-3 containing an electron withdrawing group Z (such as -CN) in the presence of a suitable base (such as DBU) at various temperatures (for example, between room temperature and 40°C) followed by in situ or a separate deprotection step which is appropriate for the selected protecting group to give compounds of the invention 15-4.

- 14 040009

Scheme 15

As shown in Scheme 16, a substituted pyrazole compound containing a sulfone or sulfoxide functional group, as in 16-6, can be obtained in a variety of ways, for example, starting from a suitably substituted bromothiophenyl ether 16-2. The thioether 16-2 can easily be obtained by alkylation of the thiophenol 16-1 with an alkyl halide, mesylate or the like. using a base such as DBU, potassium carbonate or sodium hydride. Cinnamylnitrile 16-3 can be prepared by the Heck reaction or the like using palladium acetate and triphenylphosphine in DMF at a suitable temperature using acrylonitrile. The SEM-protected intermediate 16-4 can be prepared by the methods previously described for performing the Michael addition of the pyrazole central moiety to a suitably substituted α-β-unsaturated nitrile such as 16-3. The sulfoxide 16-5 where n=1 and the sulfone 16-5 where n=2 can be prepared by methods well known in the literature for the oxidation of the thioether 16-4, such as using m-chloroperbenzoic acid (MCPBA ) in DXM. Final compounds 16-6, where n=0, 1 or 2, can be obtained by the methods described previously for the removal of the protective group SEM. Alternatively, sulfur oxidation can be carried out for compounds 16-2 or 16-3 depending on the compatibility of the substitution in the synthesis scheme.

Scheme 16

Also, as shown in Scheme 17, substituted pyrazole compounds containing a sulfonamide functional group, such as 17-6, can be prepared in a variety of ways. For example, you can start from a suitably substituted bromophenylsulfonamide 17-2, where R ^c and R ^d are suitable substituents. Compound 17-2 can easily be prepared by reacting bromophenylsulfonyl chloride 17-1 and a suitably substituted amine such as aniline or a primary or secondary amine in a suitable solvent such as DCM, THF or pyridine. Cinnamylnitrile 17-3 can be prepared by the Heck reaction or the like using palladium acetate and triphenylphosphine in DMF at a suitable temperature using acrylonitrile. Final compounds 17-6, in which R ^c and R ^d are part of the sulfonamide functional group, can be obtained by methods similar to those described for scheme 16, starting from cinnamyl nitrile 17-3.

- 15 040009

Scheme 17

Also, as shown in Scheme 18, substituted pyrazole compounds containing an alpha-allylic cyclopentylmethylene functional group, such as 18-8, can be prepared, for example, by reacting pyrazole 18-3, where P is a suitable amine protecting group, such as SEM, and X is N or C, with cyclopentyl acrylate ester 18-4 to form ester 18-5. The ester 18-5 can then be reduced to the corresponding aldehyde 18-6, for example by a two-step process involving reduction to an alcohol and selective oxidation of an intermediate alcohol to an aldehyde, for example by Swern oxidation. Aldehyde 18-6 can be converted to the corresponding olefin 18-7, for example by reaction with a Wittig reagent. The olefin 18-7 can then be deprotected as described previously to give a compound of formula 18-7. Intermediate 18-4 can be prepared, for example, as shown in Scheme 18, starting from cyclopentylaldehyde.

Scheme 18

Also, as shown in Scheme 19, the cyanoguanidine derivative 19-6 can be prepared starting from substituted pyrazole compounds such as pyrazole 18-3, where P is an appropriate protecting group and X is N or C. Compound 18-3 can, for example, be reacted with the Horner-Woodsworth-Emmons olefin 19-1 of the corresponding Boc-protected piperidone in the presence of a suitable basic catalyst in a suitable solvent to form 19-2. Intermediate 192 is deprotected using an appropriate deprotection reaction to give the amine compound 19-3 which is then reacted selectively with a cyanoimidocarbonate reagent such as 19-4 in a polar solvent at a suitable temperature, e.g., about 20°C , giving a cyanoimidocarbamate such as 19-5, which can then be reacted with any of a variety of amines at elevated temperature to give the product 19-6.

- 16 040009

Scheme 19

Intermediates 20-5 and 20-6 can be prepared using a variety of methods known in the literature, such as those summarized in Scheme 20. Intermediate 20-3 can be prepared by reacting the aldehyde compound 20-1 with a suitably substituted Wittig reagent or Horner-Emmons reagents to form the α-β-unsubstituted ester 20-3. Alternatively, 20-3 can be prepared by a Heck type reaction of a suitably substituted aryl bromide 20-2 and an acrylic ester in the presence of a palladium reagent at elevated temperature. Compound 20-4 can be prepared by the methods described previously for Michael addition of the suitably substituted pyrrole 18-3 to the α-β-unsaturated ester 20-3. The aldehyde compound 20-5 can be prepared by reducing the ester compound 20-4 using reagents such as diisobutylaluminum hydride at low temperature, such as about -78° C., in a suitable solvent. The aldehyde compound 20-5 can be further reduced to the corresponding alcohol compound 20-6 using reagents such as sodium borohydride in methanol. Alternatively, the alcohol compound 20-6 can be prepared directly by reduction of the ester 20-4 using reagents such as lithium aluminum hydride in a suitable solvent and at a suitable temperature.

Scheme 20

- 17 040009

Compounds 21-1 and 21-3 can be prepared using a variety of methods known in the literature, such as those summarized in Scheme 21. Olefin compound 21-1 can be prepared by reacting aldehyde 20-5 with a suitably substituted Wittig reagent or Horner-Emmons reagents using a base such as sodium hydride or potassium t-butoxide in a suitable solvent when carried out at elevated temperature. Olefin compound 21-1 can be reduced to saturated compound 21-2, for example, using hydrogenation conditions well known in the literature, such as hydrogenation in the presence of palladium on carbon in a solvent such as methanol. The acetylene compound 21-3 can be prepared by the methods described previously or by reacting the aldehyde 20-5 with the Bestmann-Ohira reagent (Quesada, E. et al., Tetrahedron, 62 (2006), 6673-6680) as described in the literature. Alternatively, the alcohol compound 20-6 in Scheme 20 can be oxidized to the aldehyde 20-5 using methods well known in the literature, e.g. under Swern oxidation conditions, followed by reaction with a Bestmann-Ohhir reagent, where this sequence of reactions can be carried out in as a one-pot two-step reaction sequence or as two separate reaction steps.

Scheme 21

Compounds 22-1 and 22-3 can be prepared using a variety of methods known in the literature, such as those summarized in Scheme 22. The oxygen-substituted compound 22-1 can be prepared, for example, by reaction with an appropriately substituted alcohol 20- 6 (in Scheme 20), where X is N or C and P is a protecting group, with a base such as sodium hydride and a suitable agent such as an alkyl iodide, carbonate or isocyanate, carried out in a suitable solvent and at a suitable temperature. Alternatively, the alcohol group in compound 20-6 can be converted to an LG leaving group, as in compound 222, where the leaving group can be bromide or mesylate. Compound 22-2 serves as a substrate for the subsequent reaction with a nucleophile such as, for example, sodium ethoxide (Nuc=ethoxy).

- 18 040009

Scheme 22

It should be noted that in all the schemes described, if functional groups are present in one of the substituent groups such as Y, Z, R, R ¹ , R ² , R ⁵ etc., further modifications can be made if this is appropriate and desirable. For example, a CN group can be hydrolyzed to give an amide group, a carboxylic acid can be converted to an ester, which in turn can be reduced to an alcohol. In another example, the OH group can be converted to a better leaving group, such as mesylate, which in turn is suitable for nucleophilic substitution, such as a CN group. Such subsequent modifications will be apparent to a person skilled in the art.

Methods.

The compounds of the invention may modulate the activity of one or more Janus kinases (JAKs). The term modulate is intended to mean the ability to increase or decrease the activity of one or more members of the JAK family of kinases. Accordingly, the compounds of the invention may be used in methods for modulating JAK by contacting JAK with one or more of the compounds or compositions described herein. In some embodiments, the compounds of the invention may act as inhibitors of one or more JAKs. In some embodiments, the compounds of the invention may act to stimulate the activity of one or more JAKs. In further embodiments, compounds of the invention can be used to modulate JAK activity in an individual in need of receptor modulation by administering a modulating amount of a compound of Formula Ia, Ib, or Ic.

The JAK kinases that the present compounds bind to and/or modulate include any member of the JAK family. In some embodiments, JAK is JAK1, JAK2, JAK3, or TYK2. In some embodiments, JAK is JAK1 or JAK2. In some embodiments, the JAK is JAK2. In some embodiments, the JAK is JAK3.

The compounds of the invention may be selective. By the term selective is meant that a compound binds to or inhibits a JAK with greater affinity or potency, respectively, than at least one other JAK. In some embodiments, the compounds of the invention are selective inhibitors of JAK1 or JAK2 over JAK3 and/or TYK2. In some embodiments, the compounds of the invention are selective inhibitors of JAK2 (eg, relative to JAK1, JAK3, and TYK2). Without wishing to be bound by theory, since JAK3 inhibitors may result in an immunosuppressive effect, a compound that is selective for JAK2 over JAK3 and that is used to treat cancer (such as multiple myeloma, for example) may offer the additional benefit of having less immunosuppressive side effects. The selectivity may be at least about 5-fold, 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500 times, or at least about 1000 times. Selectivity can be measured by methods that are routine in the art. In some embodiments, selectivity can be tested for the Km of each enzyme. In some embodiments, the selectivity of the compounds of the invention for JAK2 over JAK3 can be determined using cellular ATP concentration.

Another aspect of the present invention encompasses methods of treating a JAK-associated disease or disorder in an individual (eg, patient) by administering to an individual in need of such treatment a therapeutically effective amount or dose of a compound of the present invention or a pharmaceutical composition thereof. A JAK-associated disease may include any disease, disorder, or condition that is directly or indirectly associated with JAK expression or activity, including overexpression and/or abnormal levels of activity. A JAK-associated disease may also include any disease, disorder, or condition that can be prevented, reduced, or treated by modulating JAK activity.

Examples of JAK-related diseases include diseases of the immune system, including, for example, organ transplant rejection (eg, allograft rejection or graft versus host disease).

Further examples of JAK-associated diseases include autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, juvenile arthritis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, bulbospinal palsy, immunoglobulin neuropathies, autoimmune thyroid disorders, and etc. In some embodiments, the autoimmune disease is an autoimmune bullous skin disease such as pemphigus vulgaris (OP) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditions such as asthma, food allergies, atopic dermatitis, and rhinitis. Other examples of JAK-associated diseases include viral diseases such as Epstein-Barr virus (EBV), hepatitis B, hepatitis C, HIV, HTLV 1, Varicella-Zoster virus (VZV), and human papillomavirus (HPV).

Additional examples of JAK-associated diseases or conditions include skin diseases such as psoriasis (eg, psoriasis vulgaris), atopic dermatitis, skin rash, skin irritation, hypersensitivity skin reaction (eg, contact dermatitis or allergic contact dermatitis). For example, some substances, including some pharmaceuticals, can cause a skin reaction when applied topically. In some embodiments, co-administration or sequential administration of at least one JAK inhibitor of the invention together with an adverse allergic reaction agent may be useful in the treatment of such adverse allergic reactions or dermatitis. In some embodiments, the skin disorder is treated with topical application of at least one JAK inhibitor of the invention.

In further embodiments, the JAK-associated disease is a cancer, including one characterized by solid tumors (e.g., prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer glands, glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma, etc.), hematologic cancers (eg, lymphoma, leukemia such as acute lymphoblastic leukemia or multiple myeloma), and skin cancers such as cutaneous T-cell lymphoma (CTCL ) and cutaneous B-cell lymphoma. Examples of cutaneous T-cell lymphomas include Cesaria's syndrome and mycosis fungoides.

JAK-associated diseases may further include those characterized by the expression of mutant JAK2, such as having at least one mutation in the pseudokinase domain (eg, AK2V617F).

JAK-associated diseases may further include myeloproliferative disorders (MPDs) such as Wakez-Osler disease (PV), idiopathic thrombocytopenia (ET), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML) , hypereosinophilic syndrome (HES) or systemic mast cell disease (SMCD), and the like.

Other JAK-associated diseases include inflammation and inflammatory diseases. Examples of inflammatory diseases include diseases of the eye (eg, iris inflammation, uveitis, scleritis, conjunctivitis, or a related disease), inflammatory diseases of the respiratory tract (eg, upper respiratory tract, including the nose and sinuses, such as rhinitis or sinusitis, or lower respiratory pathways including bronchitis, chronic obstructive pulmonary disease, and the like), inflammatory myopathy such as myocarditis, and other inflammatory diseases.

The JAK inhibitors described herein can also be used to treat ischemic reperfusion injury, or a disease or condition related to an inflammatory ischemic event, such as stroke or cardiac arrest. The JAK inhibitors described herein can also be used to treat anorexia, cachexia, or fatigue such as that occurs with or associated with cancer. The JAK inhibitors described herein can also be used to treat restenosis, scleroderma, or fibrosis. The JAK inhibitors described herein can also be used to treat conditions associated with hypoxia or astroglioma such as diabetic retinopathy, cancer, or neurodegeneration (see, for example, Dudley, A.C. et al., Biochem. J., 2005, 390(Pt. 2):427-36;

and Sriram, K. et al., J. Biol. Chem., 2004, 279(19):19936-47, epub, 2004, Mar 2).

For use herein, the term contacting refers to the co-occurrence of said fragments in an in vitro system or in an in vivo system. For example, contacting a JAK with a compound of the invention includes administering a compound of the present invention to an individual or patient, such as a human, having a JAK, as well as, for example, administering a compound of the invention to a sample containing a cell or purified preparation containing JAK.

For use herein, the term subject or patient, used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, and most preferably to a person.

For use herein, the term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, individual, or human being considered by an investigator, veterinarian, treating physician, or other clinician, which includes one or more a few of the following:

(1) preventing a disease, such as preventing a disease, condition, or disorder, in an individual who may be predisposed to the disease, condition, or disorder, but who is not yet susceptible to, or who does not yet exhibit, the pathology or symptoms of the disease;

(2) suppression of the disease, eg, suppression of the disease, condition, or disorder, in an individual who is susceptible to or exhibits the pathology or symptoms of the disease, condition, or disorder (ie, arresting further development of the pathology and/or symptom); and (3) alleviating the course of the disease, such as alleviating the course of the disease, condition, or disorder, in an individual who is susceptible to or exhibits the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and/or symptoms).

Combined therapy.

One or more additional pharmaceutical agents, such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, and inhibitors of Bcr-Ab1, Flt-3, RAF and FAK kinases, such as those described in WO 2006/056399, or other agents can be used in combination with the compounds of the present invention for the treatment of JAK-related diseases, disorders or conditions. One or more additional pharmaceutical agents may be administered to a patient simultaneously or sequentially.

Examples of chemotherapeutic agents include proteasome inhibitors (eg, bortezomide, thalidomide, revlimide) and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.

Examples of steroids include corticosteroids such as dexamethasone or prednisone.

Examples of Bcr-Ab1 inhibitors include the compounds and their pharmaceutically acceptable salts of the general form and varieties described in US Pat.

Examples of suitable Flt-3 inhibitors include compounds and their pharmaceutically acceptable salts such as those described in WO 03/037347, WO 03/099771 and WO 04/046120.

Examples of suitable RAF inhibitors include compounds and their pharmaceutically acceptable salts such as those described in WO 00/09495 and WO 05/028444.

Examples of suitable FAK inhibitors include compounds and their pharmaceutically acceptable salts such as those described in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595 and WO 01/014402.

In some embodiments, the JAK inhibitors of the invention can be used in combination with chemotherapy in the treatment of cancer, such as multiple myeloma, and they can improve the response to treatment compared to the response to the chemotherapeutic agent by itself without excerbing its toxic effects. Examples of additional pharmaceutical agents useful in the treatment of multiple myeloma, for example, may include, without limitation, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velkad (bortezomib). Other additional agents used in the treatment of multiple myeloma include Bcr-Ab1, Flt-3, RAF and FAK kinase inhibitors. An additive or synergistic effect is a desirable result of combining a JAK inhibitor of the present invention with an additional agent. In addition, the resistance of multiple myeloma cells to agents such as dexamethasone may be reversible when treated with a JAK inhibitor of the present invention. These agents may be combined with the present compounds as a single or constant formulation, or the agents may be administered simultaneously or sequentially as separate formulations.

- 21 040009

In some embodiments, a corticosteroid, such as dexamethasone, is administered to a patient in combination with at least one JAK inhibitor, wherein the dexamethasone is administered intermittently as opposed to continuously.

In some of the following embodiments, combinations of one or more JAK inhibitors of the invention with other therapeutic agents may be administered to a patient before, during and/or after bone marrow or stem cell transplantation.

Pharmaceutical preparations and dosage forms.

When used as medicines, the compounds of the invention can be used in the form of pharmaceutical compositions. These compositions may be prepared in a manner well known in the pharmaceutical art and may be administered by a variety of routes of administration depending on whether topical or systemic treatment is required and the area being treated. Administration may be topical (including transdermal, epidermal, ophthalmic, and mucosal, including intranasal, vaginal, and rectal drug delivery), pulmonary (eg, by inhalation or insufflation of powders or aerosols, including use of a nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial, eg intrathecal or intraventricular, administration. Parenteral administration may be in the form of a single bolus dose or may be continuously delivered by a perfusion pump. Pharmaceutical compositions and topical preparations may include transdermal patches, ointments, lotions, creams, gels, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, and the like. may be necessary or desirable. Coated condoms, gloves, etc. may also be used.

This invention also includes pharmaceutical compositions which contain, as an active ingredient, one or more compounds of the invention in combination with one or more pharmaceutically acceptable carriers (excipients). In preparing the compositions of the invention, the active ingredient is usually mixed with an excipient, diluted with an excipient, or enclosed in such a carrier in the form of, for example, a capsule, sachet, paper or other container. When the excipient acts as a diluent, it may be a solid, semi-solid or liquid substance which serves as a vehicle, carrier or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments containing, for example, up to 10% by weight of the active compound, soft or hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

Upon receipt of the drug, the active compound can be crushed to obtain particles of a suitable size before combining with other ingredients. If the active compound is substantially insoluble, it may be ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to obtain a substantially uniform distribution in the formulation, for example up to about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, gum tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations may further include lubricants such as talc, magnesium stearate and mineral oil;

wetting agents;

emulsifying and suspending agents;

preservatives such as methyl and propyl hydroxybenzoates;

sweeteners and flavoring agents.

Compositions of the invention may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by methods known in the art.

The compositions may be prepared as unit dosage forms wherein each dose will contain from about 5 mg to about 1000 mg (1 g), more typically from about 100 mg to about 500 mg, of the active ingredient. The term unit dosage form refers to physically discrete units suitable as unit doses for humans and other mammals, each unit containing a predetermined amount of active agent calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient.

The active compound may be effective over a wide dosage range and is usually administered in a pharmaceutically effective amount. It should be understood that the amount of compound actually administered will be determined by the attending physician according to relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound being administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and etc.

To obtain solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to obtain a solid pre-prescription composition containing a homogeneous mixture of the compounds of the present invention. When these pre-prescription compositions are referred to as homogeneous, the active ingredient is usually evenly dispersed in the composition so that the composition can be divided into equally effective unit dosage forms such as tablets, pills and capsules. This pre-prescription composition is then subdivided into unit dosage forms of the above type, containing from, for example, about 0.1 mg to about 1000 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise formulated to provide a dosage form that has the advantage of sustained action. For example, a tablet or pill may include an internal dose component and an external dose component, the latter being in the form of an envelope for the former. The two components may be separated by an enteric layer which serves to counteract breakdown in the stomach and allow the internal component to pass into the duodenum or slow its release. A variety of materials can be used for such enteric layers, including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

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

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain pharmaceutically acceptable excipients as described above. In some embodiments, the implementation of the composition is administered through the mouth or nasal respiratory tract for local or systemic action. Compositions in a metal container can be sprayed with inert gases. Nebulized solutions can be inhaled directly from a nebulizing device, or the nebulizing device can be attached to a face mask or positive pressure swing breathing machine. Solutions, suspensions, or powder compositions can be administered orally or nasally from devices that deliver the drug in a suitable manner.

The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of administration such as prophylaxis or therapy, the patient's condition, route of administration, and the like. In therapeutic applications, the compositions may be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective dosages will depend on the disease state being treated as well as the judgment of the attending physician depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

The compositions administered to the patient may be the compositions described above. These compositions may be sterilized by conventional sterilization methods or they may be sterilized by filtration. Aqueous solutions may be packaged for use as they are or lyophilized and the lyophilized preparations combined with a sterile aqueous vehicle prior to use. The pH of compound preparations will typically be between 3 and 11, more preferably 5 to 9, and most preferably 7 to 8. It will be understood that the use of some of the aforementioned excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention may vary according to, for example, the specific application for which treatment is being carried out, the method of administration of the compound, the health and general condition of the patient, and the point of view of the attending physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition may vary depending on a number of factors including dosage, chemical characteristics (eg hydrophobicity) and route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing from about 0.1% wt./about. up to about 10% wt./about. compounds for parenteral administration. Some typical dosage ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dosage range is from about 0.011 mg/kg to about 100 mg/kg of body weight per day. The dosage will likely depend on variables such as the type and extent of progression of the disease or disorder, the general health of the individual patient, and the relative biological potency of the compound chosen.

- 23 040009 excipient formulations and routes of administration. Effective doses can be extrapolated from dose-response curves in in vitro test systems or animal models.

Compositions of the invention may further include one or more additional pharmaceutical agents, such as a chemotherapeutic agent, steroid, anti-inflammatory compound, or immunosuppressant, examples of which are listed above in this description.

Labeled compounds and methods of analysis.

Another aspect of the present invention relates to labeled compounds of the invention (radiolabeled, fluorescently labeled, etc.) which would be useful not only in imaging methods but also in assays, both in vitro and in vivo, for localization and quantification of JAK in tissue samples, including human tissues, and to identify JAK ligands by inhibiting labeled compound binding. Accordingly, the present invention includes JAK assays that contain such labeled compounds.

The present invention further includes the isotopically labeled compounds of the invention. An isotopically or radioactively labeled compound is a compound of the invention in which one or more atoms has been replaced or replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the compounds of the present invention include, but are not limited to, ² H (spelled the same as D for deuterium), ³ H (spelled the same as T for tritium), ^P C, ¹³ C , ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ¹⁸ F ^, 35 S, ³⁶ Cl, ⁸² Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and ¹³¹ I.

The nature of the radionuclide that is incorporated into the present radiolabeled compounds will depend on the specific application of such radiolabeled compound. For example, for in vitro labeling of metalloproteases and competitive assays, compounds in which ³ H, ¹⁴ C, ⁸² Br, ¹²⁵ I, ¹³¹ I, ^{35 S} are introduced will be most useful. ¹⁸ F, ¹²⁵ I, ¹²³ I, ¹²⁴ I, ¹³¹ 1, ⁷⁵ Br, ⁷⁶ Br or ⁷⁷ Br.

It should be understood that a radiolabeled or labeled compound is a compound into which at least one radionuclide has been introduced. In some embodiments, the radionuclide is selected from the group consisting of ³ H, ¹⁴ C, ^{125 1.35} S, and ⁸² Br.

The present invention may further include synthetic methods for introducing radioisotopes into the compounds of the invention. Synthetic methods for introducing radioisotopes into organic compounds are well known in the art, and methods applicable to the compounds of the invention will be apparent to one of ordinary skill in the art.

The labeled compound of the invention can be used in screening assays for the identification/evaluation of compounds. For example, the ability to bind JAK of newly synthesized or identified compounds (ie, test compounds) that are labeled can be assessed by monitoring the change in their concentration when contacted with JAK by tracking the label. For example, the ability of a test compound (labeled) to reduce the binding of another compound known to bind to JAK (ie, a standard compound) can be evaluated. Accordingly, the ability of a test compound to compete with a reference compound for binding to JAK is directly correlated with its binding affinity. Conversely, in some other screening assays, the reference compound is labeled and the test compound is unlabeled. Accordingly, the concentration of the labeled reference compound is monitored to assess competition between the reference compound and the test compound, and thus the relative binding affinity of the test compound is determined.

Sets.

The present invention also includes pharmaceutical kits that can be used, for example, for the treatment or prevention of JAK-related diseases or disorders such as cancer, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention. Such kits may further include, if desired, one or more of the various conventional components of a pharmaceutical kit, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. Instructions in the form of inserts or labels indicating quantities of components to administer, directions to administer, and/or directions to mix the components may also be included in the kit.

The invention will be described in more detail using specific examples. The following examples are given for illustrative purposes and are not intended to limit the invention in any way. Those of skill in the art will readily appreciate the many non-critical parameters that can be changed or modified to produce substantially the same results. In accordance with at least one of the assays described here, the compounds of the invention were found to be JAK inhibitors.

- 24 040009

Examples

Example 1 (3R)- and (3S)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-ul)-1H-pyrazol-1yl]propanenitrile.

Stage 1

To (2E)- and (2Z)-3-cyclopentylacrylonitrile a 1.0 M solution of potassium tert-butoxide in THF (235 ml) at 0°C was added dropwise a solution of diethylcyanomethylphosphonate (39.9 ml, 0.246 mol) in THF (300 ml). The cooling bath was removed and the reaction mixture was warmed to room temperature followed by cooling to 0° C., while a solution of cyclopentanecarbaldehyde (22.0 g, 0.224 mol) in THF (60 ml) was added dropwise. The bath was removed and the reaction mixture was warmed to ambient temperature and stirred for 64 hours. The mixture was partitioned between diethyl ether and water, the aqueous layer was extracted with three portions of ether and then two portions of ethyl acetate. The combined extracts were washed with brine, then dried over sodium sulfate, filtered and concentrated in vacuo to give a mixture containing 24.4 g of isomeric olefins, which was used without further purification (89%).

¹ H NMR (400 MHz, CDCl ₃ ): δ 6.69 (dd, 1H, trans-olefin), 6.37 (t, 1H, cis-olefin), 5.29 (dd, 1H, trans-olefin) , 5.20 (d, 1H, cis-olefin), 3.07-2.95 (m, 1H, cis-product), 2.64-2.52 (m, 1H, trans-product), 1, 98-1.26 (m, 16H).

Step 2. (3R)- and (3S)-3-cyclopentyl-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl) -1H-pyrazol-1-yl]propanenitrile.

To a solution of 4-(1H-pyrazol-4-yl)-7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidine (15.0 g, 0.0476 mol) in ACN (300 ml) was added 3-cyclopentylacrylonitrile (15 g, 0.12 mol) (as a mixture of cis and trans isomers) followed by DBU (15 ml, 0.10 mol). The resulting mixture was stirred at room temperature overnight. ACN was evaporated. The mixture was diluted with ethyl acetate and the solution was washed with l,0N HCl. The organic layer was again extracted with three portions of ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel chromatography (ethyl acetate in hexanes gradient) to give a viscous clear syrup which was dissolved in ethanol and evaporated several times to remove ethyl acetate to give 19.4 g of the racemic adduct (93%). The enantiomers were separated by preparative HPLC (OD-H, 15% ethanol/hexanes) and used separately in the next step to form their respective final product. The end products (see step 3) derived from each of the separated enantiomers were found to be active JAK inhibitors, however the end product from the second peak on prep HPLC was more active than its enantiomer.

¹ H NMR (300 MHz, CDCl3): δ 8.85 (s, 1H), 8.32 (s, 2H), 7.39 (d, 1H), 6.80 (d, 1H), 5.68 (s, 2H), 4.26 (dt, 1H), 3.54 (t, 2H), 3.14 (dd, 1H), 2.95 (dd, 1H), 2.67-2.50 ( m, 1H), 2.03-1.88 (m, 1H), 1.80-1.15 (m, 7H), 0.92 (t, 2H), -0.06 (s, 9H).

Mass spectrum (ES): 437 (M+1).

Stage 3

To a solution of 3-cyclopentyl-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-b]pyrimidin4-yl)-1H-pyrazol-1-yl]propanenitrile (6 .5 g, 0.015 mol, R or S enantiomer isolated as above) in DCM (40 ml) was added TFA (16 ml) and the mixture was stirred for 6 h. The solvent and TFA were removed in vacuo. The residue was taken up in DCM and concentrated using a rotary evaporator two more times to remove as much TFA as possible. Thereafter, the residue was stirred with ethylenediamine (4 ml, 0.06 mol) in methanol (30 ml) overnight. The solvent was removed in vacuo, water was added and the product was extracted with three portions of ethyl acetate. The combined extracts were washed with brine, dried over sodium sulfate, decanted and concentrated to give the crude product, which was purified by flash column chromatography (eluting with a methanol/DCM gradient). The resulting mixture was further purified by preparative HPLC/mass spectrometry (C18 column, eluting with an ACN/H2O gradient containing 0.15% NH ₄ OH) to give the product (2.68 g, 58%).

1H NMR (400 MHz, d ₆ -DMSO): δ 12.11 (broad s, 1H), 8.80 (s, 1H), 8.67 (s, 1H), 8.37 (s, 1H) , 7.60 (d, 1H), 6.98 (d, 1H), 4.53 (dt, 1H), 3.27 (dd, 1H), 3.19 (dd, 1H), 2.48- 2.36(m, 1H), 1.86-1.76(m, 1H), 1.68-1.13(m, 7H).

- 25 040009

MS (ES): 307 (M+1).

Example A JAK kinase in vitro assay.

These compounds were tested for inhibitory activity against JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry, 1999, 269, 94-104. Human JAK1 (aa 837-1142), JAK2 (aa 828-1132), and JAK3 (aa 781-1124) catalytic domains with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assessed by measuring the degree of phosphorylation of the biotinylated peptide. The phosphorylated peptide was detected using the time resolved homogeneous fluorescence (HTRF) method. Compound IC ₅₀ values for each kinase were measured in reaction mixtures that contained enzyme, ATP, and 500 nM peptide in 50 mM Tris buffer (pH 7.8) with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0. 01%) BSA. The ATP concentration in the reactions was 90 µl for JAK1, 30 µM for JAK2, and 3 µM for JAK3. Reactions were run at room temperature for 1 hour and then stopped using 20 μl of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the europium labeled antibody occurred within 40 min and the HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, MA). Compounds having IC ₅₀ values of 10 μM or less for any of the above JAK targets were considered active.

Example B Cellular assays.

One or more of the disclosed compounds were tested for inhibitory activity against JAK targets in accordance with at least one of the following cell assays.

Cancer cell lines dependent on cytokines and, accordingly, JAK/STAT signal transduction were plated at 6000 cells per well (96-well plate format) in RPMI 1640, 10% FBS in the presence of 1 ng/ml of the appropriate cytokine. Compounds were added to cells in DMSO/medium (final concentration 0.2% DMSO) and incubated for 72 h at 37° C., 5% CO ₂ . The effect of the compound on cell survival was assessed using the CellTiter-Glo fluorescent cell survival assay (Promega) followed by TopCount quantification (Perkin Elmer, Boston, MA). In parallel, the potential ineffective effect of the compounds was measured using a non-JAK controlled cell line with the same assay output principle. Compounds having IC ₅₀ values of 10 μM or less with selectivity for JAK-driven proliferation were considered active. All experiments were carried out in duplicate.

The above cell lines can also be used to investigate the effect of compounds on the phosphorylation of JAK kinases or potential substrates in the forward (5'-3') direction, such as STAT, AKT, Shp2 or ERK proteins. These experiments can be performed after growing the cells on minimal media without cytokines, followed by a brief period of pre-incubation with the compound (2 hours or less) and cytokine stimulation for approximately 1 hour or less. The proteins are then extracted from the cells and analyzed by methods familiar to those skilled in the art, including Western blotting or ELISA using antibodies that distinguish between phosphorylated and total proteins. Conventional or cancer cells can be used in these experiments to test the activity of compounds on tumor cell survival biology or on mediators of inflammatory disease. For example, with regard to the latter, cytokines such as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAK activation leading to phosphorylation of STAT protein(s) and potentially transcriptional profiles (as assessed by ranked series or qPCR) or the production and/or secretion of proteins such as IL-17. The ability of compounds to inhibit these cytokine-mediated actions can be measured using methods common to those skilled in the art.

These compounds can also be tested in cell models designed to evaluate their efficacy and activity against JAK mutants, such as the JAK2V617F mutation found in myeloid proliferative disorders. These experiments often use cytokine-dependent cells of hematological origin (eg, BaF/3) into which wild-type JAK kinases or mutant JAK kinases are ectopically expressed (James, C. et al., Nature, 434:1144-1148; Staerk, J. et al., JBC, 280:41893-41899). The endpoint includes the effects of the compounds on survival, cell proliferation, and phosphorylated JAK, STAT, AKT, or ERK proteins.

For some of the compounds of this invention, their activity in inhibiting T cell proliferation has been or could be evaluated. Such an analysis can be considered as a second cytokine, i.e. JAK-driven proliferation assay and also a simplified immune suppression or immune activation inhibition assay. The following is a brief description of how such experiments can be carried out. Peripheral blood mononuclear cells (PBMCs) are obtained from human whole blood samples using the Ficoll Hypaque separation method, and T cells (fraction 2000) can be obtained from PBMCs by enrichment classification. Freshly isolated human T cells can be maintained in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin) at a density of 2x106 cells/ml at 37°C to

- 26 040009 days. For IL-2 stimulated cell proliferation assay, T cells were initially treated with phytohemagglutinin (PHA) at a final concentration of 10 μg/mL for 72 h. culture medium in the presence of 100 U/ml human IL-2 (ProSpec-Tany TechnoGene; Rehovot, Israel). The plates were incubated at 37° C. for 72 h and the proliferation index was assessed using CellTiter-Glo luminescent reagents according to the manufacturer's protocol (Promega; Madison, WI).

Example C Antitumor efficacy in vivo.

The compounds described herein can be evaluated in immunocompromised human and mouse tumor xenograft models. For example, a tumorigenic variant of the plasmacytoma cell line INA-6 can be used for subcutaneous inoculation of SCID mice (Burger, R. et al., Hematol J., 2:42-53, 2001). Tumor-bearing animals can then be randomly divided into drug treatment and placebo treatment groups, and various doses of the compounds can be administered to them by any number of conventional routes, including oral, intraperitoneal, or continuous infusion using implanted pumps. Tumor growth is monitored using calipers. Next, tumor samples can be collected at any time after the start of treatment for analysis, as described above (example B), to evaluate the effect of compounds on the activity of JAK and signaling pathways in the forward direction. In addition, the selectivity of compounds can be assessed using tumor xenograft models that are driven by other known kinases (eg Bcr-Ab1), such as the K562 tumor model.

Example D Delayed hypersensitivity contact test in mouse skin.

The compounds described herein can also be tested for their efficacy (inhibition of JAK targets) in a T-cell driven mouse model of delayed hypersensitivity. Delayed-type contact hypersensitivity response in mouse skin (DTH) is regarded as a valuable model for clinical contact dermatitis and other T cell-mediated skin immune disorders such as psoriasis (Immunol. Today., 1998 Jan, 19(1):37-44) . Murine DTH shares many characteristics with psoriasis, including an immune infiltrate, a concomitant increase in inflammatory cytokines, and hyperproliferation of keratinocytes. In addition, many classes of agents that are effective in treating psoriasis in the clinic are also effective inhibitors of the DTH response in mice (Agents Actions., 1993 Jan, 38(1-2):116-21).

On days 0 and 1, Balb/c mice were sensitized by topical application of 2,4-dinitro-fluorobenzene (DNFB) antigen to their shaved abdomen. On day 5, the thickness of the ears was measured using an engineering micrometer. The measurements were recorded and used as a baseline. Both ears of the animals were then treated with DNFB by topically applying a total of 20 μl (10 μl to the inner ear and 10 μl to the outer ear) at a concentration of 0.2%. Ears were measured again between 24 and 72 hours after treatment. Treatment with the test compounds was carried out during the sensitization and antibody-producing phases (day 1-7), or before and during the antibody-producing phase (typically after day 4 to 7). Treatment with test compounds (at various concentrations) was either systemic or topically (topical drug application to the ears). The effectiveness of the test compounds was shown by reducing ear swelling compared to the situation in the absence of drug treatment. Compounds that caused a reduction of 20% or more were considered effective. In some experiments, mice were treated with a substance that causes the production of antibodies, but did not sensitize (negative control).

The inhibitory effect (inhibitory activation of JAK-STAT pathways) for test compounds can be confirmed by immunohistochemical assays. Activation of the JAK-STAT pathway(s) results in the formation and movement of functional transcription factors. In addition, immune cell influx and increased keratinocyte proliferation should also provide a unique expression profile in the ear that can be investigated and quantified. Formalin-fixed and paraffin-embedded ear sections (obtained after the antibody-producing phase of the DTH model) were subjected to immunohistochemical analysis using an antibody that specifically interacts with phosphorylated STAT3 (clone 58E12, Cell Signaling Technologies). Comparative mice ears were treated with test compounds, vehicle or dexamethasone (a clinically effective treatment for psoriasis) or left without any treatment in the DTH model. Test compounds and dexamethasone can induce similar transcriptional changes both qualitatively and quantitatively, and both test compounds and dexamethasone can reduce the number of infiltrated cells. Both systemic and local administration of test compounds can provide an inhibitory effect, ie. reduction in the number of infiltrated cells and inhibition of transcriptional changes.

Example E Anti-inflammatory activity in vivo.

The compounds described herein can be or have been evaluated in rodent models and are not

- 27 040009 rodents designed to replicate a single or complex inflammatory response. For example, a rodent model for arthritis can be used to evaluate the therapeutic potential of compounds dosed prophylactically or therapeutically. These models include, but are not limited to, murine or rat collagen-induced arthritis, rat adjuvant-induced arthritis, and collagen antibody-induced arthritis. Autoimmune diseases, including but not limited to multiple sclerosis, type I diabetes mellitus, uveoretinitis, thyroiditis, bulbospinal palsy, immunoglobulin nephropathies, myocarditis, allergic respiratory reactions (asthma), lupus, or colitis, can be used to assess the therapeutic potential of the described connections here. These models have been developed by the research community and are familiar to those skilled in the art (Current Protocols in Immunology, vol. 3., Coligan, J.E. et al., Wiley Press.; Methods in Molecular Biology, vol. 225, Inflammation Protocols., Winyard , P.G.; and Willoughby, D.A., Humana Press, 2003).

Various modifications of the invention in addition to those described here will be apparent to those skilled in the art from the above description. It is intended that such modifications fall within the scope of the appended claims. Each reference cited in this application is incorporated by reference in its entirety.

Claims (6)

CLAIM 1. A drug for the treatment of atopic dermatitis in a patient, said disease being associated with JAK activity, comprising a therapeutically effective amount of 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-b]pyrimidin-4-yl) -1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof. 2. The drug according to claim 1, wherein the compound is (3K)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1-yl] propanenitrile or a pharmaceutically acceptable salt thereof. 3. The drug according to claim 1, wherein the compound is (38)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1-yl] propanenitrile or a pharmaceutically acceptable salt thereof. 4. The use of the compound 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1yl]propanenitrile or a pharmaceutically acceptable salt thereof for the treatment of atopic dermatitis in a patient, wherein said disease is associated with JAK activity. 5. Use according to claim 4, wherein the compound is (3K)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof. 6. Use according to claim 4, wherein the compound is (3S)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof.