Novel inhibitors of proliferation and activation of signal transducer and activator of transcription (stats)

[0001] The present specification discloses pyridine compounds and compositions and their use as medicaments for use in the treatment of diseases such as cancer.

BACKGROUND OF THE INVENTION [0002] It is known that the Jak / STAT signaling pathway is activated in many types of cancer and inflammatory diseases. When activated by phosphorylation of a single tyrosine residue, STAT3 dimerizes, translocates to the nucleus, and serves as a transcription factor that drives upregulation of many proliferative and survival-determining genes including survivin, VEGF, Bcl-2 and Bcl-xL. As is known, these gene products promote the growth and metastasis of more than 20 types of tumor and are also associated with the promotion of several proliferative skin disorders, including psoriasis, T-cell lymphoma and atopic dermatitis.

[0003] Inhibition of STAT3 phosphorylation in tumor-bearing animals leads to the induction of apoptotic cell death, and in preclinical orthotopic models of several diseases, a significant reduction in tumor size. Similarly, in inflammatory skin diseases, inhibition of STAT3 phosphorylation leads to the elimination of deposits, scales and other related lesions.

[0004] Since the development of inhibitors of this pathway is currently desirable, various approaches to inhibiting them (such as small molecules, antisenses, iRNAs) have been tried with unsatisfactory results.

[0005] US 2006/0058297 discloses compounds useful in modulating abnormal cell proliferation. US 2007/0232668 discloses oral bioavailable caffeic acid related anti-cancer drugs, and WO 2005/058829 discloses compounds for the treatment of cell proliferative diseases.

SUMMARY OF THE INVENTION [0006] New pyridine compounds and pharmaceutical compositions that inhibit cancer have been developed, along with synthetic methods and the use of these compounds for use in treating patient diseases and conditions mediated by STAT3, by administering these compounds. These compounds can also be used in the treatment of STAT5 mediated diseases and conditions. These compounds have a structural formula selected from the group consisting of:

<img file="PL2307367T3_D0001.tif" />

EP 2 307 367

<img file="PL2307367T3_D0002.tif" />

[0007] The compounds described herein have useful STAT3 inhibitory activity and can be used in the treatment or prevention of a disease or condition in which STAT3 plays an active role. Thus, in a broad aspect, pharmaceutical compositions are described comprising one or more compounds of the present invention together with a pharmaceutically acceptable carrier, as well as methods of making and using these compounds and compositions.

[0008] In addition, the compounds provided herein can be used in the manufacture of a medicament for the treatment of a disease or condition alleviated by modulation of STAT3 or STAT5 activation selected from the group consisting of cancer, proliferative angiogenic conditions, proliferative skin diseases, central nervous system diseases, inflammatory and autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS [0009] The above summary, as well as the following detailed description of a preferred embodiment of the invention, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the invention is not limited to the particular systems and instruments depicted herein.

[0010] For a more complete understanding of the present invention and its advantages, reference is made to the following descriptions considered in conjunction with the accompanying drawings, in which:

Fig. 1 is data from experiments illustrating the inhibition of STAT3 phosphorylation by the compound of Example 1 in the Colo357FG tumor cell line.

Fig. 2 is a graph of data illustrating the lack of survival of U87 brain tumor cells after administration of the compound of Example 2 and Example 4.

Fig. 3 presents data illustrating the inhibition of the growth of SCOVE3 ovarian cancer cells after administration of the compounds of Example 2 and Example 4.

Fig. 4 shows data for the compound of Example 9 constitutively inhibiting IL-2-induced STAT-3 phosphorylation in D54 brain tumor cell lines.

Figure 5 is a diagram of the signaling pathways that lead to STAT activation and STAT mediated gene expression.

DETAILED DESCRIPTION OF THE INVENTION [0011] The compounds described herein have a structural formula selected from the group consisting of:

<img file="PL2307367T3_D0003.tif" />

EP 2 307 367

<img file="PL2307367T3_D0004.tif" />

<img file="PL2307367T3_D0005.tif" />

[0012] The term "pharmaceutically acceptable salts" as used herein refers to the salts of the compounds of the present invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include salts prepared by reacting the compound of the present invention with an inorganic or organic acid or organic base, depending on the substituents present in the compounds of the invention.

[0013] Examples of inorganic acids that can be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like. Examples of organic acids that can be used to form pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like . Pharmaceutically acceptable salts obtained with inorganic or organic acids include, therefore, hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydroiodide, hydrochloride, acetate, propionate , lactate, p-toluenesulfonate, methanesulfonate, maleate and the like. Other suitable salts are known to those skilled in the art.

[0014] Suitable pharmaceutically acceptable salts can be prepared by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like. Other suitable salts are known to those skilled in the art.

[0015] Pharmaceutically acceptable salts include salts formed between carboxylate or sulfonate groups present in some compounds of the present invention and inorganic cations such as sodium, potassium, ammonium or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.

[0016] It should be noted that a particular anion or cation forming part of any salt of the present invention is not critical as long as the salt as a whole is pharmaceutically acceptable and as long as the anion or cation does not give undesirable characteristics or effects. In addition, additional pharmaceutically acceptable salts are known to those skilled in the art, and can be used within the scope of the invention. Additional examples of pharmaceutically acceptable salts and methods for their preparation and use are provided in the Pharmaceutical Salts: Properties, Selection and Use - A Handbook (2002), which is incorporated herein by reference.

[0017] As used herein, the term "patient" is intended to include living organisms that may have certain conditions as described herein. Examples include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and their transgenic species. In a preferred embodiment, the patient is a primate. In an even more preferred embodiment, the primate is human. Other examples of subjects include experimental animals such as mice, rats, dogs, cats, goats, sheep, pigs, and cows. The experimental animal may be an animal model of the disorder, e.g. it may be a transgenic mouse with Alzheimer's type neuropathology. The patient may be a person suffering from a neurodegenerative disease such as Alzheimer's disease or Parkinson's disease.

[0018] The term "IC50" as used herein refers to an inhibitory dose at which 50% of the maximum response is obtained.

[0019] The term "water-soluble" as used herein means that the compound is dissolved in water at least in the amount of 0.010 mol / liter or is classified as soluble according to literature data. [0020] The term "mainly one enantiomer" as used herein means that the compound contains at least 85% of one enantiomer, or more preferably at least 90% of one enantiomer, and even more preferably at least 95% of one enantiomer, and most preferably at least 99 % one enantiomer. Similarly, the phrase "substantially free of other optical isomers" means that the composition contains at most 5% of another enantiomer or diastereomer, more preferably 2% of another enantiomer or diastereomer, and most preferably 1% of another enantiomer or diastereomer. [0021] As used herein, the singular noun may be one or more. As used in claim (s), when combined with the word "comprising" or "having," the singular noun may mean one or more than one. As used herein, the term "other" or "subsequent" may mean at least a second or more.

[0022] Other abbreviations used herein are as follows: DMSO, dimethyl sulfoxide; iNOS inducible nitric oxide synthase; COX-2, cyclooxygenase-2; NGF, nerve growth factor; IBMX, isobutylmethylxanthine; FBS fetal bovine serum; GPDH, glycerol-3-phosphate dehydrogenase; RXR, retinoid X receptor; TGF-β, transforming growth factor-β; IFN-γ, interferon-γ; LPS, endotoxic bacterial lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-β, interleukin 1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; TCA, trichloroacetic acid; HO-1, inducible heme oxygenase.

[0023] The term "combination therapy" means the administration of two or more therapeutic agents for treating the therapeutic condition or disease described in the present disclosure. Such administration includes the simultaneous administration of these therapeutic agents, in a substantially simultaneous manner, for example in one capsule with a fixed ratio of active ingredients, or in multiple separate capsules for each active ingredient. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner. In any case, the treatment regimen will provide beneficial effects of the drug combination in the treatment of the conditions or disorders described herein.

[0024] The term "therapeutically effective" has the amount of active ingredients used to treat a disease or disorder. This is the amount at which the reduction or elimination of said disease or disorder is achieved.

[0025] The term "therapeutically acceptable" refers to those compounds (or salts, prodrugs, tautomers, zwitterions, etc.) that are suitable for use in contact with tissues

Patients without excessive toxicity, irritation or an allergic reaction are commensurate with a reasonable benefit / risk ratio, and are effective in their intended use.

[0026] As used herein, reference to "treating" a patient includes prophylaxis. The term "patient" means all mammals, including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, pigs and rabbits. Preferably the patient is human.

[0027] Although it is possible for the compounds of the present invention to be administered in the form of a raw chemical, it is also possible to provide them in the form of a pharmaceutical formulation. Thus, the invention provides a pharmaceutical formulation comprising a compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, together with one or more pharmaceutically acceptable carriers and optionally with one or more other therapeutic ingredients. The carrier (s) must be "acceptable", which means that it is compatible with the other ingredients of the formulation and harmless to its recipient. The correct formulation depends on the chosen route of administration. Any methods, carriers and excipients that are known in the art can be used as suitable; e.g., as described in Remington's Pharmaceutical Sciences. The pharmaceutical compositions of the present invention can be prepared in a manner that is known per se, e.g. by conventional methods of mixing, dissolving, granulating, obtaining dragees, levigating, emulsifying, encapsulating, trapping or compressing.

[0028] The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarterial and intramedullary), intraperitoneal, mucosal, transdermal, rectal and local (including dermal, buccal, sublingual and intraocular) , although the most appropriate route of administration may depend, for example, on the patient's condition and disorder. The formulations may conveniently be provided in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the compound of the present invention or a pharmaceutically acceptable salt, ester, prodrug or solvate ("active ingredient") thereof with a carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by bringing the active ingredient uniformly and directly into contact with liquid carriers or particulate solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

[0029] Formulations suitable for oral administration may be provided as separate units, such as capsules, cachets or tablets, each containing a specific amount of the active ingredient; as a powder or granules; in the form of a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient may also be present as a bolus, jam or paste.

[0030] Pharmaceutical preparations that can be used orally include tablets, snap-on capsules made of gelatin, as well as soft, closed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets can be made by compression or molding, optionally with one or more additional ingredients. Compressed tablets can be made by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, inert diluents, or lubricants, surface-active or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the wetted powdered compound

EP 2 307 367 inert liquid diluent. The tablets may optionally be coated or have a dividing line and may be formulated to give slow or controlled release of the active ingredient. All formulations for oral administration should be in dosages suitable for such administration. Snap-on capsule capsules may contain the active ingredients in admixture with a filler such as lactose, binders such as starches and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Dragee cores are supplied in suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes may be added to the tablets or dragee coatings. or pigments to identify or differentiate different combinations of active compound doses.

[0031] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injectable preparations may be provided in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. The formulations can be provided in single-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored as a powder or in lyophilized form, requiring only the addition of a sterile liquid carrier, e.g. physiological saline or sterile, pyrogen-free water immediately before use . Injectable solutions and suspensions prepared immediately before use can be prepared from sterile powders, granules and tablets of the type described above.

[0032] Preparations for parenteral administration include aqueous and non-aqueous (oily) sterile injectable active compound solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes that make the formulation isotonic with the recipient's blood; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, enabling the preparation of highly concentrated solutions.

[0033] Preferred unit formulations are formulations containing an effective dose of the active ingredient as defined below, or an appropriate portion thereof.

[0034] It should be understood that in addition to the ingredients specifically mentioned above, the formulations may further contain other agents conventional in the art taking into account the type of formulation concerned, and for example formulations suitable for oral administration may contain flavoring agents.

[0035] The compounds described herein can be administered by various methods, eg, orally, topically, or by injection. The exact amount of compound administered to the patient will be at the discretion of the physician. The specific dose level for each particular patient will depend on a variety of factors, including the activity of the particular compound being used, age, body weight, overall health, sex, diet,

The time of administration, route of administration, rate of excretion, combination with other drugs, specific disease being treated, and severity of the symptom or condition being treated. Also, the route of administration may vary depending on the condition and severity.

[0036] Briefly, the compounds of the invention may be administered orally, topically, or by injection at a dose of 0.1 to 500 mg / kg per day. Tablets or other delivery forms contained in separate units may conveniently contain an amount of a compound of the invention that is effective at such a dose or as a multiple thereof, for example as units containing 5 mg to 500 mg.

[0037] The amount of active ingredient that can be combined with carrier materials to form a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0038] In some cases, it may be appropriate to administer at least one of the compounds described herein (or a pharmaceutically acceptable salt thereof) in combination with another therapeutic agent. For example, if hypertension is one of the side effects experienced by a patient after receiving one of the compounds described herein, then administration of the antihypertensive agent in combination with the first therapeutic agent may be appropriate. Optionally, by way of example only, the therapeutic efficacy of one of the compounds described herein can be increased by administering an adjuvant (i.e., the adjuvant alone can have only minimal therapeutic effect, but when combined with another therapeutic agent, the overall therapeutic benefit for the patient is increased). Alternatively, by way of example only, the benefit experienced by the patient may be increased by administering one of the compounds described herein together with another therapeutic agent (which also includes a therapy regimen) that also has therapeutic benefit. For example, in the treatment of cancer involving the administration of one of the compounds described herein, an increased therapeutic benefit may also be obtained by administering to the patient another therapeutic agent for the treatment of cancer. In any case, regardless of the disease, disorder or condition being treated, the total benefit experienced by the patient may simply be the sum of the benefits of the two therapeutic agents or the patient may experience a synergistic benefit.

[0039] In any case, many therapeutic agents (of which at least one is a compound described herein) can be administered in any order or even simultaneously. If the administration is simultaneous, many therapeutic agents may be provided in a single, combined form or in multiple separate forms (by way of example only, or as a single tablet or as two separate tablets). One of the therapeutic agents may be administered in multiple doses or both may be administered in multiple doses. When not administered simultaneously, the intervals between multiple doses may be of any length, ranging from a few minutes to four weeks.

[0040] Thus, treatment of STAT3 mediated diseases or conditions (as well as STAT5 mediated disorders or conditions) in a human or animal in need of such treatment includes administering to the subject an compound of the present invention in an amount effective to reduce or prevent said a disorder in an individual, in combination with at least one additional agent for treating said disorder, which is known in the art. In a related aspect, the therapeutic compositions contain at least one of these compounds in combination with one or more additional agents for use in the treatment of STAT3 and STAT5 mediated disorders. [0041] The compounds described herein may be useful in the treatment of various disorders or conditions in which inhibition or modulation of the STAT3 pathway is needed. These compounds reduce and can have therapeutic effects

EP 2 307 367 for disease states associated with proliferation. Uses of these compounds include compounds as agents for: reducing STAT3 activity; STAT3 phosphorylation; and expression of proteins controlled by transcriptional activation of activated STAT3. Accordingly, the compounds may be specifically used as agents for lowering the level of VEGF, MMP9, MMP2, survivin, c-Myc, MMP-1, MEK-5, cFOS, COX-2, Bcl-xl, MMP-10, HSP- 27 and Jmjdla.

[0042] Diseases or conditions that can be prevented or treated using the compounds described herein include the prevention or treatment of cancer such as T-cell skin leukemia, head and neck tumors, pancreatic cancer, bladder cancer, high grade glioma ( high grade gliomas), metastatic brain tumors, melanoma, skin cancer, lung cancer, breast cancer, prostate cancer, colon cancer, leukemia, myelodysplastic syndrome (pre-leukemia), and multiple myeloma.

Generally, metastasis of any cancer can be treated or prevented using the compounds described herein. These compounds can also be used to prevent or treat angiogenic proliferative conditions, including telangiectasia, venous hemangiomas, and hemangioblastomas.

[0043] These compounds can also be used to prevent or treat proliferative skin diseases or disorders, including local dermatitis, psoriasis and rosacea.

[0044] In addition, these compounds can be used to prevent or treat diseases and conditions of the central nervous system ("CNS"), such as CNS inflammation, e.g., multiple sclerosis and progressive multifocal leukoencephalopathy.

[0045] In addition, the compounds described herein can be used to prevent or treat inflammatory diseases and conditions such as osteoarthritis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and autoimmune diseases such as lupus and mixed disease autoimmune.

[0046] Diseases and conditions such as telangiectasia, venous hemangiomas, hemangioblastoma and polycythemia may also be advantageously prevented or treated using the compounds described herein.

[0047] These compounds can affect stem cell survival and differentiation by maintaining 'motherhood' by stem cells, eg, by preventing stem cell differentiation.

[0048] The compounds indicated herein can also be used to increase the immune response, in particular when increasing the immune response leads to the expression of co-stimulatory molecules on peripheral macrophages and tumor infiltrating microglia. These compounds are also useful when the immune response leads to proliferation of effector T cells and / or upregulation of several key intracellular signaling molecules that critically regulate the activation of T lymphocytes and monocytes. These compounds are useful when the immune response leads to upregulation of several key intracellular signaling molecules that critically regulate the activation of T lymphocytes and monocytes, especially the phosphorylation of Syk (Tyr (352)), in monocytes and ZAP-70 (Tyr (319)) in T. cells

[0049] Table 1, immediately below, describes some of the diseases and conditions that can be treated using the compounds described herein:

EP 2 307 367

Table 1

<td colspan="2">Activation of STAT in Human Cancers</td>

<td>Type of cancer</td><td>activated STAT</td>

<td>Blood tumors</td><td></td>

<td>Multiple myeloma</td><td>STAT1, STAT3</td>

<td>leukemias: Dependent on HTLV-1</td><td>STAT3, STAT5</td>

<td>erythroleukemia</td><td>STAT1, STAT5</td>

<td>Acute myeloid leukemia (AML)</td><td>STAT1, STAT3, STAT5</td>

<td>Chronic Myelogenous Leukemia (CML)</td><td>STAT5</td>

<td>Large Granular Lymphocyte Leukemia (LGL)</td><td>STAT3</td>

<td>lymphomas: Dependent on EBV / Burkitt</td><td>STAT3</td>

<td>Mycosis fungoides</td><td>STAT3</td>

<td>Cutaneous T cell lymphoma</td><td>STAT3</td>

<td>Non-Hodgkin's lymphoma (NHL)</td><td>STAT3</td>

<td>Anaplastic large cell lymphoma (ALCL)</td><td>STAT3</td>

<td>Solid tumors</td><td></td>

<td>Breast cancer</td><td>STAT1, STAT3, STAT5</td>

<td>Head and neck cancer</td><td>STAT1, STAT3, STAT5</td>

<td>Melanoma</td><td>STAT3</td>

<td>Ovarian cancer</td><td>STAT3</td>

<td>Lung cancer</td><td>STAT3</td>

<td>Pancreatic cancer</td><td>STAT3</td>

<td>Prostate cancer</td><td>STAT3</td>

<td>glioma</td><td>STAT3, STAT5</td>

<td colspan="2">Based on the footnotes cited in REFERENCES 12, 17, EBV, Epstein-Barr virus; HTLV-1, human virus</td>

<td>T-lymphotropic 1.</td><td></td>

[0050] The STAT protein family ("signal transducer and activator of transcription") includes transcription factors that are specifically activated to regulate gene transcription when cells contact cytokines and growth factors. Mammals have seven known members of the STAT protein family: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105 (2004). These seven proteins have a size in the range of 750 to 850 amino acids. STAT5a and STAT5b proteins, collectively referred to herein as "STAT5", are closely related but encoded by different genes. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97105 (2004).

[0051] STAT proteins act as signal transducers in the cytoplasm and transcriptional activators in the nucleus. Kisseleva T., Bhattacharya S., Braunstein J., Schindler CW, Signaling Through the JAK / STAT Pathway, Recent Advances and Future Challenges, Gene 285: 1-24 (2002). For example, STAT proteins transmit signals from cytokine receptors, growth factor receptors, as well as non-receptor cytoplasmic tyrosine kinases, such as Src and Abl, to the nucleus of the cell in which they bind DNA and regulate gene transcription. Citation as above. As a result, STAT proteins regulate physiological functions such as immune response, inflammation, proliferation, differentiation, survival, metastasis, apoptosis and immune tolerance (e.g. cancer escape from immune surveillance). Xie, T. et al., Stat3 Activation Regulates the Expression of Matrix Metalloproteinase-2 and Tumor Invasion and Metastasis, Oncogene 23: 3550-3560 (2004); Levy, DE, Inghirami, G., STAT3: A Multifaceted Oncogene, PNAS 103: 10151-52 (2006).

[0052] STATs have structurally and functionally conserved domains including: an N-terminal domain that enhances interactions between STAT dimers at adjacent DNA binding sites; domain

EP 2 307 367

Coiled-coil STAT that is involved in protein-protein interactions; DNA binding domain with immunoglobulin-like folding similar to the p53 tumor suppressor protein; EF-hand similar linker domain joining DNA and SH2 binding domains; an SH2 domain that acts as a phosphorylation dependent switch to control receptor recognition and DNA binding; and the C-terminal transactivation domain. Chen X., Vinkemeier U., Zhao Y., Jeruzalmi D., Darnell JE, Kuriyan J., Crystal Structure of a Tyrosine Phosphorylated STAT-1 Dimer Bound to DNA, Cell 93: 827-839 (1998). In order to bind DNA, STAT proteins must dimerize. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105 (2004), Darnell, J. Jr., Validating Stat3 in Cancer Therapy, Nature Medicine, 11: 595-96 (2005). Dimer formation is mediated by the interaction between domain 2 of SRC (SH2) homology of one STAT molecule with the phosphotyrosine residue of the other STAT molecule. [0053] STAT proteins have been shown to be overly active and / or continuously activated in cancer cells, including solid tumors and blood malignancies, proliferative skin diseases, and inflammatory diseases. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105 (2004). For example, STAT signaling is involved in many different cancers. Song, JI and Grandis, JR, STAT Signaling in Head and Neck Cancer, Oncogene, 19: 2489-2495, 2000; Nikitakis, NG et al., Targeting the STAT Pathway in Head and Neck Cancer: Recent Advances and Future Prospects, Current Cancer Drug Targets, 4: 639-651. The increased presence of activated STAT3 and / or STAT5 in the nucleus is associated with deregulation of gene transcription Yu, H., Jove, R., The STATS of Cancer-New Molecular Targets Come of Age, Nature Reviews, 4: 97-105. In addition, the increased presence of STAT3 and / or STAT5 is associated with increased transcription of genes that contribute to cell proliferation, survival, angiogenesis, and tumor-induced immunotolerance. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105. For example, STAT5 is activated in certain leukemias, breast cancer and head and neck cancer. Yu, H., Jove, R., The STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105. Similarly, STAT3 activation is known to reduce growth factor dependence, which contributes to the growth of certain cancerous tumors. Kijima, T., Niwa, H., Steinman, RA, Drenning, SD, Gooding, W. E., Wentzel, AL, Xi, S., and Grandis, JR, STAT3 Activation Abrogates Growth Factor Dependence And Contributes To Head And Neck Squamous Cell Carcinoma Tumor Growth In Vivo, Cell Growth Differ, 13: 355-362, 2002. also that STAT3 activation regulates the survival of human non-small cell carcinoma cells. Song, L., Turkson, J., Karras, JG, Jove, R., and Haura, E. B., Activation Of Stat3 By Receptor Tyrosine Kinases And Cytokines Regulates Survival In Human Non-Small Cell Carcinoma Cells, Oncogene, 22: 4150-4165, 2003. Accordingly, STAT3 and STAT5 are useful targets for treating diseases mediated by STAT3 and STAT5.

[0054] Both STAT3- and STAT5 signaling pathways involve the binding of cytokines or growth factors to cell surface receptors, which leads to the activation of cytoplasmic tyrosine kinases such as the JAK family, which in turn leads to phosphorylation of STAT monomers. Gadina, M., Hilton, D., Johnston, JA, Morinobu, A., Lighvani, A., Zhou, YJ, Visconti, R., O'Shea, JJ Signaling by Type I and Type II Cytokine Receptors: Ten Years After, Curr. Opin. Immunol. 2001, 13: 363. STAT proteins are activated by phosphorylation, which causes them to dimerize and move to the nucleus, where they bind to specific promoter sequences of the target genes. Horvath, CM, The Jak-STAT Pathway Stimulated by Interferon Gamma, Science, STKE, 2004, 260: tr8.

[0055] As shown schematically in Figure 5, growth factor receptors, cytokine receptors and non-receptor tyrosine kinases have signaling pathways that converge to STAT3 and STAT5.

EP 2 307 367

For STAT3, these receptors and kinases include IL-2, IL-6, IL-7, IL-9, IL-10, IL-11, IL-15, IL-21, EGF, OSM, G-CSF, TPO, LIF and GH. For STAT5, these receptors and kinases include IL-2, IL-3, IL-5, IL-7, IL-9, IL-15, G-CSF, GM-CSF, EPO, TPO, GH and PRL.

[0056] More specifically, it is known that STAT3 activation is mediated by EGFR EPO-R, and IL-6R via cSrc or JAK2. See e.g., Lai, SY, Childs, EE, Xi, S., Coppelli, FM, Gooding, WE, Wells, A., Ferris, RL, and Grandis, JR, Erythropoietin-Mediated Activation of JAK-STAT Signaling Contributes to Cellular Invasion in Head and Neck Squamous Cell Carcinoma, Oncogene, 24: 4442-4449, 2005; Siavash, H., Nikitakis, NG, and Sauk, J. J., Abrogation of IL-6-Mediated JAK Signaling by the Cyclopentenone Prostaglandin 15d-PGJ (2) in Oral Squamous Carcinoma Cells, Br J Cancer, 91: 1074-1080, 2004; and Quadros, MR, Peruzzi, F., Kari, C., and Rodeck, U., Complex Regulation of Signal Transducers and Activators of Transcription 3 Activation in Normal and Malignant Keratinocytes, Cancer Res, 64: 3934-3939, 2004. Activation MAPK may lead to a reduction in STAT3 phosphorylation. In solid tumors, PDGFR and c-Met can also activate STAT3 via c-Src. IGFR1 and EGFR can activate STAT3 independently of the JAK. Activation of STAT3 can lead to activation of many underlying target genes, including Bcl-XL, cyclin D1 and VEGF.

[0057] The ligand binding to the cytokine receptor on the cell surface triggers the activation of JAK kinases. As the kinase increases, JAK phosphorylates the tyrosine residue on the receptor and forms sites of interaction with proteins that contain the phosphotyrosine binding SH2 domain. STATs having SH2 domains capable of binding these phosphotyrosine residues are recruited to receptors and are phosphorylated on tyrosine by JAK. Phosphotyrosine then acts as a docking site for other STAT SH2 domains, mediating their dimerization. Different STATs form hetero- as well as homodimers. Activated STAT dimers accumulate in the cell nucleus and activate the transcription of their target genes. Hebenstreit D. et al. (2005) Drug News Perspect. Volume 18 (4), pp. 243-249. STAT can be phosphorylated on tyrosine by other non-receptor tyrosine kinases, such as c-src, as well as receptor tyrosine kinases, such as epidermal growth factor receptor.

[0058] Similarly, binding of the IL-6 family of cytokines (including IL-6, oncostatin M and leukemia inhibitory factor) to the gp130 receptor triggers phosphorylation of STAT3 by JAK2. Boulton, TG, Zhong, Z, Wen, Z, Darnell, Jr, JE, Stahl, N, and Yancopoulos, GD, STAT3 Activation by Cytokines Utilizing gp130 and Related Transducers Involves a Secondary Modification Requiring an H7-Sensitive Kinase Proc Natl Acad Sci USA. 92 (15): 6915-6919. EGF-R and some other receptor tyrosine kinases such as c-MET phosphorylate STAT3 in response to their ligands. Yuan ZL et al. (2004) Mol. Cell Biol. Volume 24 (21), pages 9390-9400. STAT3 is also a target for c-src non-receptor tyrosine kinases. Silva CM (2004) Oncogene Volume 23 (48), pages 8017-8023. In addition to STAT3 activation by IL-6 binding, STAT3 is additionally activated by IL-2, IL-7, IL-9, IL-10, IL-11, IL-15, IL-21, EGF, OSM, G- binding CSF, TPO, LIF or GH to the appropriate receptor. Similarly, STAT5 is activated by binding IL-2, IL-3, IL-5, IL-7, IL-9, IL-15, G-CSF, GM-CSF, EPO, TPO, GH or PRL to the appropriate receptor .

[0059] In fact, Janus kinases ("JAK") play an important role in the initial stages of cytokine receptor signaling. In contrast, the specificity of four members of the JAK family (Jak1, Jak2, Jak3 and Tyk2) for various cytokine receptors is not fully understood, studies show that certain specific cytokine receptors can activate one or more Jak kinases. O'shea, JJ, Pesu, M., Borie, DC, Changelian, P. S., A New Modality for Immunosuppression: Targeting the JAK / STAT Pathway, Nature Rev. Drug Disc. 2004 (3): 555-564. For example, the growth hormone receptor (GHR) may interact

EP 2 307 367 mainly from Jak2, and has been shown to induce phosphorylation of Jak1 and Jak3. Jansson, JO, Carlsson, LM, Carlsson, B., The Growth Hormone Receptor Associates with Jak1, Jak2 and Tyk2 in Human Liver, Growth Horm. IGF Res. 1999 9 (3): 212-8.

[0060] The JAK-STAT pathway can be negatively regulated at many levels. Protein tyrosine phosphatases remove phosphates from cytokine receptors as well as from activated STATs. Hebenstreit D. et al. (2005) Drug News Perspect. Volume 18 (4), pages 243-249. Recently identified Cytokine Signaling Suppressors (SOCS) inhibit STAT phosphorylation by binding and inhibiting JAK kinases or competing with STAT for phosphotyrosine binding sites on cytokine receptors. Krebs, L. et al. (2001) Stem Cells Volume 19, pp. 378-387. STAT are also negatively regulated by STAT Activated Protein Inhibitors (PIAS), which act in the nucleus through several mechanisms. Shuai, K. (2006) volume 16 (2), pages 196-202. For example, PIAS1 and PIAS3 inhibit transcriptional activation by STAT1 and STAT3, by binding and blocking access to recognized DNA sequences, respectively.

[0061] The JAK-STAT signaling pathway is involved in the regulation of cellular responses to cytokines and growth factors. Using Janus kinases (JAK kinases) and Signal Relays and Transcription Activators (STAT), the pathway transmits the signal carried by these extracellular polypeptides to the cell nucleus, where activated STAT proteins modify gene expression. Although STAT was originally discovered to be Janus kinase targets, some stimuli may activate them independent of JAK kinases. DW Leaman, S Pisharody, TW Flickinger, MA Commane, J Schlessinger, IM Kerr, DE Levy, and GR Stark Roles of JAKs in Activation of STATs and Stimulation of c-fos Gene Expression by Epidermal Growth Factor, Mol Cell Biol. 1996 16 (1): 369-375. By regulating cell proliferation, differentiation and apoptosis, this pathway plays a key role in key decisions about the fate of the cell. Phosphorylation of the STAT activator requires JAK phosphorylation to perform phosphorylation. In other words, the JAK must first phosphorylate and bind to STAT. STAT is then able to phosphorylate and dimerize with another STAT.

[0062] The compounds described herein by modulating STAT can control cell growth and survival. Cell activity is regulated by "signals". Signals enter cell walls through receptors. Signals are transmitted by STAT. STATs complete the pathway to DNA. Signals activate DNA transcription (also known as gene activation). Bcl-X1, MCL1, survivin and p53 are involved in cell survival. MYC, Cyclin D1 / D2 and p53 are involved in cell proliferation. Downstream of STAT3 phosphorylation, signaling pathways cause angiogenesis as a result of VEGF, H1F1 and p53 regulation and immune escape regulated by immunosuppressive agents, proinflammatory cytokines and proinflammatory chemokines.

[0063] The compounds and methods described herein can directly or indirectly modulate the activation of STAT3 and / or STAT5. For example, the compounds and methods can inhibit STAT3 activation by: (1) affecting signaling pathways, which leads to STAT3 or STAT5 activation, such that STAT3 or STAT5 activation is prevented or reduced; (2) directly preventing or reducing STAT3 or STAT5 activation, e.g., by preventing STAT3 or STAT5 phosphorylation; and (3) interfering with the complexation of STAT3 or STAT5 with any number of cofactors and / or macromolecules in which these complexes are responsible for regulating STAT3 or STAT5 signaling, and similarly activating STAT5.

[0064] Inhibition of STAT3 and / or STAT5 activation can be accomplished, directly or indirectly, by any strategy currently known in the art targeting STAT. By way of example, STAT3 activity can be reduced by: (1) reducing the availability of STAT3 or STAT5 molecules for recruitment; (2) blocking recruitment of STAT3 or STAT5 to receptors, (3) blocking phosphorylation

EP 2 307 367 STAT3 or STAT5 molecules; (4) interfering with the formation of STAT3 or STAT5 dimers; (5) interfering with translocation of STAT3 or STAT5 dimers to the nucleus; (6) interfering with STAT3 or STAT5 binding to DNA; (7) interfering with the interaction of STAT3 or STAT5 with transcription activators; and / or (8) other methods for preventing STAT-mediated transcriptional activation. See, e.g., NG Nikitakis, et al., Targeting the STAT Pathway in Head and Neck Caner: Recent Advances and Suture Prospects, Current Cancer Drug Targets, 4: 637-651 (2004).

[0065] Direct or indirect modulation of STAT3 or STAT5 activity by the compounds provided herein can prevent, reduce or eliminate STAT3- and / or STAT5-dependent activation of genes responsible for proliferation, survival, angiogenesis, metastasis, immune response and tumor immune escape. As shown schematically in Figures 5 and 6, activation of STAT3 and / or STAT5 affects the transcription of many genes, including, but not limited to, VEGF, MMP9, MMP2, survivin, c-Myc, MMP-1, MEK-5, c-FOS, COX-2, Bcl-xL, MMP-10, HSP27, Jmj for, PGE-2.

[0066] These effects of activated STAT on the transcription of some genes are also associated with physiological effects. For example, as reported by Yu and Jove on pages 99-101, which are incorporated herein by reference, activated STAT promotes cell survival by upregulation of Bcl-xL, MCL1 and survivin, and down regulation of p53. Yu, H., et al., STATS of Cancer - New Molecular Targets Come of Age, Nature Reviews, 4: 97-105 (2004). In addition, activated STAT supports angiogenesis by upregulating MYC and cyclin D1 / D2 and down regulating p53. In addition, activated STATs support angiogenesis by upregulating VEGF and HIF1 and downregulating p53. In addition, activated STAT also help in the tumor's immune escape by upregulating immunosuppressive factors and downregulating pro-inflammatory cytokines and chemokines.

[0067] In addition to being useful in the treatment of humans, the compounds and compositions of the invention are also useful for the veterinary treatment of domestic animals, exotic animals and farm animals, including mammals, rodents, and the like. More preferably animals include horses, dogs and cats.

General synthetic methods for preparing compounds [0068] The following schemes may be used to practice the present invention.

Synthesis and characterization

Synthesis of (E) -3- (6-bromopyridin-2-yl) acrylaldehyde [0069] A solution of 6-bromo-2-carboxypyridine (3 g, 16.1 mmol) was dissolved in dichloromethane (100 mL). (Triphenylphosphoranylidene) acetaldehyde (4.9 g, 16.1 mmol) was added and the resulting mixture was stirred at room temperature for 5 hours. The dichloromethane was partially evaporated (to a volume of about 50 ml) and the reaction mixture was introduced into a chromatography column (silica gel 60). The products were eluted with dichloromethane. The product containing fractions were combined and the solvent was evaporated, yielding 2.3 g of a white powder (67% yield).

[0070] ¹HNMR (CDCl3, δ) ppm: 9.81 (d, 1H, J = 7.6 Hz, CHO), 7.63 (dd, 1H, J = J = 7.7 Hz, H4), 7.51 ( dd,

2H, J = J = 7.7 Hz, H-3.5), 7.45 (d, 1H, J = 15.8 Hz, Ar-CH = CH-CHO), 7.12 (dd, 1H, 1H, J = 15.8 Hz, J = 7.6 Hz, Ar-CH = CH-CHO)

General procedure: Synthesis of (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N-substituted-2,4-dieneamides [0071] A solution of compound (1) was prepared (2.1 g, 9, 9 mmol) and piperidine (0.2 mL) in anhydrous ethanol. Aldehyde (10.6 mmol) was added and the reaction mixture was stirred at room temperature. After 3 hours

The resulting solid was filtered off, washed with ethanol and dried. Compounds were prepared as shown in the examples below.

Example 1 (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N - [(1S) -1-phenylethyl] penta-2,4-dienamide [0072]

<img file="PL2307367T3_D0006.tif" />

[0073] Yield 67% (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N - ((S) -1-phenylethyl) penta-2,4-dieneamide ¹HNMR (CDCl3, δ) ppm: 8.07 (d, 1H, J = 12.1 Hz, H-3), 7.71 (dd, 1H, J = 12.1 Hz, J = 15.0 Hz, H4), 7.59 (dd, 1H, J = J = 7.7 Hz, H-4 '), 7.47 (d, 1H, J = 7.5 Hz, H-3'), 7.41 (d, 1H, J = 7.63 Hz, H-5 '), 7.39 7.29 (m, 5H, H aroma), 7.15 (d, 1H, J = 15.0 Hz, H- 5), 6.45 (d, 1 H, J = 7.5 Hz, NH), 5.23 (dt, 1 H, J = 7.0 Hz, J = 7.5 Hz, H-1 ") , 1.61 (d, 1H, J = 7.0 Hz, Me).

Example 2 (2E, 4E) -5- (6-chloropyridin-2-yl) -2-cyano-N - [(1S) -1-phenylethyl] penta-2,4-dienamide [0074]

<img file="PL2307367T3_D0007.tif" />

[0075] ¹HNMR (CDCl3, δ) ppm: 8.07 (d, 1H, J = 12.1 Hz, H-3), 7.73 (dd, 1H, J = 12.0 Hz, J = 15.0 Hz, H-4), 7.70 (dd, 1H, J = J = 7.81 Hz, H-4 '), 7.43 - 7.29 (m, 7H, H-3', H-5 ', H aroma), 7.18 (d, 1H, J = 15.0 Hz, H-5), 6.45 (d, 1H, J = 7.3 Hz, NH), 5.25 (dt, 1H, J = 7.0 Hz, J = 7.5 Hz, H-1 "), 1.61 (d, 1 H, J = 7.0 Hz, Me).

Example 3 (2E, 4E) -5- (6-chloropyridin-2-yl) -2-cyano-N - [(1R) -1-phenylethyl] penta-2,4-dienamide [0076]

<img file="PL2307367T3_D0008.tif" />

[0077] ¹HNMR (CDCl3, δ) ppm: 8.07 (d, 1H, J = 12.1 Hz, H-3), 7.71 (dd, 1H, J = 12.1 Hz, J = 15.0 Hz, H-4), 7.59 (dd, 1H, J = J = 7.7 Hz, H-4 '), 7.47 (d, 1H, J = 7.5 Hz, H-3'), 7 , 41 (d, 1H, J = 7.63 Hz, H-5 '), 7.39 7.29 (m, 5H, aromatic H), 7.15 (d, 1H, J = 15.0 Hz, H-5), 6.45 (d, 1 H, J = 7.5 Hz, NH), 5.23 (dt, 1 H, J = 7.0 Hz, J = 7.5 Hz, H-1 "), 1.61 (d, 1H, J = 7.0 Hz, Me).

Example 4 (2E, 4E) -N-benzyl-5- (6-bromopyridin-2-yl) -2-cyanopenta-2,4-dienoamide [0078]

<img file="PL2307367T3_D0009.tif" />

[0079] ¹HNMR (CDC13, δ) ppm: 8.10 (d, 1H, J = 12.1 Hz, H-3), 7.70 (dd, 1H, J = 12.1 Hz, J = 15.0 Hz, H-4), 7.57 (d, 1H, J = 7.7 Hz, H-4 '), 7.46 - 7.29 (m, 7H, H-3', H-5 ', aromatic H ), 7.16 (d, 1H, J = 15.0 Hz, H-5), 6.52 (bs, 1H, NH), 4.58 (d, 1H, J = 5.8 Hz, 1 " ).

Example 5 (2E, 4E) -5- (6-chloropyridin-2-yl) -2-cyano-N - [(1R) -1-phenylethyl] penta-2,4-dienamide [0080]

EP 2 307 367

<img file="PL2307367T3_D0010.tif" />

[0081] ¹HNMR (CDCl3, δ) ppm: 8.07 (d, 1H, J = 12.1 Hz, H-3), 7.73 (dd, 1H, J = 12.0 Hz, J = 15.0 Hz, H-4), 7.70 (dd, 1H, J = J = 7.81 Hz, H-4 '), 7.43 - 7.29 (m, 7H, H-3', H-5 ', aromatic H), 7.18 (d, 1H, J = 15.0 Hz, H-5), 6.45 (d, 1H, J = 7.3 Hz, NH), 5.25 (dt, 1H, J = 7.0 Hz, J = 7.5 Hz, H-1 "), 1.61 (d, 1 H, J = 7.0 Hz, Me),

Example 6 (2E, 4E) -N-benzyl-5- (6-chloropyridin-2-yl) -2-cyanopenta-2,4-dienoamide [0082]

<img file="PL2307367T3_D0011.tif" />

[0083] ¹HNMR (CDCl3, δ) ppm: 8.09 (d, 1H, J = 12.1 Hz, H-3), 7.72 (dd, 1H, J = 12.1 Hz, J = 15.0 Hz, H-4), 7.68 (dd, 1H, J = J = 7.8 Hz, H-4 '), 7.40 - 7.28 (m, 7H, H-3', H-5 ', aromatic H), 7.17 (d, 1H, J = 15.0 Hz, H-5), 6.45 (bs, 1H, NH), 4.58 (d, 1H, J = 5.75 Hz, H-1 ").

Example 7 (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N - [(S) -cyclopropyl (phenyl) methyl] penta-2,4-dienoamide [0084]

<img file="PL2307367T3_D0012.tif" />

[0085] ¹HNMR (CDC13, δ) ppm: 8.06 (d, 1H, J = 12.1 Hz, H-3), 7.72 (dd, 1H, J = 12.1 Hz, J = 15.0 Hz, H-4), 7.59 (dd, 1H, J = J = 7.9 Hz, H-4 '), 7.47 - 7.29 (m, 7H, H-3', H-5 ', aromatic H), 7.15 (d, 1H, J = 15.0 Hz, H-5), 6.68 (d, 1H, J = 7.8 Hz, NH), 4.49 (dd, 1H, J = 8.0 Hz, J = 8.8 Hz, H-1 "), 1.33-1.24 (m, 1 H, H-2"), 0.69 - 0.41 (m, 4H , 3 "-CH2, 4" -CH2).

Example 8 (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N - [(1S) -1-phenylpropyl] penta-2,4-dienamide [0086]

<img file="PL2307367T3_D0013.tif" />

[0087] ¹HNMR (CDCl3, δ) ppm: 8.03 (dd, 1H, J = 12.1 Hz, J = 0.7 Hz, H-3 '), 7.69 (dd, 1H, J = 12.1 Hz , J = 15.0 Hz, H-4), 7.57 (dd, 1H, J = 7.5 Hz, J = 7.9 Hz, H-4 '), 7.45 (dd, 1H, J = 7.9 Hz, J = 0.8 Hz, H-3), 7.39 (dd, 1H, J = 7.5 Hz, J = 0.8 Hz, H-5), 7.39 - 7 , 27 (m, 5H, aromatic H), 7.1 (d, 1H, J = 15.0 Hz, H-5), 6.43 (d, 1H, J = 7.8 Hz, NH), 4 , 96 (dd, 1H, J = 15.2 Hz, J = 7.5 Hz, H-1 "), 1.99 - 1.85 (m, 2H, H-2"), 0.93 (t , 3H, J = 7.4 Hz, H-3 ").

Example 9 (2E, 4E) -5- (6-bromopyridin-2-yl) -2-cyano-N - [(1S) -1-phenylbutyl] penta-2,4-dienamide [0088]

<img file="PL2307367T3_D0014.tif" />

EP 2 307 367 [0089] ¹HNMR (CDCl3, δ) ppm: 8.03 (dd, 1H, J = 12.1 Hz, J = 0.7 Hz, H-3), 7.68 (dd, 1H, J = 15.0 Hz, J = 12.1 Hz, H-4), 7.55 (dd, 1H, J = J = 7.7 Hz, H-4 '), 7.45 (dd, 1H, J = 7.9 Hz, J = 0.8 Hz, H-3 '), 7.39 (dd, 1H, J = 7.5 Hz, J = 0.8 Hz, H -5'), 6.66 - 7.28 (m , 6H, H-4, aromatic H, 7.13 (d, 1H, J = 14.9 Hz, H-5), 6.42 (d, 1H, J = 8.0 Hz, NH), 5, 05 (dd, 1H, J = 15.3, J = 7.6 Hz, H-1 "), 1.91 - 1.82 (m, 2H, H-2"), 1.41 - 1.24 (m, 2H, H-3 "), 0.95 (t, 3H, J = 7.3 Hz, H-4").

[0090] The antiproliferative activity of the Compound of Example 1 and Example 7 was demonstrated using the Colo357FG pancreatic cancer cell line as follows:

<td></td><td>Colo357FG</td>

<td>Relationship</td><td>IC50 μΜ</td>

<td>Example 1</td><td> 3,2</td>

<td>Example 7</td><td> 5,4</td>

[0091] The activity of the compounds as STAT inhibitors in Examples 1, 2, 4 and 9 was demonstrated using at least one of the following tests. Related activity data are shown in Figures 1 to 4. It is anticipated that these tests will show the activity of other compounds described above that have not yet been prepared.

Biological Activity Test

MTT Method for Determination of Cell Viability and Drug Dose Response [0092] Various cell types were plated at a density of 50,000 cells / 100 μΐ per well in 96 well plates and grown for 24-48 hours. in DMEM medium until they reach confluence. Confluent, 25 μΐ of fresh media containing WP1220 at various concentrations (from 0 to 100 mM) was added to the wells and incubated for 72 hours at 37 ° C. The complex was removed, 100 μΐ fresh medium was added and the cells were incubated overnight. The medium was removed and fresh medium (100 μΐ) along with (20 μ godzin) MTT solution (5 mg / ml in PBS) was added to each well before incubation of the cells for a further 4 hours at 37 ° C. The reaction product was solubilized in sodium dodecyl sulfate (100 μΐ, 10% w / v) in 0.01 N HCl overnight, and then the product was quantified using a microplate reader at 590 nm and compared with control cells. The experiment was carried out in triplicate (n = 3).

Biological Activity Test

Western Blot Methods for Determining Protein and Phosphoprotein Content [0093] Western blots were carried out by the following methods using anti-STAT3 antibodies with phosphorylated tyrosine (Y705), Jak2 with phosphorylated tyrosine (Y1007 / 1008), total STAT3, total Jak2, Bcl-XL , actin (Cell Signaling Technology, Danvers, MA) and survivin (R&amp;D Systems, Minneapolis, MN) .. Briefly, (1) samples were prepared from cells that were homogenized in a buffer protecting proteins of interest against degradation; (2) The sample was then separated using SDS-PAGE (10-15 μg protein / well) and then transferred to the membrane for detection; and (3) The membrane was incubated with non-specific protein (milk proteins) to bind to all other sites on the membrane. Then the primary antibody was added to a solution that is capable of binding to its specific protein; (4) A secondary antibody-enzyme conjugate that recognizes the primary antibody was then added to find the site where the primary antibody was bound.

EP 2 307 367