RU2772645C2 - Aminothiazole compounds as protein kinase inhibitors - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to a compound of the formula (I) or to its pharmaceutically acceptable salt, where R₁ is C_1-6alkyl; X is NR_a; Y is CR_bR_c or NR_d, in which R_c is H, N(CH₃)₂, R_b, together with R_a, carbon atom related to R_b, and nitrogen atom related to R_a, is piperidinyl, and R_d, together with R_a and nitrogen atoms related to R_d and R_a, is piperazinyl; R₂ is -CH₂CH₂R_e or NR_fR_g, in which R_e is H, halogen or OR_h, and each of R_f and R_g is C_1-6alkyl, R_h is H, and R₃ is pyridyl or pyrimidinil. The invention also relates to a pharmaceutical composition for the treatment of cancer associated with tyrosine kinase, based on the compound of the formula (I).

EFFECT: new compounds and a pharmaceutical composition based on them are obtained that can be used in medicine for the treatment of cancer.

13 cl, 1 tbl, 5 ex

Description

Background of the Invention

Protein kinases are important in cellular signaling pathways that regulate various cellular functions, including differentiation, proliferation, migration, and apoptosis. Dysregulation of protein kinases has been implicated in the development of cancer and several other diseases.

Tyrosine kinases, a subclass of protein kinases, regulate the function of a target protein by transferring phosphate from ATP to the hydroxyl group of tyrosine in the target protein. FMS-like tyrosine kinase 3 (“FLT3”), vascular endothelial growth factor receptor (“VEGFR”), and tyrosine protein kinase Kit (“c-Kit”) are three tyrosine kinases that have been studied as attractive therapeutic targets for cancer treatment. .

Mutations in FLT3, a receptor tyrosine kinase, can lead to the development of cancers such as acute myeloid leukemia. See Pratz et al., Current Drug Targets, 2010, 11(7), 781-9.

When bound to VEGFR and activated via transphosphorylation, vascular endothelial growth factor, a signaling protein, stimulates the growth of new blood vessels. VEGFR has been identified as a major regulator of tumor angiogenesis. See Hicklin et al., J Clin Oncol, 2005, 23, 1011-1027.

c-Kit, also a receptor tyrosine kinase, is involved in intracellular signaling. The mutated form of c-Kit plays a critical role in some types of cancer. Inhibition of c-Kit has been shown to be effective in the treatment of gastrointestinal stromal tumor, acute myeloid leukemia, and melanoma. See Babaei et al., DrugDesDevel Ther., 2016 10, 2443-2459.

Aminothiazole compounds, extensively studied as effective inhibitors of tyrosine kinases, have several problems as drug candidates. They have poor kinase selectivity, often cause animal death in toxicity studies, and generally lack sufficient in vivo activity to exhibit the desired efficacy in preclinical or clinical studies.

There is a need to develop new aminothiazole compounds that specifically inhibit certain tyrosine kinases, exhibit desirable safety profiles, and exhibit sufficient in vivo efficacy in the treatment of their target cancers.

Brief summary of the present invention

The present invention is based on the unexpected discovery that certain aminothiazole compounds effectively inhibit several tyrosine kinases, eg FLT3, VEGFR and c-Kit.

According to one aspect, the present invention relates to aminothiazole compounds of formula (I):

in which R ₁ represents C _1-6 alkyl or C _1-6 thioalkyl; X is O or NR _a , where R _a is H or C _1-6 alkyl; Y is CR _b R _c or NR _d wherein R _b and R _c are each independently H, halogen, C _1-6 alkyl, C _1-6 alkoxy or amino, or R _b together with R _a , the carbon atom associated with R _b and the nitrogen atom associated with R _a is C _3-10 heterocycloalkyl and R _d is H or C _1-6 alkyl, or R _d together with R _a and the nitrogen atoms associated with R _d and R _a , is C _3-10 heterocycloalkyl; R ₂ is -CH ₂ CH ₂ R _e or NR _f R _g , wherein R _e is H, halogen, C _1-6 alkyl, or OR _h and R _f and R _g are each independently C _1-6 alkyl or C _3-8 cycloalkyl, R _h is H or C _1-6 alkyl, or R _h , together with R _d , the oxygen atom associated with R _h and the nitrogen atom associated with R _d , is C _{3 -10} heterocycloalkyl; and R ₃ is heteroaryl.

The term "alkyl" as used herein refers to a saturated, straight or branched hydrocarbon moiety such as -CH ₃ or branched -C ₃ H ₇ . The term "cycloalkyl" refers to a non-aromatic, monocyclic, bicyclic, tricyclic or tetracyclic hydrocarbon moiety such as cyclohexyl, cyclohexen-3-yl or adamantyl. The term "alkoxy" refers to an -O-alkyl radical. Examples of alkoxy include, without limitation, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. The term "thioalkyl" refers to a -S-alkyl radical. Examples of thioalkyl include, without limitation, methylthiol, ethylthiol, and benzylthiol. The term "heterocycloalkyl" refers to a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic moiety containing one or more ring heteroatoms (eg, N, O, or S). Examples of heterocycloalkyl include, without limitation, 4-morpholinyl, 1-piperazinyl, 4-tetrahydropyranyl, and 4-pyranyl. The term "heteroaryl" refers to a moiety containing one or more aromatic rings that contain at least one heteroatom (eg, N, O, or S). Examples of heteroaryl moieties include furyl, furylene, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolinyl, isoquinolyl, and indolyl.

Alkyl, thioalkyl, alkoxyl, cycloalkyl, heterocycloalkyl and heteroaryl mentioned in the present invention include both substituted and unsubstituted fragments, unless otherwise noted. Possible substituents on cycloalkyl, heterocycloalkyl and heteroaryl include C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-20 cycloalkyl, C _3-20 cycloalkenyl, C _1-20 heterocycloalkyl, C _{1- 20} heterocycloalkenyl, C _1-10 alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C _1-10 alkylamino, C _1-20 dialkylamino, arylamino, diarylamino, hydroxyl, halogen, thio, C _1-10 alkylthio, arylthio, C _1-10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy, carboxyl, and carboxylic acid esters. On the other hand, possible substituents on alkyl include all of the above substituents except C _1-10 alkyl, C _2-10 alkenyl, and C _2-10 alkynyl. Cycloalkyl, heterocycloalkyl, aryl and heteroaryl can also be fused to each other.

Aminothiazole compounds described above include the compounds themselves, as well as their salts, prodrugs and solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (eg, amino) on an aminothiazole compound. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, and maleate. Similarly, a salt can also be formed between a cation and a negatively charged group (eg, a carboxylate) on an aminothiazole compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium cation such as tetramethylammonium ion. Aminothiazole compounds also include those salts containing quaternary nitrogen atoms. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives which, upon administration to a subject, are capable of providing active aminothiazole compounds. Solvate refers to a complex formed between an active aminothiazole compound and a pharmaceutically acceptable solvent. Examples of the pharmaceutically acceptable solvent include water, ethanol, isopropanol, ethyl acetate, acetic acid, and ethanolamine.

In accordance with another aspect, the present invention relates to a method for inhibiting tyrosine kinase, for example, FLT3, VEGFR and c-Kit. The method includes contacting the tyrosine kinase with an effective amount of one or more of the aminothiazole compounds described above.

Also within the scope of the present invention is a method of treating tyrosine kinase associated cancer. The method includes administering to a subject in need thereof an effective amount of one or more of the aminothiazole compounds of formula (I) described above.

The cancer-associated tyrosine kinase may be a wild-type tyrosine kinase or a mutant tyrosine kinase. Examples of tyrosine kinase include, without limitation, FLT3, FLT4, VEGFR, platelet growth factor receptor (PDGFR) A, PDGFR B, c-Kit, c-Src (SRC), tyrosine protein kinase Lyn (LYN) A, LYN B, rearranged during transfection, tyrosine kinase (RET), lymphocyte-specific protein tyrosine kinase, Gardner-Rashid strain feline sarcoma virus viral oncogene homologue, discoidin 1 domain receptor, receptor containing kinase insertion domain, B-lymphocyte kinase, tyrosine protein kinase Yes, viral oncogene homologue 1 Abelson murine leukemia virus (ABL1), Tek tyrosine protein kinase, RET V804L, RET Y791F, FLT3 D835Y, PDGFR A V561D, or ABL1 T315I.

In an exemplary method, aminothiazole compounds of formula (I) are used to treat cancer associated with FLT3, VEGFR, or c-Kit.

Examples of cancers include acute myeloid leukemia, chlorleukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, Waldenström's macroglobulinemia, myelodysplastic syndrome, pancreatic cancer, bladder cancer , colorectal cancer, breast cancer, male genital tract cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, uterine cancer, gestational trophoblastic disease, gastric cancer, bile duct cancer, bile duct cancer bladder cancer, small intestine cancer, esophageal cancer, oropharyngeal cancer, hypopharyngeal cancer, eye cancer, nervous tissue cancer, head and neck cancer, melanoma, plasmacytoma, endocrine gland neoplasm, neuroendocrine cancer, brain tumor, bone cancer, and sarcoma (eg, gastrointestinal stromal tumor or GIST).

Additionally, within the scope of the present invention, there is provided a pharmaceutical composition containing one or more of the above-described aminothiazole compounds of formula (I). The pharmaceutical composition may be used to treat cancer.

The present invention also encompasses the use of one or more of the above-described aminothiazole compounds of formula (I) for the manufacture of a medicament for the treatment of cancer.

The term "treatment" or "treatment" refers to the administration of one or more aminothiazole compounds to a subject who has the above-described disease, i.e., cancer, a symptom of such a disease, or a predisposition to such a disease, with the aim of providing a therapeutic effect, for example, for treatment, alleviate, change, defeat, improve the condition or prevent the above-described disease, its symptom or predisposition to it. "Effective amount" refers to the amount of active compound that is necessary to produce a therapeutic effect. Effective doses will vary, as recognized by those skilled in the art, depending on the types of disease being treated, the route of administration, the use of the excipient, and the possibility of co-administration with other therapeutic treatments.

For the practical implementation of the method of the present invention, the composition containing one or more of the above-described aminothiazole compounds can be administered parenterally, orally, nasally, rectally, topically or buccally. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

The sterile injectable composition may be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as the solution used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, fatty oils have traditionally been used as a solvent or suspending medium (eg, synthetic mono- or diglycerides). Fatty acids such as butyric acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil and castor oil, in particular in their polyoxyethylated versions. Such oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersants. Other conventionally used surfactants, such as those that have traditionally been used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be applicable to the composition.

The composition for oral administration may be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets, conventionally used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. For oral administration of aqueous suspensions or emulsions, the active ingredient may be suspended or dissolved in an oil phase combined with emulsifying or suspending agents. Certain sweetening, flavoring or coloring agents may be added as needed.

Nasal aerosol or inhalation composition can be obtained according to methods well known from the field of pharmaceutical composition. For example, such a composition can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance biological activity, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

A composition containing one or more of the aminothiazole compounds described above may also be administered in the form of rectal suppositories.

The carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient of the composition (and preferably capable of stabilizing the active ingredient) and not harmful to the subject being treated. One or more solubilizing agents may be used as pharmaceutical excipients to deliver the active 1,5-diphenylpenta-1,4-dien-3-one compound. Examples of other carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

The details of one or more embodiments of the present invention are set forth in the description below. Other features, objects and advantages of the present invention will be apparent from the description or from the claims.

Detailed description of the present invention

Aminothiazole compounds of formula (I) are disclosed in detail:

in which the variables R ₁ , R ₂ , R ₃ , X and Y are defined in the summary section above.

Typically, a compound of formula (I) contains R ₃ which is a 5- or 6-membered heteroaryl substituted with one or more (CH ₂ ) _n Z moieties independently, wherein n is 0 or 1 and Z is H, halogen, CN, OH, CF ₃ , C _1-6 alkyl or C _1-6 alkoxy; or contains R ₃ which is a 5- or 6-membered heteroaryl fused to a phenyl ring, substituted with one or more substituents independently selected from the group consisting of H, halogen, CN, OH, CF ₃ , C _1-6 alkyl and C _1-6 alkoxy. Exemplary compounds contain R ₃ which is a 6-membered heteroaryl substituted with one or more (CH ₂ ) _n Z moieties independently, wherein n is 0 or 1 and Z is H, halogen, CN, OH, CF ₃ . C _1-6 alkyl or C _1-6 alkoxy. Two examples of R ₃ are pyridyl and pyrimidyl.

The group of novel aminothiazole compounds described above are compounds of formula (II):

in which R ₁ represents C _1-6 alkyl.

In one subgroup, compounds of formula (II) contain X which is O, Y which is NR _d and R ₂ which is -CH ₂ CH ₂ R _e in which R _e is OR _h , R _h , together with R _d , the oxygen atom associated with R _h , and the nitrogen atom associated with R _d , is a C _3-10 heterocycloalkyl. Compounds of this subgroup may contain R ₃ which is a 5- or 6-membered heteroaryl substituted with one or more (CH ₂ ) _n Z moieties independently, in which n is 0 or 1 and Z is H, halogen, CN, OH, CF ₃ , C _1-6 alkyl or C _1-6 alkoxy; or is a 5- or 6-membered heteroaryl fused to a phenyl ring, substituted with one or more substituents independently selected from H, halo, CN, OH, CF ₃ , C _1-6 alkyl, and C _1-6 alkoxy. For example, R ₃ may be pyridyl or pyrimidyl. Exemplary compounds include, without limitation, the following compounds:

In another subgroup, compounds of formula (II) contain X which is NR _a and Y which is CR _b R _c or NR _d in which R _a is together with R _b the nitrogen atom associated with R _a , and the carbon atom associated with R _b is C _3-10 heterocycloalkyl; R _c is H, halogen, C _1-6 alkyl, C _1-6 alkoxy or amino; and R _a , together with R _a and the nitrogen atoms associated with R _a and R _d , is C _3-10 heterocycloalkyl.

It should be noted that such compounds may contain X which is NR _a , Y which is CR _b R _c , and R ₂ which is NR _f R _g in which R _a , together with R _b , is a nitrogen atom , associated with R _a , and a carbon atom associated with R _b , is C _3-10 heterocycloalkyl; R _c is H, halogen, C _1-6 alkyl, C _1-6 alkoxy or amino; and each of R _f and R _g is C _1-6 alkyl. They typically contain R ₃ which is a 5- or 6-membered heteroaryl substituted with one or more (CH ₂ ) _n Z moieties independently, in which n is 0 or 1 and Z is H, halogen, CN, OH, CF ₃ , C _1-6 alkyl or C _1-6 alkoxy; or is a 5- or 6-membered heteroaryl fused to a phenyl ring, substituted with one or more substituents independently selected from H, halo, CN, OH, CF ₃ , C _1-6 alkyl, and C _1-6 alkoxy. R ₃ may be pyridyl or pyrimidyl. Exemplary compounds include, without limitation, the following compounds:

On the other hand, compounds in such a subgroup may contain X which is NR _a , Y which is NR _d and R ₂ which is -CH ₂ CH ₂ R _e in which R _a together with R _d and nitrogen atoms associated with R _a and R _d is C _3-10 heterocycloalkyl; and R _e is H, halogen, or OR _h , R _h is H or C _1-6 alkyl. In general, such compounds contain R ₃ which is a 5- or 6-membered heteroaryl substituted with one or more (CH ₂ ) _n Z moieties independently, in which n is 0 or 1 and Z is H, halogen, CN , OH, CF ₃ , C _1-6 alkyl or C _1-6 alkoxy; or is a 5- or 6-membered heteroaryl fused to a phenyl ring, substituted with one or more substituents independently selected from H, halo, CN, OH, CF ₃ , C _1-6 alkyl, and C _1-6 alkoxy. For example, R ₃ is pyridyl or pyrimidyl. Exemplary compounds include, without limitation, the following compounds:

Another group of new aminothiazole compounds set forth above is the compound of formula (III):

in which R ₁ represents C _1-6 alkyl.

An exemplary compound of formula (III) is

Below are exemplary compounds of the present invention, each labeled with a compound number.

Also within the scope of the present invention is a pharmaceutical composition containing one or more aminothiazole compounds of formula (I) for the treatment of cancer.

Additionally, the present invention discloses a method for treating cancer, the method comprising administering to a subject in need of such treatment an effective amount of a compound of formula (I).

The transformations in synthesis chemistry and the protecting group techniques (introduction and deprotection) used to synthesize compounds of formula (I) are well known in the art. See, for example, R. Larock, Comprehensive Organic Transformations (2nd ^Ed ., VCH Publishers 1999); PGM Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis (4th ^Ed ., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd ^ed ., John Wiley and Sons 2009); and GJ Yu et al., J. Med. Chem. 2008, 51, 6044-6054.

Compounds of formula (I) thus obtained may first be screened using biochemical assays, for example the kinase assays described in Examples 2-4 below, or cellular assays, for example the in vitro anticancer activity assay described in Example 5 below, for their activity of inhibiting tyrosine kinases or inhibiting the growth of cancer cells expressing certain tyrosine kinases. They can then be evaluated using in vivo assays, eg, xenograft animal model assays, for their tumor suppression activity in a mammal. Selected compounds can be further tested to confirm their effectiveness in the treatment of cancer. For example, a compound can be administered to an animal (eg, mouse) having cancer and then its therapeutic effect is evaluated. Based on the results, appropriate dosage ranges and routes of administration can be studied and determined.

Without further specification, it is assumed that a person skilled in the art, based on the above description, can use the present invention to its fullest extent. Thus, the following specific examples should be construed as illustrative only, and not limiting the rest of the present disclosure in any way. All publications cited in this document are incorporated by reference in their entirety.

Example 1 below provides the synthesis and characterization of 13 exemplary compounds of formula (I). The assay data for the compounds thus obtained are also set forth in Example 1, and the procedures for testing these compounds are described in the following Examples 2-5.

All chemicals and solvents were purchased from commercial suppliers and used immediately upon receipt. All reactions were carried out under a dry nitrogen atmosphere. Reactions were monitored by TLC (Thin Layer Chromatography) using silica gel plates on a Merck 60 F254 glass substrate (5 x 10 cm) and zones were identified visually under ultraviolet light (254 nm) or by spraying with phosphomolybdic acid reagent (Aldrich) followed by heating up to 80°С. All flash column chromatography experiments were performed using silica gel ASTM 230-400 mesh Merck Kieselgel 60, #9385, as the stationary phase. Nuclear magnetic resonance spectra for protons ( ¹ H) were measured on a Varian Mercury-300 or Varian Mercury-400 spectrometer. Chemical shifts were recorded in parts per million (ppm) on a delta(δ) scale relative to solvent peak resonance. The following abbreviations have been used to describe the bond: s = singlet; d = doublet; t = triplet; q = quartet; quin = quintet; br = wide; and m = multiplet. LCMS data were measured on Agilent MSD-1100 ESI-MS/MS, Agilent 1200 series LC/MSD VL and Waters Acquity UPLC-ESI-MS/MS systems.

Example 1 Synthesis of Compounds 1-13

Compounds 1-13 were prepared according to the synthetic route shown in Scheme 1 below. Among the reagents listed, TEA is triethylamine, KOAc is potassium acetate, Pd(PPh ₃ ) ₄ is tetrakis(triphenylphosphine)palladium(0), DMAc is N,N-dimethylacetamide, CsF is cesium fluoride, HCl is hydrochloric acid, NaHCO ₃ is sodium bicarbonate, NaH is sodium hydride, NMP is 1-methyl-2-pyrrolidinone, KOH is potassium hydroxide, and DMSO is dimethyl sulfoxide.

Scheme 1. Reagents and conditions: (a) TEA, CH ₂ Cl ₂ , from 0°C to rt; (b) KOAc, Pd(PPh ₃ ) ₄ , DMAc, 150°C (KOAc replaced CsF for Py=pyridin-2-yl); (c) 12 N HCl, H ₂ O, heating, with arr. cold.; (d) NaHCO ₃ , H ₂ O, k.t.; (f) NaH, NMP, 0°C; (f) DMSO, RH, 100° C. or diglyme, KOH, 160° C. for RH=4-(2-hydroxyethyl)-morpholine; and (g) 6N HCl, 0°C.

Stage I. Synthesis of 2,2-dimethyl-N-thiazol-2-ylpropionamide B

Trimethylacetyl chloride (310 mmol) was added to a mixture of 2-aminothiazole A (300 mmol) and triethylamine (330 mmol) in anhydrous CH ₂ Cl ₂ (250 ml) at 0°C, and the mixture was stirred at room temperature under argon for 1 h. The mixture was washed with 6 N HCl (60 ml) and the organic layer was separated, dried over magnesium sulfate (MgSO ₄ ) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20% EtOAc/hexane) to give the title product B as an off-white solid (72%). ¹ H NMR (300 MHz, DMSO-d6): δ 11.75 (s, 1H), 7.46 (d, J=6.0 Hz, 1H), 7.17 (d, J=6.0 Hz, 1.22 (s, 9H); MS ( ES ⁺ ) m/z calculated for C ₈ H ₁₂ N ₂ OS: 184.07, obtained: 185.1 (M+H ⁺ ).

Stage II. Synthesis of compound C (Py = pyridin-3-yl, pyridin-4-yl and pyrimidin-5-yl)

A mixture of 2,2-dimethyl-N-thiazol-2-ylpropionamide B (30 mmol), chloropyridine (30 mmol), potassium acetate (120 mmol) and tetrakis(triphenylphosphine)palladium(0) (1.5 mmol) in N, N-dimethylacetamide (60 ml) was heated at 150° C. under argon for 24 hours. Most of the solvent was removed by distillation (120° C./160 mm Hg) and the residue was washed with water (250 ml). The precipitate was collected by filtration, redissolved in 10% CH ₃ OH/CH ₂ Cl ₂ (200 ml) and filtered through a pad of celite. The filtrate was concentrated under reduced pressure and purified by silica gel chromatography (1% MeOH/CdH ₂ Cl ₂ ) to give the desired product as an off-white solid (40-85%).

Stage II. Synthesis of compound C (Py = pyridin-2-yl)

A mixture of 2,2-dimethyl-N-thiazol-2-ylpropionamide B (10 mmol), chloropyridine (10 mmol), cesium fluoride (20 mmol) and tetrakis(triphenylphosphine)palladium (0) (0.5 mmol) in dimethyl sulfoxide ( 20 ml) was heated at 160° C. under argon for 16 hours. The resulting mixture was partitioned between 0.5 N HCl (150 ml) and CH ₂ Cl ₂ (150 ml). The organic layer was separated, dried over MgSO ₄ , concentrated under reduced pressure and purified by silica gel chromatography (3% acetone/CH ₂ Cl ₂ ) to give the desired product as a pale brown solid (20%)

Only one of the C compounds was chosen to display its NMR spectrum and mass spectrum.

2,2-Dimethyl-N-(5-pyridin-4-ylthiazol-2-yl)-propionamide. ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.28 (bs, 1H), 8.61 (dd, J=4.8, 1.6 Hz, 2H), 7.84 (s, 1H), 7.42 (dd, J=4.8, 1.6 Hz , 2Н), 1.38 (s, 9Н); MS (ES ⁺ ) m/z calcd. for C ₁₃ H ₁₅ N ₃ OS: 261.09; obtained: 262.1 (M+H ⁺ ).

Stage III. Synthesis of compound D

A mixture of compound C (5 mmol) and 12 N HCl (5 ml) in water (5 ml) was heated to reflux for 2 h. Most of the solvent was removed under reduced pressure and the residue was diluted with CH ₃ OH (15 ml). Most of the solvent was removed by distillation and the residue was dried in vacuo to give the hydrochloride compound D as a pale brown solid.

The pH of the above suspension of the above solid in water (30 ml) at room temperature was adjusted to 7 with sodium bicarbonate and the mixture was stirred at 50° C. for 2 hours. The precipitate was collected by filtration and dried in vacuo to give the desired product D as a pale -brown solid (85-90%).

Only one of the compounds D was chosen to display its NMR spectrum and mass spectrum.

5-Pyridine-4-ylthiazol-2-ylamine. ¹ H NMR (300 MHz, DMSO-d6): δ 8.41 (dd, J=4.8.1.5 Hz, 2H), 7.73 (s, 1H), 7.48 (s, 2H), 7.35 (dd, J=4.8, 1.5 Hz, 2H); MS (ES ⁺ ) m/z calcd. for C ₈ H ₇ N ₃ S: 177.04; obtained: 178.1 (M+H ⁺ ).

Stage IV Synthesis of compound E

To a mixture of compound D or commercially available 4-pyridin-3-ylthiazol-2-ylamine (4 mmol) and 4,6-dichloro-2-methylpyrimidine (8 mmol) in 1-methyl-2-pyrrolidinone (20 ml) at 0 °C sodium hydride (60% in oil, 10 mmol) was added and the mixture was stirred at 0°C under argon for 1 h. The reaction was quenched with water (100 ml) at 0°C and the pH was adjusted to 2 with 6N HCl. The pH of the slurry was adjusted to 7 with sodium bicarbonate and the precipitate was collected by filtration, washed with water (50 ml) and dried in vacuo. The residue was purified by silica gel chromatography (20% EtOAc/CH ₂ Cl ₂ then 5% to 10% MeOH/CH ₂ Cl ₂ gradient) to give the desired product E as a brown solid (45-60%).

Only one of the E compounds was selected to display its NMR spectrum and mass spectrum.

(6-Chloro-2-methylpyrimidin-4-yl)-(5-pyridin-4-ylthiazol-2-yl)-amine. ¹ H NMR (300 MHz, DMSO-d6): δ 12.15 (s, 1H), 8.53 (dd, J=4.5, 1.5 Hz, 2H), 8.18 (s, 1H), 7.59 (dd, J=4.5, 1.5 Hz, 2H), 6.90 (s, 1H), 2.59 (s, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₃ H ₁₀ ClN ₅ S: 303.03; obtained: 304.1 (M+H ⁺ ).

Stage V. Synthesis of compounds 1-4 and 7-13

A mixture of compound E (2 mmol) and 1-ethylpiperazine (8 mmol) in dimethyl sulfoxide (2 ml) was heated at 100° C. for 1 h. After cooling to room temperature, the mixture was diluted with water (50 ml). The precipitate was collected by filtration, washed with water (10 ml) and dried in vacuo. The residue was purified by alumina chromatography (0.5% to 1.5% MeOH/CH ₂ Cl ₂ gradient) to give the free base of each of compounds 1-4 and 7-13 as an off-white solid.

The above solid was added to stirred 6 N HCl (10 ml) at 0° C. and the solution was filtered through a 0.45 μm PVDF membrane. Acetone (40 ml) was added dropwise to the stirred filtrate over 1 h and stirred an additional 1 h at 0°C. The precipitate was collected by filtration, washed with acetone (15 ml) and dried in vacuo to give the HCl salt of each of compounds 1-4 and 7-13 as a yellow solid (90-95%).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyridin-4-ylthiazol-2-yl)-amine hydrochloride salt (compound 1). ¹ NMR (400 MHz, DMSO-d6): δ 11.55 (bs, 1H), 8.72 (d, J=5.6Hz, 2H), 8.61 (s, 1H), 8.14 (d, J=5.2 Hz, 2H), 6.27 (s, 1H), 4.35 (d, J=13.2 Hz, 2H), 3.55 (d, J=12.0 Hz, 2H), 3.45 (t, J=13.0 Hz, 2H), 3.13 (t, J=5.8 Hz, 2H), 3.02 (q, J=10.0 Hz, 2H), 2.50 (s, 3H), 1.28 (t, J=6.8 Hz, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₃ N ₇ S: 381.17; obtained: 382.2 (M+H ⁺ ).

{6-[4-(2-Fluoroethyl)-piperazin-1-yl]-2-methylpyrimidin-4-yl}-(5-pyridin-4-ylthiazol-2-yl)-amine hydrochloride salt (compound 2). ¹ H NMR (300 MHz, DMSO-d6): δ11.89 (bs, 1H), 8.73 (d, J=6.3 Hz, 2H), 8.62 (s, 1H), 8.15 (d, J=5.7 Hz, 2H ), 6.26 (s, 1H), 4.95 (d, J=47.4 Hz, 2H), 4.38 (s, 2H, water peak overlap), 3.70-3.35 (m, 6H), 3.18 (bs, 2H), 2.50 ( s, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₂ FN ₇ S: 399.16; obtained: 400.1 (M+H ⁺ ).

2-{4-[2-Methyl-6-(5-pyridin-4-ylthiazol-2-ylamino)-pyrimidin-4-yl]-piperazin-1-yl}-ethanol hydrochloride salt (compound 3). ¹ H NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 8.73 (d, J=7.2 Hz, 2H), 8.63 (s, 1H), 8.15 (d, J=7.2 Hz, 2H) , 6.26 (s, 1H), 4.34 (d, J=12.4 Hz, 2H), 3.82 (t, J=5.2 Hz, 2H), 3.62 (d, J=12.0 Hz, 2H), 3.43 (t, J= 12.4 Hz, 2H), 3.30-3.09 (m, 4H), 2.49 (s, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₃ N ₇ OS: 397.17; obtained: 398.1 (M+H ⁺ ).

[6-(4-Dimethylaminopiperidin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyridin-4-ylthiazol-2-yl)-amine hydrochloride salt (compound 4). ¹ H NMR (300 MHz, DMSO-d6): δ 11.07 (s, 1H), 8.73 (d, J=6.9 Hz, 2H), 8.62 (s, 1H), 8.15 (d, J=6.9 Hz, 2H) , 6.25 (s, 1H), 4.43 (d, J=12.9 Hz, 2H), 3.44 (quin., J=5.2 Hz, 1H), 2.94 (t, J=12.5 Hz, 2H), 2.69 (d, J =4.5 Hz, 6H), 2.49 (s, 3H), 2.15 (d, J=10.5 Hz, 2H), 1.60 (q, J=11.0 Hz, 2H); MS (ES ⁺ ) m/z calcd. for C ₂₀ H ₂₅ N ₇ S: 395.19; obtained: 396.1 (M+H ⁺ ).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyridin-3-ylthiazol-2-yl)-amine hydrochloride salt (compound 7). ¹ H NMR (400 MHz, DMSO-d6): δ 11.23 (bs, 1H), 9.15 (s, 1H), 8.68 (d, J=5.2Hz, 1H), 8.60 (d, J=8.0 Hz, 1H) , 8.19 (s, 1H), 7.93 (t, J=6.2 Hz, 1H), 6.21 (s, 1H), 4.35 (d, J=14.4 Hz, 2H), 3.55 (d, J=11.6 Hz, 2H) , 3.40 (t, J=13.2 Hz, 2H), 3.13 (t, J=5.8 Hz, 2H), 3.01 (q, J=6.9 Hz, 2H), 2.50 (s, 3H), 1.28 (t, J= 6.6 Hz, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₃ N ₇ S: 381.17; obtained: 382.2 (M+H ⁺ ).

{6-[4-(2-Fluoroethyl)-piperazin-1-yl]-2-methylpyrimidin-4-yl}-(5-pyridin-3-ylthiazol-2-yl)-amine hydrochloride salt (compound 8). ¹ H NMR (400 MHz, DMSO-d6): δ 11.80 (bs, 1H), 9.16 (s, 1H), 8.68 (d, J=5.6 Hz, 1H), 8.62 (d, J=8.0 Hz, 1H) , 8.20 (s, 1H), 7.95 (t, J=6.8 Hz, 1H), 6.22 (s, 1H), 4.98 (d, J=46.8 Hz, 2H), 4.34 (bs, 2H), 3.70-3.35 ( m, 6H), 3.16 (bs, 2H), 2.49 (s, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₂ FN ₇ S: 399.16; obtained: 400.1 (M+H ⁺ ).

2-{4-[2-Methyl-6-(5-pyridin-3-ylthiazol-2-ylamino)-pyrimidin-4-yl]-piperazin-1-yl}-ethanol hydrochloride salt (compound 9). ¹ H NMR (400 MHz, DMSO-d6): δ 11.02 (bs, 1H), 9.18 (s, 1H), 8.69 (s, 1H), 8.64 (d, J=7.6 Hz, 1H), 8.22 (s, 1H), 7.96 (d, J=5.2 Hz, 1H), 6.25 (s, 1H), 4.33 (d, J=11.2 Hz, 2H), 3.80 (s, 1H), 3.60 (d, J=11.6 Hz, 2Н), 3.19 (s, 2Н), 3.13 (s, 2Н), 2.48 (s, 3H); MS (ES ⁺ ) rn/z calc. for C ₁₉ H ₂₃ N ₇ OS: 397.17; obtained: 398.1 (M+H ⁺ ).

[6-(4-Dimethylaminopiperidin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyridin-3-ylthiazol-2-yl)-amine hydrochloride salt (compound 10). ¹ H NMR (400 MHz, DMSO-d6): δ 11.27 (bs, 1H), 9.18 (s, 1H), 8.69 (d, J=5.2 Hz, 1H), 8.64 (d, J=8.0 Hz, 1H) , 8.23 (s, 1H), 7.97 (t, J=6.8 Hz, 1H), 6.36 (bs, 1H), 4.42 (d, J=8.8 Hz, 2H), 3.43 (bs, 1H), 2.99 (t, J=12.4 Hz, 2H), 2.68 (s, 3H), 2.67 (s, 3H), 2.55 (s, 3H), 2.17 (d, J=10.8 Hz, 2H), 1.64 (q, J=9.2 Hz, 2H); MS (ES ⁺ ) m/z calcd. for C ₂₀ H ₂₅ N ₇ S: 395.19; obtained: 396.2 (M+H ⁺ ).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyridin-2-ylthiazol-2-yl)-amine hydrochloride salt (compound 11). ¹ H NMR (300 MHz, DMSO-d6): δ 11.34 (s, 1H), 8.54 (d, J=4.8 Hz, 1H), 8.32 (s, 1H), 8.01-7.96 (m, 2H), 7.40- 7.34 (m, 1H), 6.31 (s, 1H), 4.37 (d, J=13.2 Hz, 2H), 3.62-3.38 (t, 4H), 3.20-2.90 (t, 4H), 2.47 (s, 3H) , 1.26 (t, J=7.4 Hz, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₃ N ₇ S: 381.17; obtained: 382.2 (M+H ⁺ ).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(5-pyrimidin-5-ylthiazol-2-yl)-amine hydrochloride salt (compound 12). ¹ H NMR (400 MHz, DMSO-d6): δ 11.35 (bs, 1H), 9.20-9.03 (m, 3H), 8.09 (s, 1H), 6.28 (s, 1H), 4.40 (s, 2H), 3.56 (d, J=12.4 Hz, 2H), 3.44 (d, J=7.4 Hz, 2H), 3.13 (bs, 2H), 3.02 (d, J=8.0 Hz, 2H), 1.28 (bs, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₈ H ₂₂ N ₈ S: 382.17; obtained: 383.3 (M+H ⁺ ).

[6-(4-Ethylpiperazin-1-yl)-2-methylpyrimidin-4-yl]-(4-pyridin-3-ylthiazol-2-yl)-amine hydrochloride salt (compound 13). ¹ H NMR (400 MHz, DMSO-d6): δ 11.80 (bs, 1H), 11.54 (bs, 1H), 9.28 (s, 1H), 8.94 (d, J=8.4 Hz, 1H), 8.84 (d, J=5.2 Hz, 1H), 8.15-8.07 (t, 2H), 6.31 (bs, 2H), 4.35 (d, J=14.0 Hz, 2H), 3.55 (d, J=12.0 Hz, 2H), 3.45 ( t, J=13.0 Hz, 2H), 3.15-3.07 (m, 2H), 3.00 (q, J=10.0 Hz, 2H), 2.49 (s, 3H), 1.27 (t, J=7.4 Hz, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₃ N ₇ S: 381.17; obtained: 382.1 (M+H ⁺ ).

Stage V. Synthesis of compounds 5 and 6

Potassium hydroxide (10 mmol) was added to a mixture of compound E (1 mmol) and 4-(2-hydroxyethyl)-morpholine (4 mmol) in diglyme (1 ml) at 100°C, and the mixture was stirred at 160°C under argon in within 10 min. The reaction was quenched with water (20 ml) at 0° C. and the pH was adjusted to 2 with 6 N HCl. The slurry was adjusted to pH 7 with sodium bicarbonate and the precipitate was collected by filtration, washed with water (10 ml) and dried in vacuo. The residue was purified by alumina chromatography (0.5% to 1.5% MeOH/CH ₂ Cl ₂ gradient) to give the free base of compound 6 as an off-white solid.

To a suspension of the above solid in MeOH (10 ml) at 0° C. was added 6 N HCl (1 ml) with stirring. Most of the solvent was removed under reduced pressure and the residue was treated with EtOH (10 ml). The precipitate was collected by filtration, washed with acetone (10 ml) and dried in vacuo to give the HCl salt of each of compounds 5-6 as a yellow solid (45-50%).

[2-Methyl-6-(2-morpholin-4-yl-ethoxy)-pyrimidin-4-yl]-(5-pyridin-4-ylthiazol-2-yl)-amine hydrochloride salt (compound 5). ¹ H NMR (400 MHz, DMSO-d6): δ 11.61 (s, 1H), 8.74 (d, J=5.2 Hz, 2H), 8.64 (s, 1H), 8.17 (d, J=5.2 Hz, 2H) , 6.37 (s, 1H), 4.72 (s, 2H), 4.00-3.80 (m, 4H), 3.64-3.42 (m, 4H), 3.16 (bs, 2H), 2.59 (s, 3H); MS (ES ⁺ ) rn/z calc. for C ₁₉ H ₂₂ N ₆ O ₂ S: 398.15; obtained: 399.2 (M+H ⁺ ).

[2-Methyl-6-(2-morpholin-4-yl-ethoxy)-pyrimidin-4-yl]-(5-pyridin-3-ylthiazol-2-yl)-amine hydrochloride salt (compound 6). ¹ H NMR (400 MHz, DMSO-d6): δ 11.51 (bs, 1H), 9.20 (s, 1H), 8.71 (d, J=5.6 Hz, 1H), 8.66 (d, J=8.4 Hz, 1H) , 8.24 (s, 1H), 7.98 (dd, J=8.0, 5.6 Hz, 1H), 6.35 (s, 1H), 4.72 (t, J=4.8 Hz, 2H), 3.96 (d, J=10.8 Hz, 2Н), 3.85 (t, J=12.0 Hz, 2Н), 3.55 (bs, 2Н), 3.47 (d, J=12.4 Hz, 2Н), 3.19 (bs, 2Н), 2.59 (s, 3H); MS (ES ⁺ ) m/z calcd. for C ₁₉ H ₂₂ N ₆ O ₂ S: 398.15; obtained: 399.1 (M+H ⁺ ).

Example 2 Inhibition of FLT3 Activity

To study certain compounds obtained in accordance with example 1, in relation to the inhibition of FLT3 activity, the following study was carried out.

GST-FLT3-KD^wt, containing the FLT3 kinase catalytic domain (residues Y567-S993), was expressed in Sf9 insect cells transfected with a baculovirus containing the plasmid pBac-PAK8-GST-FLT3-KD. Kinase-Glo FLT3 kinase assay^wt was performed in 96-well plates at 30°C for 4 hours to test the compound in a final volume of 50 μl, including the following components: 75 ng of GST-FLT3-KD proteins^wt, 25 mM HEPES, pH 7.4, 4 mM MnCl₂, 10 mM MgCl₂, 2 mM DTT, 0.02% Triton X-100, 0.1 mg/ml bovine serum albumin, 25 μM Her2 peptide substrate, 0.5 mM Na₃VO_four and 1 μM ATP. After incubation, 50 μl of Kinase-Glo Plus Reagent (Promega, Madison, WI, USA) was added to each well and the mixture was incubated at 25° C. for 20 minutes. A 70 μl aliquot of each reaction mixture was transferred to a black microtiter plate and luminescence was measured on a Wallac Vector 1420 multi-label counter (PerkinElmer, Shelton, CT, USA).

Several compounds were investigated. Surprisingly, compounds 1-11 showed IC ₅₀ values (the inhibitor concentration at which the response is halved) below 100 nM.

Example 3 Inhibition of VEGFR2 activity

To study certain compounds obtained in accordance with example 1, in relation to the inhibition of VEGFR2 activity, the following study was carried out. It should be noted that VEGFR2 is one of the three major VEGFR subtypes.

Recombinant GST-VEGFR2 (residues V789-V1356) containing the kinase domain was expressed in Sf9 insect cells. The kinase assay was performed in 96-well test compound plates in a final volume of 50 μl of the reaction mixture at 30°C for 120 minutes with the following components: 25 Mm HEPES, pH 7.4, 10 Mm MgCl ₂ , 4 mM MnCl ₂ , 0 .5 mM Na ₃ VO ₄ , 2 mM DTT, 0.02% Triton X100, 0.01% BSA, 1 μM ATP, 2 μM polyGlu4:Tyr peptide, 50~100 ng recombinant VEGFR2. After incubation, 50 μl of Kinase-Glo Plus Reagent (Promega, Madison, WI, USA) was added to each well and the mixture was incubated at 25° C. for 20 minutes. A 70 μl aliquot of each reaction mixture was transferred to a black microtiter plate and luminescence was measured on a Wallac Vector 1420 multi-label counter (PerkinElmer, Shelton, CT, USA).

Several compounds were tested in the VEGFR2 assay. Compounds 1, 9 and 10 unexpectedly showed IC ₅₀ values below 30 nM.

Example 4 Inhibition of c-Kit activity

To study certain compounds obtained in accordance with example 1, in relation to the inhibition of c-Kit activity, the following study was carried out.

N-terminally His-tagged recombinant human c-KIT proteins (residues T544-V976) expressed in expression systems based on baculovirus-transfected Sf9 insect cells were purified for c-Kit ADP Kinase-Glo analysis. The c-Kit-ADP Kinase-Glo assay was performed in 96-well plates at 30° C. for 150 minutes using a final volume of 10 μl containing 40 mM Tris, pH 7.4, 20 mM MgCl ₂ , 2 mM MnCl ₂ , 2 mM DTT, 0.01% BSA, 20 μM ATP, 20 μM poly(Glu, Tyr) 4:1 peptide, 0.1 mM Na ₃ VO ₄ , 250 ng c-Kit recombinant proteins and test compound as indicated concentration. Reactions were stopped by adding 5 µl of ADP-Glo™ reagent (Promega, Madison, Wisconsin, USA) at 25° C. with a 40 minute incubation followed by the addition of 10 µl of kinase detection reagent for an additional 30 minutes. Finally, a 30 μl aliquot of each reaction mixture was transferred to a black microtiter plate and luminescence was measured on a Wallac Vector 1420 multi-label counter (Perkin-Elmer, Shelton, CT, USA).

Several compounds were investigated. Surprisingly, compounds 1-7 and 11-12 showed IC ₅₀ values below 100 nM.

Example 5 In Vitro Anticancer Activity

To evaluate the in vitro anticancer activity of certain compounds prepared according to Example 1, the following study was performed using cell lines and MTS cell viability assays (MTS is 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

The leukemic cell lines MOLM-13, MV4:11 and Kasumin-1 were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). GIST-T1 gastrointestinal stromal tumor cell line was purchased from Cosmo Bio Co., LTD (Tokyo, Japan). All leukemic cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin and 10 g/ml streptomycin at 37° C. and 5% CO ₂ . The GIST-T1 cell line was cultured in DMEM supplemented with 10% FBS, 0.01% non-essential amino acids, 10 U/ml penicillin and 10 g/ml streptomycin.

All of the cells GIST882, GIST48 and GIST430 were cultured in incubators maintained at 37°C and 5% CO ₂ . GIST882 was cultured in RPMI-1640 supplemented with 20% fetal bovine serum (FBS). GIST48 was cultured with F10 supplemented with 20% FBS, 0.5% Mito serum enhancer (BD Bioscience, 355006) and 1% bovine pituitary extract (BD Bioscience 354123). GIST430 was cultured in IMDM supplemented with 20% FBS. GIST882, GIST430 and GIST48 cells were provided by Dr. Jonathan A. Fletcher (Harvard Medical School, USA).

MTS analyzes of MOLM-13, MV4:11 and Kasumin-1

Cells were seeded in 96-well culture plates at a density of 1×10 ⁴ cells/100 μl/well in triplicate and treated for 72 hours with the indicated concentration of test compound in the range of 1 nM to 10 μM. Colorimetric CellTiter 96® Aqueous One Solution Cell Proliferation assay (MTS assay; Promega, Madison, WI, USA) was used to determine cytotoxicity. Absorbance at 492 nm was measured using a microplate photometer (Victor2; Perkin-Elmer, Waltham, MA, USA). IC ₅₀ values were determined by MTS analysis when cells were treated with test compound for 72 hours and calculated using GraphPad Prism 6. Each experiment was performed in triplicate.

MTS analysis of GIST-T1

GIST-T1 cells were seeded in 96-well culture plates at a density of 8,000 cells/100 μl/well in triplicate and treated for 72 hours with the indicated concentration of test compound ranging from 1 nM to 10 μM. Colorimetric CellTiter 96® Aqueous One Solution Cell Proliferation assay (MTS assay; Promega, Madison, WI, USA) was used to determine cytotoxicity. Absorbance at 492 nm was measured using a microplate photometer (Victor2; Perkin-Elmer, Waltham, MA, USA). IC ₅₀ values were determined by MTS analysis when cells were treated with test compound for 72 hours and calculated using GraphPad Prism 6. Each experiment was performed in triplicate.

MTS analyzes GIST882, GIST48 and GIST430

GIST cells (4×10 ⁴ ) were treated with different doses of compounds. Treated GIST882 cells were incubated for 144 hours, and GIST48 and GIST430 cells were incubated for 120 hours at 37°C in 5% CO ₂ . Cell proliferation was determined by incubating the cells with methylene blue (Clontech, CA, USA) for 1 hour. The absorbance was measured at a wavelength of 450 nm using a SpectraMax M5 microplate reader (Molecular Devices, USA).

The GI50 values (concentration for 50% of the maximum inhibition of cell proliferation) for certain compounds of formula (I) are presented in the table below:

Other embodiments

All features disclosed in this application may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

From the above description, a person skilled in the art can easily ascertain the essential features of the present invention, and without deviating from its spirit and scope, can make various changes and modifications to the present invention to adapt it to various applications and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims (25)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

where R ₁ is C _1-6 alkyl; X is NR _a ; Y is CR _b R _c or NR _d , wherein R _c is H, N(CH ₃ ) ₂ , R _b together with R _a , the carbon atom associated with R _b , and the nitrogen atom associated with R _a , is piperidinyl and R _d together with R _a and the nitrogen atoms associated with R _d and R _a is piperazinyl; R ₂ is -CH ₂ CH ₂ R _e or NR _f R _g , wherein R _e is H, halogen or OR _h and R _f and R _g are each C _1-6 alkyl, R _h is H ; and R ₃ is pyridyl or pyrimidinyl. 2. The compound or salt according to claim 1, where the compound is a compound of formula (II):

3. The compound or salt according to claim 2, where X is NR _a , Y is CR _b R _c and R ₂ is NR _f R _g , where R _a together with R _b , the nitrogen atom associated with R _a , and the carbon atom attached to R _b is piperidinyl. 4. The compound or salt according to claim 2, where X is NR _a , Y is NR _d and R ₂ is -CH ₂ CH ₂ R _e , where R _a together with R _d and the nitrogen atoms associated with R _a and R _d is piperazinyl; and R _e is H, halo, or OR _h , R _h is H. 5. The compound or salt according to claim 3, where the compound is

6. The compound or salt according to claim 4, where the compound is one of the following compounds:

7. The compound or salt according to claim 1, where the compound is one of the following compounds:

8. The compound or salt according to claim 1, where the compound is a compound of formula (III):

9. Pharmaceutical composition for the treatment of tyrosine kinase-associated cancer, containing a pharmaceutically acceptable carrier and an effective amount of a compound or salt according to claim 1. 10. A method of inhibiting a tyrosine kinase, the method comprising contacting the tyrosine kinase with an effective amount of a compound or salt of claim 1, and the tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3), vascular endothelial growth factor receptor (VEGFR), or tyrosine protein kinase Kit (with -KIT). 11. A method of treating a tyrosine kinase associated cancer, the method comprising administering to a subject in need of such treatment an effective amount of a compound or salt of claim 1, and the tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3), vascular endothelial growth factor receptor (VEGFR), or tyrosine protein kinase Kit (c-KIT). 12. The method of claim 11 wherein the cancer is leukemia or sarcoma. 13. The pharmaceutical composition of claim 12 wherein the tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3), vascular endothelial growth factor receptor (VEGFR), or tyrosine protein kinase Kit (c-KIT).