SOLID PHASE SYNTHESES OF AMINO PYRIMIDINE COMPOUNDS

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. provisional application serial number 60/912,853, filed April 19, 2007, which is incorporated herein by reference in its entirety.

Technical Field

[0002] The invention relates to protein kinase inhibitors, and methods of making such compounds.

Background Art

[0003] The protein kinases represent a large family of proteins, which play a central role in the regulation of a wide variety of cellular processes and maintaining control over cellular function. Kinase activity is usually regulated by additional phosphorylation events, cellular localization, inhibitory or activating protein partners, protein degradation or gene transcription. Deregulation of protein kinase activity through mutation to constitutively active alleles, loss of negative regulators, and chromosomal rearrangements that lead to the formation of oncogenic fusion proteins are associated with disorders ranging from leukemias to diabetes.

[0004] The clinical success of imatinib, an inhibitor of Bcr-Abl for the treatment of chronic myelogenous leukemia (CML) has provided the paradigm for cancer therapy via kinase inhibition. Imatinib is a member of the ATP-competitive 2-phenylamino pyrimidine (PAP) class of kinase inhibitor and also inhibits the platelet-derived growth factor receptor (PDGFR) kinase, Abl-related kinase (ARG) and the stem cell factor receptor tyrosine kinase (c-kit). (Druker et al., Nature Med. 1996, 2, 561; Carroll et al., Blood 1997, 90, 4947; Beran et al., Clin. Cancer. Res. 1998, 4, 1661; Gambacorti-Passerini et al., Blood Cells MoI. Dis. 1997, 23, 380; Deininger et al., Blood 1997, 90, 3691; Dan et al., Cell Death Differ. 1998, 5, 710; and Le Coutre et al., J. Natl. Cancer Inst. 1999, 91, 163). The "multi-targeted" nature of imatinib has resulted in additional clinical applications for the drug such as the treatment of gastro-intestinal stromal tumors (GIST) where c-kit appears to be the target and hypereosinophilic syndrome (HES) where an aberrant fusion protein involving PDGFRβ is the target. (Griffin et al., Proc. Natl. Acad. Sci. USA 2003, 100, 7830).

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Disclosure of the Invention

[0005] The invention provides methods for preparing amino pyrimidine compounds, such as imatinib and analogs thereof.

[0006] In one aspect, the present invention provides a method for preparing a compound of Formula (1) on a solid support:

wherein R ¹ is H or Ci_6 alkyl;

R ² , R ³ , R ⁴ and R ⁵ are independently H, an optionally halogenated C _1-6 alkyl or C _1-6 alkoxy, NR ⁶ R ⁷ , or (CR ₂ ) _n R ⁸ ;

R and R ⁷ are independently H, an optionally halogenated Ci_6 alkyl, C ₂ -6 alkenyl or C ₂ -6 alkynyl; Ci_ ₆ alkanol, Ci_ ₆ alkoxy or (CR ₂ ) _q -R ⁸ ; or R ⁶ and R ⁷ together with N in NR ⁶ R ⁷ may form an optionally substituted ring;

R ⁸ is an optionally substituted NR >6 ⁰ rR _> 7', C ₃ - ₇ cycloalkyl, 5-7 membered aryl, heterocyclic or heteroaryl; each R in (CR ₂ ) is H or Ci_ ₆ alkyl; n and q are independently 0-4; comprising a) contacting a resin-bound compound of Formula (2)

wherein S is a solid support; L is a linker; and

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X is a leaving group; with a heterocyclic amine of Formula (3) in the presence of a palladium catalyst

to form a support-bound compound of Formula (4)

[0007] In one embodiment, the methods of the invention further comprise b) removing said compound of Formula (1) on the solid support. In some examples, the resin-bound amine of Formula (2) and the heterocyclic amine of Formula (3) are contacted in the presence of a phosphine ligand and a base; and in other examples, in the presence of phenol. Examples of bases which may be used in the methods of the invention include but are not limited to tert- butoxide or phenoxides , such as sodium t-butoxide or sodium phenoxide. Examples of phenols which may be used in the methods of the invention include but are not limited to 2,6-di-tert- butyl-4-methylphenol. The reaction may proceed in the presence of a solvent at a temperature ranging from room temperature to 120 ⁰ C. In some examples, the solvent is dioxane.

[0008] In the above Formula (2), X may be chloro, bromide, iodide or triflate. In some examples, X is bromide.

[0009] In the above Formula (2) and (4), L may be a (CR ₂ ) _1-2 linker.

[0010] In the methods of the invention, the palladium catalyst may be tris(dibenzylideneacetone)-dipalladium(0) (Pd ₂ dba ₃ ). In some examples, the solid support is an aldehyde resin. For example, the aldehyde resin may be (4-formyl-3,5- dimethoxyphenoxymethyl) functionalized polystyrene resin.

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[0011] In the methods of the invention, the resin bound amine may be prepared by 1

contacting a support-bound aldehyde with to form a support-bound amine, and

contacting the support-bound amine with a compound of formula R ⁴ wherein Y is chloro (acyl chloride) or hydroxyl (carboxylic acid) in the presence of appropriate coupling or activating reagents.

[0012] In some embodiments, the methods of the invention are used to prepare the compounds below

[0013] Compounds described herein may modulate the activity of kinases and, as such, may be useful for treating diseases or disorders in which kinases contribute to the pathology and/or symptomology of the disease. Examples of kinases that may be inhibited by the compounds and compositions described herein and against which the methods described herein may be useful include, but are not limited to, c-kit, AbI, Lyn, MAPK14 (p38α), PDGFRα, PDGFRβ, ARG, BCR-AbI, BRK, EphB, Fms, Fyn, KDR, LCK, B-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinase.

Definitions

[0014] "Alkyl" refers to a moiety and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, and may be straight-chained or branched. An optionally substituted alkyl, alkenyl or alkynyl as used herein may be optionally halogenated (e.g., CF ₃ ), or

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may have one or more carbons that is substituted or replaced with a heteroatom, such as NR, O or S (e.g., -OCH ₂ CH ₂ O-, alkylthiols, thioalkoxy, alkylamines, etc).

[0015] "Aryl" refers to a monocyclic or fused bicyclic aromatic ring containing carbon atoms. For example, aryl may be phenyl or naphthyl. "Arylene" means a divalent radical derived from an aryl group.

[0016] "Heteroaryl" as used herein is as defined for aryl above, where one or more of the ring members is a heteroatom. Examples of heteroaryls include but are not limited to pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[l,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

[0017] A "carbocyclic ring" as used herein refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring containing carbon atoms, which may optionally be substituted, for example, with =0. Examples of carbocyclic rings include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclohexanone, etc.

[0018] A "heterocyclic ring" as used herein is as defined for a carbocyclic ring above, wherein one or more ring carbons is a heteroatom. For example, a heterocyclic ring may contain N, O, S, -N=, -S-, -S(O), -S(O) ₂ -, or -NR- wherein R may be hydrogen, Ci^alkyl or a protecting group. Examples of heterocyclic rings include but are not limited to morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.

Modes of Carrying Out the Invention

[0019] The invention provides methods for preparing amino pyrimidine compounds, such as imatinib and analogs thereof.

[0020] The published synthetic route for imatinib relies on pyrimidine formation by cyclization of an enaminone with an aryl-guanidine. The enaminone is prepared by the reaction of l-pyridin-3-yl-ethanone with neat N, N-dimethylformamide dimethyl acetal. Imatinib is obtained following hydrogenation and acylation of the nitro group with 4-methylpiperizin-l- ylmethylbenzoyl chloride. (Zimmermann, et al., Bioorg. Med. Chem. Lett. 1997, 7, 187). Although this route is well suited for gram-scale synthesis, it requires isolation and purification of intermediates and does not utilize "building blocks" for which there is commercial access to a

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diverse array of functionality (e.g., aryl guanidines, acetyl-heterocycles). Thus, new synthetic procedures for the efficient synthesis of imatinib analogs are desirable.

[0021] New imatinib analogs may show activity against clinically relevant imatinib resistant Bcr-Abl mutants. This has recently been demonstrated by the development of AMN107 which is second-generation imatinib analog with potent anti-proliferative activity against a large number of imatinib-resistant Bcr-Abl mutants. (See e.g., O'Hare et al., Cancer Res. 2005, 65, 4500; Manley et al., Biochimica et Biophysica Acta (Proteins and Proteomics) 2005, 1754, 3; Manley et al., Bioorg. Med. Chem. Lett. 2004, 14, 5793). Another imatinib analog, NS-187, that also inhibits a variety of imatinib resistant Bcr-Abl mutants was also recently disclosed. (Asaki et al., Bioorg. Med. Chem. Lett. 2006, 16, 1421). Highly selective imatinib analogs could also serve as "tool" compounds to elucidate the molecular targets responsible for the activity of imatinib in several clinical and non-clinical indications such as pulmonary hypertension, asthma, and diabetes. The preparation of diverse imatinib analogs could also determine if kinases other than AbI, c-kit, PDGFR, and ARG can be potently inhibited by this compound class.

[0022] The present invention provides new methods for the synthesis of imatinib and analogs, particularly, solid phase synthesis, and demonstrates its application to the construction of a 126-membered combinatorial library using IRORI RF-tagged MicroKan™ reactors. (Xiao et al., Biotechnol. Bioeng. (Comb. Chem.) 2000, 71, 44; Nicolaou et al., Angew. Chem. Int. Ed. Engl. 1995, 34, 2289; Xiao et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 780; Xiao et al., J. Org. Chem. 1997, 62, 6029; Wilson et al., Combinatorial Chemistry, Synthesis and Application, Wiley-Interscience, New York, 1997; Gallop et al., J. Med. Chem. 1994, 37, 1233; Dolle et al., J. Comb. Chem. 1999, 1, 235).

[0023] In one aspect, the present invention provides a method for preparing a compound of Formula (1) on a solid support:

wherein R ¹ is H or Ci_6 alkyl;

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R ² , R ³ , R ⁴ and R ⁵ are independently H, an optionally halogenated Ci_ ₆ alkyl or Ci_ ₆ alkoxy, NR ⁶ R ⁷ , or (CR ₂ ) _n R ⁸ ;

R ⁶ and R ⁷ are independently H, an optionally halogenated C _1-6 alkyl, C ₂ -6 alkenyl or C ₂ -6 alkynyl; Ci_ ₆ alkanol, Ci_ ₆ alkoxy or (CR ₂ ) _q -R ⁸ ; or R ⁶ and R ⁷ together with N in NR ⁶ R ⁷ may form an optionally substituted ring;

R ⁸ is an optionally substituted NR R ⁷ , C ₃ _ ₇ cycloalkyl, 5-7 membered aryl, heterocyclic or heteroaryl; each R in (CR ₂ ) is H or Ci_ ₆ alkyl; n and q are independently 0-4; comprising a) contacting a resin-bound compound of Formula (2)

wherein S is a solid support; L is a linker; and X is a leaving group; with a heterocyclic amine of Formula (3) in the presence of a palladium catalyst

to form a support-bound compound of Formula (4)

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[0024] In some examples, the invention provides a solid phase synthesis, comprising the steps of: a) loading the resin by reductive amination with a bromoaniline; b) acylation of the resulting support-bound secondary amine; c) palladium catalyzed N-arylation of a bromoaryl- functionalized solid-support with a 2-aminopyrimidine; and optionally followed by acid- mediated cleavage from the support (Scheme 1).

1 a: R ₁ = H ; 1 b: R ₁ = Me. 9b-1 : imatinib R ₁ = Me,

NRR' = W-methylpiperazine

Scheme 1

[0025] A variety of suitable phosphine ligands known to those skilled in the art may be used in the methods of the invention. In particular examples, 4,5-bis(diphenylphosphino)-9,9-

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dimethyl-xanthene (Xantphos) is used in the palladium catalyzed N-arylation of a bromoaryl- functionalized solid-support. In other examples, sodium ?-butoxide (νaO'Bu) is used to drive the reaction to completion, and 2,6-di-tert-butyl-4-methylphenol (BHT) is added to minimize the quantity of debromination by-product.

[0026] In Scheme 1, loading of resin may be accomplished by reacting aniline Ib (3 eq) with a Pal aldehyde resin (4-formyl-3,5-dimethoxyphenoxymethyl functionalized polystyrene resin, 2% divinylbenzene cross linked, loading capacity 1.05 mmol/1 g) in the presence of 4 equivalents of sodium triacetoxyborohydride and acetic acid (5 v/v %) in NN- dimethylformamide (DMF). Resin 2b is reacted with 3 equivalents of 4-chloromethylbenzoyl chloride in the presence of NN-diisopropylethylamine (DIEA) in dichloromethane (DCM), followed by S _ν 2 amination with 1-methylpiperazine in DMSO to give resin 6b- 1. Aminopyrimidine 7 is synthesized from enaminone by reaction with guanidinum carbonate. The N-arylation of 7 is performed in 1,4-dioxane at 100 ⁰ C in the presence of tris(dibenzylideneacetone)-dipalladium(0) (Pd ₂ dba ₃ ), Xantphos, BHT, and νaO'Bu to afford resin 8b- 1. After each reaction step, the resin is washed extensively with appropriate solvents to remove unbound reagents and impurities. The final product imatinib (9b- 1) is cleaved from the solid support using a solution of trifluoroacetic acid/water/dichloromethane (TFA/H ₂ 0/DCM) (45/5/50, v/v/v). The LC-MS chromatogram of the crude product monitoring at UV 220 and 254 nm as well as electrospray positive-ion mode mass spectrum showed imatinib being produced with a purity of greater than 80%. Analytically pure imatinib is isolated by mass-triggered preparative HPLC. Imatinib analogs may be synthesized using the same protocol.

[0027] In the amination between aryl bromide and the heterocyclic amine on the solid support, the effect of different bases in the reaction was investigated. A suitable base for the reaction includes but is not limited to sodium ?-butoxide (νaO'Bu). In the presence of sodium phenoxide (NaOPh), the reaction yield of desired products increased with decrease of debromonation in the reactions. Weak bases such as potassium phosphate (K ₃ PO ₄ ) and cesium carbonate (CS ₂ CO ₃ ) gave debromo byproducts.

[0028] It was further found that the addition of phenol type compounds in combination with phosphine and sodium ?-butoxide (NaO'Bu) could decrease the bromo-reduction byproducts and hence increase the overall reaction yield. The bulkiest 2, 4, 6-tributylphenolic additive (TBP) gave the optimal result among all the cases, with less than 10 % of debromonated side products.

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The general trend was that the more substitutional groups on the aromatic ring of the phenolic additives, the higher the yield of desired products. In all cases, the amount of uncoupled starting aromatic bromides decreased to neglectable level (all less than 5%). Phenolic additives' beneficial effects and the size-matter trend were also applied to the aniline with methoxy group on the ortho-position.

[0029] More reactions were run between phenyl bromides on Pal resin and anilines with different electronic properties in the presence of NaO ¹ Bu, Pd ₂ dba ₃ , and DPEphos in 1, 4-dioxane at 100 ⁰ C (Scheme 2 and 3). In these reactions, the phenolic additive not only improved the coupling yield, but also suppressed the undesired dehalonation on this side group. For example, in the presence of phenolic additive, the yield of desired coupling product went up more than 50 % without Br-reduced coupling product. In the case of electron-rich aniline as substrate, the yield of the desired product also increased by more than 50 %.

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Scheme 2. The coupling reaction between phenyl bromides on Pal resin and anilines

R = H 2a R = H 3c

Ic α 2d Cl 3d

OCH ₃ 2e OCH ₃ 3e

NO ₂ 2f NO ₂ 3f

4c

Scheme 3. The coupling reactions between phenyl bromide on Pal resin and anilines

[0030] In another embodiment, the methods of the invention may be used to design and produce a compound library, for example, a 126-member library using split-pool synthesis enabled by the IRORI RF-tagged MicroKan™ system. The diversity elements may include two bromoanilines, nine acyl chlorides and seven 2-aminopyrimidines (Scheme 4).

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l; ; ; ; yl-thιazol-5-yl AC7: R _{1 3} = H,

R ₂ = morpholιn-4-ylmethyl; AC8: R _{1 3} = H,

R ₂ = 4-methyl-pιperazιn-1 -ylmethyl; AC9: R _{1 3} = H, R ₂ = CH ₂ NEt ₂

Scheme 4

[0031] In Scheme 4, AC7, AC8 and AC9 are synthesized on resin by acylation with 3 followed by S _N 2 displacement by morpholine, 4-methyl piperazine, and diethylamine, respectively. Aminopyrimidines HC2, HC3, HC4, HC6 and HC7 are easily prepared on gram scale in a similar manner to HC5 (compound 7 in Scheme 1). Crude library compounds are characterized by LC-MS and the purity of each product is estimated by UV chromatogram integration at 254 nm without calibration. Approximately 35% of the library showed purity greater than 75%, and approximately 13% of the products exhibited purities less than 50%.

[0032] To obtain products suitable for biological testing (>95 % purity), the entire library is subject to purification by mass-triggered preparative LC-MS. Approximately 1 to 3 mg is obtained for most library members. To investigate the selectivity of compounds relative to the kinase targets of imatinib, the library is screened in cellular assays dependent on Bcr-Abl, PDGFRβ and c-kit. These assays allow the identification of analogs that inhibit PDGFRβ and c- kit dually but not Bcr-Abl, for example:

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IC ₅₀

Bcr-Abl 5.0 μM PDGFR-b 0.299 μM C-Wt 0.272 μM

General Techniques

[0033] The methods of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of synthetic organic chemistry, solid phase synthetic organic chemistry, including cleavage and purification techniques related to solid phase synthesis using resin or solid supports, and the use of polystyrene based resins, including modified and commercially available resins. (See e.g., Burgess, Solid Phase Organic synthesis, John Wiley & Sons (2000); and Kates et al., Solid-Phase Synthesis: A Practical Guide, Marcel Dekker (2000)).

[0034] Synthesis on a solid support and solid phase synthesis may be performed such that the synthesis from starting material to intermediates to final product is accomplished by linking at least one of the starting materials to a solid support during one of the initial synthetic steps, such as a resin bead, during synthesis from the relevant starting materials. Methods for isolation, purification and characterization of the intermediates and products of the processes as described herein are known to those of skill in the art.

[0035] Detailed examples for synthesizing compounds of Formula (1) in a solid support may be found in the Examples, infra. Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the Examples hereinafter.

[0036] The present invention also includes all suitable isotopic variations of the compounds of the invention, or pharmaceutically acceptable salts thereof. An isotopic variation of a compound of the invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that may be

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incorporated into the compounds of the invention and pharmaceutically acceptable salts thereof include but are not limited to isotopes of hydrogen, carbon, nitrogen and oxygen such as as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ 0, ³⁵ S, ¹⁸ F, ³⁶ Cl and ¹²³ I. Certain isotopic variations of the compounds of the invention and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as ³ H or ¹⁴ C is incorporated, are useful in drug and/or substrate tissue distribution studies. In particular examples, ³ H and ¹⁴ C isotopes may be used for their ease of preparation and detectability. In other examples, substitution with isotopes such as ² H may afford certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Isotopic variations of the compounds of the invention or pharmaceutically acceptable salts thereof can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

[0037] In general, purity of compounds are assessed by reverse-phase liquid chromatography-mass spectrometer (Agilent Series 1100 LC-MS) with an UV detector at λ = 254 nm and an API -ES ionization source. LC elution method (using a Betabasic-18 column) is set to: a linear gradient lml/min flow from 10% to 90% of acetonitrile with 0.035% trifluoroacetic acid in water with 0.05% trifluoroacetic acid over 3 minutes. Purification of compounds by high pressure liquid chromatography is achieved using a Waters 2487 series with Ultra 120 5μm C18Q column with a linear gradient from 10% solvent A (acetonitrile with 0.035% trifluoroacetic acid) in solvent B (water with 0.05% trifluoroacetic acid) to 90% A in seven and half minutes, followed by two and half minutes elution with 90% A. NMR spectra are recorded on Bruker-400MHz instrument and calibrated using residual undeuterated solvent as an internal reference. The following abbreviations were used to designate the multiplicities: 5 = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. Reagent-grade chemicals and solvents were used as purchased from Aldrich.

[0038] The following examples are offered to illustrate but not to limit the invention.

Example 1 Preparation of Intermediates

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Reductive amination for synthesis of PAL-resin-bound aniline (2b) [0039] To a suspension of 4-formyl-3,5-dimethoxyphenoxymethyl functionalized polystyrene resin (PAL) (1Og, Midwest Biotech, 2% divinylbenzene cross linked, 1.05 mmol/g) in N, N-dimethylformamide (DMF), 3-bromo-4-methylaniline Ib (5.86 g, 3 equiv.) and acetic acid (4.80 mL, 8 equiv.) are added. After shaking at 22 ⁰ C for 2 hrs, sodium triacetoxyborohydride (8.80 g, 4 equiv.) is added in one portion to the reaction and the reaction mixture is shaken at 22 ⁰ C for an additional 16 hrs. The resin is washed thoroughly with methanol (MeOH), DMF, and dichloromethane (DCM), and dried in vacuo. The complete consumption of PAL aldehyde is confirmed by disappearance of the IR stretching of aldehyde peak (1710 cm ^"1 ).

N-(3-Bromo-4-methyl-phenyl)-4-chloromethyl-benzamide resin (4b) [0040] Resin 2b (4.0 g) is immersed in DCM (40 mL) completely. N, N-Diisopropyl ethylamine (DIEA, 4.2 mL, 6 equiv.) and 4-chloromethylbenzoyl chloride 3 (2.27 g, 3 equiv.) are added to the reaction. The reaction mixture is shaken at 22 ⁰ C for 3 hrs. Around 5 mg resins are taken out, washed, and cleaved with a mixture of trifluoroacetic acid (TFA): H ₂ O: DCM (45:5:50/v:v:v). LC-MS analysis of the cleavage solution revealed clean and complete conversion to product (UV254, t = 2.767 min). The rest of the resins are washed thoroughly with MeOH and DCM, and are dried in vacuo.

N-(3-Bromo-4-methyl-phenyl)-4-(4-methyl-piperazin- l-ylmethyl)benzamide resin (6b-l) [0041] Resin 4b (1.0 g) is immersed in DMSO (5 mL). N-Methyl-piperazine (554 μl, 5 equiv.) is added to the reaction mixture. The mixture is shaken at 22 ⁰ C for 2 hrs. Around 5 mg of resins are taken out, washed, and cleaved by a mixture of TFA: H ₂ O: DCM (45:5:50/v:v:v). LC-MS analysis of this cleavage solution revealed a complete conversion to product (UV254, t = 1.805 min). The rest of the resins are washed thoroughly with MeOH, DMF, and DCM, and dried in vacuo.

4-Pyridin-3 - yl-pyrimidin-2- ylamine (7)

[0042] A mixture of 3-acetylpyridine (20.72 g, 171.0 mmol) and N, N-dimethylformamide dimethyl acetal (60 mL, 450 mmol) is heated at 110 ⁰ C. After reaction overnight, the reaction mixture is cooled to room temperature and concentrated. Ethyl ether (20 mL) and hexanes (60

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mL) are added to the residue. The resulting solid is collected by filtration, washed with hexanes, and dried to afford 3-dimethylamino-l-pyridin-3-yl-propenone which is used for next reaction without further purification. A mixture of 3-dimethylamino-l-pyridin-3-yl-propenone (17.6 g, 100 mmol) and guanidine carbonate (10.8 g, 60.0 mmol) in 2-butanol (100 mL) is heated at reflux. After 24 hours, the reaction mixture is cooled to room temperature and concentrated. The residue is triturated in water (50 mL). The solid is collected by filtration, washed with water and dried to afford the desired product 4-pyridin-3-yl-pyrimidin-2-ylamine as a white solid. C ₂₉ H ₃₁ N ₇ O Exact MS: 172.07. Found MS m/z 173.1 (M+l). ¹ H NMR (DMSO-J ₆ ): δ 9.23 (s, IH), 8.69 (m, IH), 8.37 (m, 2H), 7.54 (d, IH), 7.20 (d, IH), 6.80 (s, 2H).

Example 2

N-r4-Methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl l-4-(4-methyl-piperazin-l- ylmethylbenzamide resin (8b-l)

[0043] Resin (6b-l) (0.2 g) is immersed in 1,4-dioxane (3 mL). Aminopyrimidine 7 (172 mg, 5 equiv.), tris(dibenzylideneacetone)-dipalladium(0) (Pd ₂ dba ₃ , 9 mg, 0.05 equiv.), 4,5- bis(diphenylphosphino)-9,9-dimethyl-xanthene (Xantphos, 14 mg, 0.12 equiv.), 2,6-di-tert- butyl-4-methylphenol (BHT, 264 mg, 6 equiv.) and sodium ?-butoxide (96 mg, 5 equiv.) are added into the reaction mixture. The reaction mixture is shaken at 100 ⁰ C for 17 hrs. Around 5 mg of resins are taken out, washed, dried, and cleaved by a mixture of TFA: H ₂ O: DCM (45:5:50/v:v:v). LC-MS analysis of this cleavage solution revealed a complete conversion to product. LC-MS retention time: (UV254, t = 1.348 min).The rest of the resins are washed thoroughly with MeOH, DMF, and DCM, and dried in vacuo.

Example 4

4-(4-Methyl-piperazin-l-ylmethyl)-ν-r4-methyl-3-(4-pyrid in-3-yl-pyrimidin-2-ylamino)- phenyll-benzamide (9b- 1)

[0044] Resin (8b-l) is cleaved by a mixture of TFA: H ₂ O: DCM (45:5:50/v:v:v). The resin is removed by filtration and the filtrate is concentrated in vacuo, affording the crude 9b-l. The crude product is purified by preparative HPLC equipped with a MS triggered fraction collector and the fractions containing product 9b- 1 are combined and lyophilized, affording pure 9b- 1 as TFA salt. C ₂₉ H ₃ ]N ₇ O Exact MS: 493.26. Found MS m/z 494.3 (M+l). ¹ H NMR (DMSO-J ₆ ): δ

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10.29 (s, IH), 9.39 (s, IH), 9.11 (s, IH), 8.71 (J, IH, J = 8.4 Hz), 8.69 (J, IH, J = 5.2 Hz), 8.13 (J, IH, J = 1.6 Hz), 8.02 (J, 2H, J = 8.0 Hz), 7.73 (dd, IH), 7.58 (J, 2H, J = 8.4 Hz), 7.50 ~ 7.48 (m, 2H), 7.23 (d, IH, J = 8.4 Hz), 4.08 (5, 2H), 3.62 ~ 2.85 (m, 8H), 2.85 (5, 3H), 2.24 (5, 3H). ¹³ C NMR (DMSO-J ₆ ): δ 164.84, 160.99, 160.74, 159.57, 159.15, 158.80, 158.45, 158.10, 149.12, 146.15, 137.60, 137.03, 134.98, 133.13, 129.98, 127.91, 124.78, 117.26, 116.88, 107.63, 59.07, 10.96, 48.38, 42.00, 17.60.

Assays

[0045] The compounds described herein may be assayed to measure their capacity to selectively inhibit the proliferation of wild type Ba/F3 cells and Ba/F3 cells transformed with Tel c-kit kinase and Tel PDGFR fused tyrosine kinases. In addition, compounds described herein may selectively inhibit SCF dependent proliferation in Mo7e cells. Further, compounds may be assayed to measure their capacity to inhibit c-kit, AbI, Lyn, MAPK14 (p38α), PDGFRα, PDGFRβ, ARG, BCR-AbI, BRK, EphB, Fms, Fyn, KDR, LCK, B-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinase.

Proliferation Assay : BaF3 Library -Bright glo Readout Protocol [0046] The compounds described herein may be tested for their ability to inhibit the proliferation of wt Ba/F3 cells and Ba/F3 cells transformed with Tel fused tyrosine kinases. Untransformed Ba/F3 cells are maintained in media containing recombinant IL3. Cells are plated into 384 well TC plates at 5,000 cells in 50ul media per well and test compound at 0.06 nM to 10 μM is added. The cells are then incubated for 48 hours at 37 °C, 5% CO ₂ . After incubating the cells, 25 μL of BRIGHT GLO® (Promega) is added to each well following manufacturer's instructions and the plates are read using Analyst GT - Luminescence mode - 50000 integration time in RLU. IC ₅₀ values, the concentration of compound required for 50% inhibition, are determined from a dose response curve.

Mo7e Assay

[0047] The compounds described herein may be tested for inhibition of SCF dependent proliferation using Mo7e cells which endogenously express c-kit in a 96 well format. Briefly, two-fold serially diluted test compounds (Cmax=10 μM) are evaluated for their antiproliferative

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activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37°C, cell viability is measured by using a MTT colorimetric assay from Promega.

Inhibition of cellular BCR-AbI dependent proliferation (High Throughput method) [0048] The murine cell line 32D hemopoietic progenitor cell line may be transformed with BCR-AbI cDNA (32D-p210). These cells are maintained in RPMI/10% fetal calf serum (RPMI/FCS), supplemented with penicillin 50 μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM. Untransformed 32D cells are similarly maintained with the addition of 15% of WEHI conditioned medium as a source of IL3.

[0049] 50 μl of a 32D or 32D-p210 cells suspension are plated in Greiner 384 well microplates (black) at a density of 5000 cells per well. 50 nl of test compound (1 mM in DMSO stock solution) is added to each well (STI571 is included as a positive control). The cells are incubated for 72 hours at 37 °C, 5% CO ₂ . 10 μl of a 60% Alamar Blue solution (Tek diagnostics) is added to each well and the cells are incubated for an additional 24 hours. The fluorescence intensity (Excitation at 530 nm, Emission at 580 nm) is quantified using the Acquest™ system (Molecular Devices).

Inhibition of cellular BCR-AbI dependent proliferation

[0050] 32D-p210 cells are plated into 96 well TC plates at a density of 15,000 cells per well. 50 μL of two fold serial dilutions of the test compound (C _max is 40 μM) are added to each well (STI571 is included as a positive control). After incubating the cells for 48 hours at 37 °C, 5% CO ₂ , 15 μL of MTT (Promega) is added to each well and the cells are incubated for an additional 5 hours. The optical density at 570 nm is quantified spectrophotometrically and IC ₅₀ values, the concentration of compound required for 50% inhibition, is determined from a dose response curve.

Effect on cell cycle distribution

[0051] 32D and 32D-p210 cells are plated into 6 well TC plates at 2.5xlO ⁶ cells per well in 5 ml of medium and test compound at 1 or 10 μM is added (STI571 is included as a control). The cells are then incubated for 24 or 48 hours at 37 °C, 5% CO ₂ . Two mL of cell suspension is washed with PBS, fixed in 70% EtOH for 1 hour and treated with PBS/EDTA/RNase A for 30

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minutes. Propidium iodide (Cf= 10 μg/ml) is added and the fluorescence intensity is quantified by flow cytometry on the FACScalibur™ system (BD Biosciences). In some embodiments, test compounds of the present invention may demonstrate an apoptotic effect on the 32D-p210 cells but not induce apoptosis in the 32D parental cells.

Effect on Cellular BCR-AbI Autophosphorylation

[0052] BCR-AbI autophosphorylation is quantified with capture Elisa using a c-abl specific capture antibody and an antiphosphotyrosine antibody. 32D-p210 cells are plated in 96 well TC plates at 2x10 cells per well in 50 μL of medium. 50 μL of two fold serial dilutions of test compounds (C _max is 10 μM) are added to each well (STI571 is included as a positive control). The cells are incubated for 90 minutes at 37 °C, 5% CO ₂ . The cells are then treated for 1 hour on ice with 150 μL of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA and 1% NP-40) containing protease and phosphatase inhibitors. 50 μL of cell lysate is added to 96 well optiplates previously coated with anti-abl specific antibody and blocked. The plates are incubated for 4 hours at 4 °C. After washing with TBS-Tween 20 buffer, 50 μL of alkaline-phosphatase conjugated anti-phosphotyrosine antibody is added and the plate is further incubated overnight at 4 °C. After washing with TBS-Tween 20 buffer, 90 μL of a luminescent substrate are added and the luminescence is quantified using the Acquest™ system (Molecular Devices). In some embodiments, test compounds described herein may inhibit the proliferation of the BCR-AbI expressing cells, inhibiting the cellular BCR-AbI autophosphorylation in a dose-dependent manner.

Effect on proliferation of cells expressing mutant forms of Bcr-abl [0053] Compounds prepared using the methods of the invention may be tested for their antiproliferative effect on Ba/F3 cells expressing either wild type or the mutant forms of BCR- AbI (G250E, E255V, T315I, F317L, M351T) that confers resistance or diminished sensitivity to STI571. The antiproliferative effect of these compounds on the mutant-BCR-Abl expressing cells and on the non transformed cells may be tested at 10, 3.3, 1.1 and 0.37 μM as described above (in media lacking IL3). The IC ₅₀ values of the compounds lacking toxicity on the untransformed cells are determined from the dose response curves obtained as described above.

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FGFR3 (enzymatic assay)

[0054] Kinase activity assay with purified FGFR3 (Upstate) is carried out in a final volume of 10 μL containing 0.25 μg/mL of enzyme in kinase buffer (30 mM Tris-HCl pH7.5, 15 mM MgCl ₂ , 4.5 mM MnCl ₂ , 15 μM Na ₃ VO ₄ and 50 μg/mL BSA), and substrates (5 μg/mL biotin- poly-EY(Glu, Tyr) (CIS-US, Inc.) and 3 μM ATP). Two solutions are made: the first solution of 5 μl contains the FGFR3 enzyme in kinase buffer is first dispensed into 384- format ProxiPlate® (Perkin-Elmer) followed by adding 50 nL of compounds dissolved in DMSO, then 5 μl of second solution contains the substrate (poly-EY) and ATP in kinase buffer is added to each wells. The reactions are incubated at room temperature for one hour, stopped by adding 10 μL of HTRF detection mixture, which contains 30 mM Tris-HCl pH 7.5, 0.5 M KF, 50 mM ETDA, 0.2 mg/mL BSA, 15 μg/mL streptavidin-XL665 (CIS-US, Inc.) and 150 ng/mL cryptate conjugated anti-phosphotyrosine antibody (CIS-US, Inc.). After one hour of room temperature incubation to allow for streptavidin-biotin interaction, time resolved florescent signals are read on Analyst GT (Molecular Devices Corp.). IC ₅₀ values are calculated by linear regression analysis of the percentage inhibition of each compound at 12 concentrations (1:3 dilution from 50 μM to 0.28 nM). In this assay, compounds described herein have an IC ₅₀ in the range of 10 nM to 2 μM.

FGFR3 (cellular assay)

[0055] Compounds described herein may be tested for their ability to inhibit transformed Ba/F3-TEL-FGFR3 cells proliferation, which is dependent on FGFR3 cellular kinase activity. Ba/F3-TEL-FGFR3 are cultured up to 800,000 cells/mL in suspension, with RPMI 1640 supplemented with 10% fetal bovine serum as the culture medium. Cells are dispensed into 384- well format plate at 5000 cell/well in 50 μL culture medium. Compounds described herein are dissolved and diluted in dimethylsulfoxide (DMSO). Twelve points 1:3 serial dilutions are made into DMSO to create concentration gradients ranging typically from 10 mM to 0.05 μM. Cells are added with 50 nL of diluted compounds and incubated for 48 hours in cell culture incubator. AlamarBlue® (TREK Diagnostic Systems), which can be used to monitor the reducing environment created by proliferating cells, are added to cells at final concentration of 10%. After additional four hours of incubation in a 37 ⁰ C cell culture incubator, fluorescence signals from reduced AlamarBlue® (Excitation at 530 nm, Emission at 580 nm) are quantified on

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Analyst GT (Molecular Devices Corp.). IC ₅₀ values are calculated by linear regression analysis of the percentage inhibition of each compound at twelve concentrations.

FLT3 and PDGFRβ (cellular Assay)

[0056] Compounds described herein may be tested for their activity against FLT3 and PDGFRβ using identical methods as described above for FGFR3 cellular activity, except that instead of using Ba/F3-TEL-FGFR3, Ba/F3-FLT3-ITD and Ba/F3-Tel-PDGFRβ are used, respectively.

B-Raf (enzymatic assay)

[0057] Compounds described herein may be tested for their ability to inhibit the activity of b-Raf. The assay is carried out in 384-well MaxiSorp plates (NUNC) with black walls and clear bottom. The substrate, IκBα is diluted in DPBS (1:750) and 15 μl is added to each well. The plates are incubated at 4°C overnight and washed 3 times with TBST (25 mM Tris, pH 8.0, 150 mM NaCl and 0.05% Tween-20) using the EMBLA plate washer. Plates are blocked by Superblock (15 μl/well) for 3 hours at room temperature, washed 3 times with TBST and pat- dried. Assay buffer containing 20μM ATP (10 μl) is added to each well followed by lOOnl or 500nl of compound. B-Raf is diluted in the assay buffer (1 μl into 25 μl) and 10 μl of diluted b- Raf is added to each well (0.4μg/well). The plates are incubated at room temperature for 2.5 hours. The kinase reaction is stopped by washing the plates 6 times with TBST. Phosph- IκBα (Ser32/36) antibody is diluted in Superblock (1:10,000) and 15 μl is added to each well. The plates are incubated at 4°C overnight and washed 6 times with TBST. AP-conjugated goat- anti-mouse IgG is diluted in Superblock (1:1,500) and 15μl is added to each well. Plates are incubated at room temperature for 1 hour and washed 6 times with TBST. 15 μl of fluorescent Attophos AP substrate (Promega) is added to each well and plates are incubated at room temperature for 15 minutes. Plates are read on Acquest or Analyst GT using a Fluorescence Intensity Program (Excitation 455 nm, Emission 580 nm).

B-Raf (cellular assay)

[0058] Compounds described herein may be tested in A375 cells for their ability to inhibit phosphorylation of MEK. A375 cell line (ATCC) is derived from a human melanoma patient

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and it has a V599E mutation on the B-Raf gene. The levels of phosphorylated MEK are elevated due to the mutation of B-Raf. Sub-confluent to confluent A375 cells are incubated with compounds for 2 hours at 37 ⁰ C in serum free medium. Cells are then washed once with cold PBS and lysed with the lysis buffer containing 1% Triton XlOO. After centrifugation, the supernatants are subjected to SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes are then subjected to western blotting with anti-phospho-MEK antibody (ser217/221) (Cell Signaling). The amount of phosphorylated MEK is monitored by the density of phospho-MEK bands on the nitrocellulose membranes.

Upstate KinaseProfiler™ - Radio-enzymatic filter binding assay

[0059] Compounds prepared using the methods of the invention may be assessed for their ability to inhibit individual members of a panel of kinases (a partial, non-limiting list of kinases includes: c-kit, AbI, Lyn, MAPK14 (p38α), PDGFRα, PDGFRβ, ARG, BCR-AbI, BRK, EphB, Fms, Fyn, KDR, LCK, B-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinase). The compounds are tested in duplicates at a final concentration of 10 μM following this generic protocol. Note that the kinase buffer composition and the substrates vary for the different kinases included in the "Upstate KinaseProfiler™" panel. Kinase buffer (2.5 μL, 10x - containing MnCl ₂ when required), active kinase (0.001-0.01 Units; 2.5μL), specific or Poly(Glu4-Tyr) peptide (5-500 μM or .01mg/ml) in kinase buffer and kinase buffer (50 μM; 5 μL) are mixed in an eppendorf on ice. A Mg/ATP mix (10 μL; 67.5 (or 33.75) mM MgCl ₂ , 450 (or 225) μM ATP and 1 μCi/μl [γ- ³² P]-ATP (3000Ci/mmol)) is added and the reaction is incubated at about 30 ⁰ C for about 10 minutes. The reaction mixture is spotted (20 μL) onto a 2 cm x 2 cm P81 (phosphocellulose, for positively charged peptide substrates) or Whatman No. 1 (for Poly (Glu4-Tyr) peptide substrate) paper square. The assay squares are washed 4 times, for 5 minutes each, with 0.75% phosphoric acid and washed once with acetone for 5 minutes. The assay squares are transferred to a scintillation vial, 5 ml scintillation cocktail are added and ³² P incorporation (cpm) to the peptide substrate is quantified with a Beckman scintillation counter. Percentage inhibition is calculated for each reaction.

[0060] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this

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application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.