HK1242677A1 - Benzimidazole analogues and related methods - Google Patents

Description

Benzimidazole analogs and related methods

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority from U.S. provisional patent application No. US62/007,321, filed 6/3/2014, the disclosure of which is incorporated herein by reference.

Background

Protein Kinases (PKs) are enzymes that catalyze the phosphorylation of specific serines, threonines or tyrosines in cellular proteins. Post-translational modifications of these substrate proteins function as molecular switches that play key roles in various biological processes, such as controlling cell growth, metabolism, tumor microenvironment (e.g., VEGFR), differentiation, and apoptosis. Abnormal, excessive, or more generally inappropriate PK activity has been observed in several disease states, including malignant proliferative disorders such as functional mutations in Medullary Thyroid Carcinoma (MTC) and other human malignancies, ITD (inter-junction repeat) -mutations in Flt3 of Acute Myeloid Leukemia (AML), c-Kit mutations in gastrointestinal stromal tumors (GIST), and RET acquisition of Bcr-abl rearrangement in Chronic Myeloid Leukemia (CML). Furthermore, activation and/or overexpression of tyrosine kinases (e.g., Trk-A, Trk-B, Trk-C and RET) is associated with severe pain in cancer patients, particularly pancreatic cancer patients. Many tyrosine kinases are homologous to each other; inhibition of one tyrosine kinase can also result in certain inhibitory activity against other tyrosine kinases. For example, imatinib has been used as a therapeutic agent not only in CML patients (based on inhibition of Bcr-abl kinase), but also in GIST cancer patients (based on inhibition of c-Kit kinase). Recent advances in basic and clinical studies on tyrosine kinases have demonstrated that many tyrosine kinases can be targeted by drugs. For example, over twelve new drugs have been approved over the last decade that target VEGFR2, Bcr-abl, Flt3, Platelet Derived Growth Factor Receptor (PDGFR), and c-Kit. Briefly described are several targets for cancer therapy and the problems involved therewith.

RET

In 1985, RET (rearrangement during transfection) gene was identified as a novel oncogene activated by DNA rearrangement (Takahashi, M.cell,1985,42, 581-588).

MTC (medullary thyroid carcinoma) is a malignant tumor of C cells in the thyroid gland. MTC may be sporadic or familial as multiple endocrine neoplasia type 2 syndromes MEN2A and MEN 2B. Familial (about 95%) and sporadic (about 50%) MTC exhibited gain-of-function point mutations in the RET proto-oncogene, resulting in increased pro-survival signaling and cell growth. RET signaling maintains tumorigenesis. As a result, blocking this RET signaling provides an "Achilles)' heel" therapeutic approach for MTC patients. Multiple endocrine adenomatosis 2B is a hereditary syndrome caused primarily by mutation of M918T in the RET receptor kinase domain, while multiple endocrine malignancy 2A is caused primarily by mutation of C634 (Santoro, m. et al Science,1995,267,381). None of the mutations are located at the ATP binding site. Isolated familial MTC results from different mutations in the extracellular or intracellular domains of RET, including mutations V804M and V804L, which target a gatekeeper residue in the kinase ATP binding site. Systemic treatment for MTC is often difficult to be effective, with 56% of patients experiencing postoperative recurrence due to early metastasis (Wells, jr.s.a. et al, clin.cancer res.2009,15, 7119-.

PTC (papillary thyroid carcinoma) is derived from follicular thyroid cells. In PTC, RET is targeted by chromosomal rearrangements, resulting in an in-frame fusion of the components of the intracellular domain of the heterologous gene at its 5' -end. It is estimated that there are approximately 60,000 new cases in the us in 2012. Distant metastasis is observed in less than 5% of patients with differentiated thyroid cancer, and the incidence of recurrent disease is 10-15%. The treatment of recurrent diseases mainly includes surgery and iodine radiation. However. There remains an unmet medical need for the treatment of radiation refractory PTC diseases (Schlumberger, m.thyroid,2009,19, 1393-.

Lung cancer is the leading cause of cancer-related mortality. Paradigms for the treatment of non-small cell lung cancer (NSCLC), which accounts for a major portion of all lung cancers, have shifted from histological diagnosis to the use of molecular subtype diagnosis. Some "molecular driven mutations" can produce constitutively active mutant signaling proteins, such as EGFR and ALK. Recently, several studies have identified RET kinase fusions (KIF5B-RET and the more rare RET/PTC variants) in about 1% of patients with adenocarcinoma-type NSCLC. As soon as 4 years after identification of ALK fusions in lung cancer, the FDA approved an ALK inhibitor for NSCLC. Thus, RET-positive NSCLC patients can similarly benefit from specific targeting therapy (Hutchinson, k.e.nat. med.2012,18, 349-.

CMML is a myeloproliferative disease presenting mononucleosis. Myeloproliferative tumors are often associated with aberrant constitutive tyrosine kinases resulting from chimeric fusion genes or point mutations. Two novel fusion genes among CMML, BCR-RET and FGFR1OP-RET have been reported. Blocking RET activity in CMML is important for reestablishing signaling homeostasis, regenerating appropriate committed hematopoietic stem cell differentiation, and controlling aberrant oncogenic signaling (Ballerini, p. et al, leukamia 2012, 1-6).

RET overexpression in the ER-positive breast cancer panel has also recently been identified. In situ hybridization of a panel of 245 breast invasive carcinomas (a technique used to determine the presence or absence of genetic sequences in tissues) detected RET and GFR α 1 mRNA in 29.7% and 59.4% of tumors, respectively. The majority of these tumors were ER-positive. Similar findings were reported in the observations of breast cancer cell lines. In addition, qPCR analysis of a small panel of breast tumor primary cells detected preferential expression of the retmrnas in ER-positive samples. Finally, these studies were enhanced by microarray studies, i.e., RET expression was positively correlated with ER expression in a set of 36 breast cancer samples. The role of RET in tamoxifen resistant breast cancer was further validated in a cell-based assay format for dr. In ER α -positive breast cancer cells, activation of the receptor tyrosine kinase RET by its ligand GDNF leads to increased ER α phosphorylation on Ser118 and Ser167, as well as estrogen-independent activation of ER α transcriptional activity. In vitro RET down-regulation resulted in a 6.2-fold increase in the sensitivity of MCF7 cells to the antiproliferative effects of tamoxifen, whereas GDNF stimulation had a protective effect on the drug. Targeting RET restores tamoxifen sensitivity in tamoxifen resistant MCF7 cells. Finally, examination of two independent tissue microarrays of major human breast cancers revealed that RET protein expression was clearly associated with era-positive tumors and that the number of RET-positive tumors increased 2-fold in patients with immediate invasive recurrence following adjuvant tamoxifen treatment (Morandi, a. trends in mol. med.2011,17, 149-.

VEGFR

Vascular Endothelial Growth Factor (VEGF) is an important signaling protein involved in angiogenesis (vasculogenesis) and angiogenesis (angiogenesis). As its name suggests, VEGF activity is primarily limited to vascular endothelial cells, although it has an effect on a limited number of other cell types. In vitro, VEGF has been shown to stimulate mitogenesis and cell migration in endothelial cells. VEGF also promotes microvascular permeability and is sometimes referred to as vascular permeability factor. VEGFR kinases have been used as targets for solid tumors, for example, highly vascularized malignancies such as renal, glioblastoma and liver cancers (bhragova, p. curr Oncol Rep,2011, 103-.

FLT3

Although the cure rate for Acute Myeloid Leukemia (AML) has improved over the past forty years, the survival rate is still poor. The 5-year survival rate for 60-year-old patients is only 40%. The standard of care for the newly diagnosed patients with AML consists of induction of chemotherapy and infusion of cytarabine and anthracyclines.

Mutations in the FMS-like tyrosine kinase 3(FLT3) gene are characterized in 30% of AML cases. Adapter repeat (ITD) mutations within FLT3 (accounting for about 23% of AML cases) are associated with particularly poor prognosis. The problem of prognostic involvement of FLT3/D835 point mutations found in approximately 7% of cases at diagnosis has not been established. It may be advantageous to inhibit FLT3 and its mutations.

c-KIT

c-Kit is a receptor tyrosine kinase that normally controls primitive hematopoietic, melanocyte and germ cell functions. It is evident that uncontrolled activity of c-Kit contributes to the formation of a large number of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loop or mutational activation. This makes c-Kit an excellent target for Cancer therapy in these tumors, especially GIST and AML (Von Mehren, m.clin.colorectal Cancer,2006, S30-40).

TRK

Tropomyosin-related kinases (Trks) are receptor tyrosine kinases that are commonly expressed in neuronal tissue, where they play an important role in development and function. The Trk receptor family consists of 3 members (A, B and C) activated by specific ligands called neurotrophins. Each Trk receptor comprises an extracellular domain, a transmembrane region and an intracellular domain which, upon binding to their respective ligands (nerve growth factor (NGF) of TrkA, brain derived growth factor (BDNF) and NT-4/5 of TrkB and NT3 of TrkC), triggers the receptor oligomerization, specific tyrosine residue phosphorylation in the kinase domain and downstream signal transduction pathways, including survival, proliferation and differentiation in normal and tumor neuronal cells. Dysregulation of TrkA and TrkB and their cognate ligands has been described in a number of types of cancer, including prostate, breast, colorectal, ovarian, lung, pancreatic, melanoma, thyroid, and neuroblastoma, and occurs primarily through wild-type receptor overexpression, activation, amplification, and/or mutation. Importantly, increased Trks activation in tumor tissues correlates with a aggressive phenotype and poor clinical outcome. In addition, Trks and RET play a role in peripheral nerve impingement and associated cancer pain.

PDGFR

Platelet-derived growth factors function as potent mitogens and chemokines for a variety of cells, such as fibroblasts, smooth muscle cells, mesenchymal cells, and glial cells. It has been proposed that aberrant PDGF-induced cell proliferation leads to proliferative disorders. There is a need for PDGFR inhibitors that provide therapeutic benefit for proliferative disorders such as gastrointestinal stromal tumors (GIST), gliomas, and melanomas.

Brief Description of Drawings

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of necessary fee.

FIG. 1: blot photographs showing inhibition of RET/C634Y and RET/M918T phosphorylation in intact cells with Pz-1 or vehicle (NT: untreated). Serum-starved NIH3T3 cells expressing RET/C634Y or the RET/M918T mutant were treated with Pz-1 at the indicated concentrations for 2 hr. 50 μ g of total cell lysate was immunoblotted with anti-phospho-Y1062 (. alpha.p 1062) or anti-phospho-Y905 (. alpha.p 905) RET antibody. Blots were normalized using anti-RET (α RET) antibodies.

FIG. 2: blot photographs showing inhibition of protein phosphorylation of the gatekeeper RET point mutations and rearranged RET oncogenes with Pz-1 or vehicle (NT: untreated). Cells HEK293 were transiently transfected with vectors expressing either the RET/C634R-V804M mutant (V804 is the gatekeeper RET residue) or RET/PTC1(CCDC6-RET, found in papillary thyroid carcinomas and lung adenocarcinomas), RET/PTC3(NCOA4-RET, found in papillary thyroid carcinomas and lung adenocarcinomas) or KIF5B-RET (found in lung adenocarcinomas). 36hr post transfection, cells were serum starved for 12hr and then treated with Pz-1 at the indicated concentrations for 2 hr. 50 μ g of total cell lysate was subjected to immunoblotting using anti-phospho-Y1062 (. alpha.p 1062) and anti-phospho-Y905 (. alpha.p 905) RET antibodies. Blots were normalized using anti-RET (α RET) antibodies.

FIG. 3: blot photographs showing the effect of inhibiting the reduction in TT cells (RET/C634W expressing human MTC cell line) of endogenously expressed RET protein and thyroid non-transformed Nthy-ori-3-1 cells with indicated concentrations of Pz-1 or vehicle (NT: untreated). Serum-starved human TT and thyroid untransformed Nthy-ori-3-1 cell lines were treated with Pz-1 at the indicated concentrations for 2 hr. Mu.g of total cell lysate was immunoblotted with anti-phospho-Y1062 (. alpha.p 1062) and anti-phospho-905 (. alpha.pY 905) RET antibodies, anti-phospho-MAPK (. alpha.pMAPK, T302/Y304) and anti-phospho-SHC (alpha. pSHC, Y317) antibodies. Blots were normalized using anti-RET (α RET) and anti-SHC (α SHC).

FIG. 4: blots showing inhibition of abnormal phosphorylation of VEGFR2 expressed in HEK293 cells with Pz-1 or vehicle (NT: untreated) at the doses indicated. HEK293 cells were transiently transfected with a vector expressing VEGFR 2. 36hr post transfection, cells were serum starved for 12hr, treated with Pz-1 at the indicated concentrations for 2hr, and finally treated with VEGF-A (100ng/ml) for 15 min. 50 μ g of total cell lysate were subjected to immunoblotting using anti-phospho-Y1175 (α pVEGFR2) VEGFR2 antibody. Blots were normalized using anti-VEGFR 2(α VEGFR 2).

FIG. 5: the indicated concentrations of Pz-1 or vehicle (NT: untreated) exerted an effect on the proliferation of TT (human MTC cell line expressing RET/C634W), Lc-2/ad (human NSCLC expressing CCDC6-RET) and Nthy-ori-3-1 (thyroid untransformed) cell lines. Data are mean ± SD (standard deviation) of one experiment performed in triplicate. Bottom, right) Pz-1 growth inhibition IC on different cell lines₅₀: the 95% CI (confidence interval) is shown in parentheses. Cells were plated in triplicate on 60-mm dishes and maintained in either 5% (Nthy-ori-3-1) or 10% (TT and Lc-2/ad) fetal calf serum. On the day after plating, different concentrations of Pz-1 or vehicle were added to the medium and changed every 2-3 days. Cells were counted every 2-3 days and cell numbers were reported as ± SD (standard deviation). IC was calculated by curve-fitting analysis of the last 1 day from growth curves using the PRIZM Software program (Graphpad Software Inc)₅₀Dose (with confidence interval).

FIG. 6: pz-1 activity on NCOA4-RET oncogene-driven IL-3-independent proliferation of murine Ba/F3 cells. Cell growth curves show Pz-1 inhibition of Ba/F3-NCOA4-RET, but not of parental Ba/F3The preparation method comprises the following steps. Ba/F3 and Ba/F3NCOA4-RET cells were incubated with vehicle (NT) or Pz-1 at the indicated concentrations in complete medium and counted at different time points. Data are mean ± SD of one experiment performed in triplicate. Murine interleukin-3 (IL-3) dependent pro-B Ba/F3 cells were from ATCC. Ba/F3 cells stably expressing the protein of NCOA4-RET (RET/PTC3) were generated by infecting the long isoform of NCOA4-RET (RET-51) with electroporation. Parental and Ba/F3-NCOA4-RET cells were cultured in RPMI 1640 with 10% FBS; the parent cells also required 10ng/ml IL-3. Display IC₅₀Dose (with confidence interval).

FIG. 7: pz-1 anti-tumorigenic activity against TT cells xenografted into SCID mice. The figure shows the effect of Pz-1 on subcutaneous tumors of SCID mice implanted with TT cells and treated daily with vehicle (n.10 mice, 18 tumors) or with 0.3, 1.0 or 3.0mg/kg Pz-1(n.29 mice, 52 tumors) by oral gavage. Mean ± SD (standard deviation) of tumor volumes are reported. Mixing TT cells (7.5x 10)⁶Mice) were inoculated subcutaneously into the dorsal part (bilateral) of 39 female SCID mice (Jackson Laboratories, Bar Harbor, Maine). After 5 weeks, there was at least one tumor in each mouse and a total of 70 tumors were visible: 56 tumors are 40-150mm³And 14 tumors are<40mm³. Tumors were evident in 2 injection sites of 31 mice; only 1 site exhibited tumor formation in the remaining 8 mice. Tumor-bearing animals were randomly grouped to receive Pz-1(0.3, 1.0 or 3.0mg/kg daily) (29 mice, 52 tumors) or vehicle (10 mice, 18 tumors) by oral gavage. Treatment was administered for 28 consecutive days. Tumor diameter was measured weekly using calipers. Tumor volume (V) was calculated by the ellipsoid rotation formula: v is AxB²And/2 (a ═ shaft diameter; B ═ rotation diameter) and reported as mean volume ± standard deviation. To compare tumor growth, the gram-Wacker test (nonparametric ANOVA) and Dunn's multiple comparison test (InStat program, GraphPad software) were used. P value is in P<It was statistically significant at 0.05.

FIG. 8: pz-1 anti-tumorigenic activity in nude mice implanted with RET/C634Y or HRAS (G12V) transformed NIH3T3 fibroblasts. The FIGURE shows the effect of Pz-1 on subcutaneous tumors of mice treated by oral gavage for the indicated time periods with Pz-1(1.0, 3.0 or 10mg/kg daily) or vehicle. The mean tumor volume (. + -. SD: standard deviation) is reported. The dorsal part (bilateral) of 6-week-old female BALB/C nu/nu mice (n.31 mice/cell line) (Jackson Laboratories, Bar Harbor, Maine) was inoculated subcutaneously with NIH3T3RET/C634Y (200,000 cells) or NIH3T3HRAS/G12V (50,000 cells). After 4 days, animals were randomized to receive Pz-1(1.0, 3.0 or 10mg/kg daily) by oral gavage (23 mice/cell line: 8 mice/group, 1.0 and 3.0mg/kg daily dose; and 7 mice, 10dose mg/kg daily) or vehicle control (8 mice) before tumor appearance. Tumor diameters were measured every 1-2 days using calipers. Tumor volume (V) was calculated by the ellipsoid rotation formula and reported as mean volume ± standard deviation. To compare tumor growth, the gram-Wacker test (nonparametric ANOVA) and Dunn's multiple comparison test (InStat program, GraphPad software) were used. P values were statistically significant at P < 0.05.

FIG. 9: photographs of blots showing the effect of Pz-1 on cellular phosphorylation events (from figure 8) in tumors induced in nude mice by injection of NIH3T3 fibroblasts transformed with RET/C634Y or HRAS/G12V. Blots showing inhibition of Pz-1 treatment of pvgfr 2 in RET/C634Y-and HRAS/G12V-induced tumors and inhibition of RET phosphorylation and intracellular signaling (SHC, MAPK, p70S6K and S6RP) only in RET/C634Y-induced tumors. A portion of the vehicle-treated tumors at the end of the tumor growth experiment described in FIG. 8 were treated with varying doses of Pz-1(TR) for 48hr or left untreated (NT). At the end of the treatment, 50 μ g of tumor protein lysate was immunoblotted with anti-phospho-Y1062 (α p1062) and anti-phospho-905 (α pY905) RET antibodies, anti-phospho-MAPK (α pMAPK, T302/Y304), anti-phospho-SHC (α pSHC, Y317), anti-phospho-p 70S6K (α pp70S6K, T389), anti-phospho-S6 RP (α pS6RP, S235/S236) and anti-phospho-VEGFR 2(α pvfr 2pY1175) antibodies. Blots were normalized using anti-RET (α RET), anti-SHC (α SHC), anti-MAPK (α MAPK), anti-p 70S6K (α p70S6K), anti-S6 RP (α S6RP), or anti-VEGFR 2(α VEGFR2) antibodies.

FIG. 10: a docking model of Pz-1(N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide) with red RET kinase and green VEGFR2 kinase. There are 4 amino acids differences between RET and VEGFR2, which can be used to design selective RET inhibitors. The amino acid sequence of the VEGFR-2DFG outer crystal structure (PDB #2OH4) and RET (PDB #2IVU) was obtained. The RET sequence was used to construct a RET DFG outer homology MODEL using the VEGFR 2DFG outer structure as a template using SWISS-MODEL Automatic modeling Mode (swissmodel. expasy. org). Using AutoDock Tools: 1) all hydrogen was added as 'only polar'; 2) a gate-box for ATP binding site was generated (center x-25.881, center y 9.55, center z-10.927/size x 16, size y 44, size z 18). AutoDock Tools were used to specify the appropriate rotatable bond of the computer modeled compound. AutoDock Vina was then used to model the compound. The modeling results were visible and analyzed using Discovery studio 3.5.

Summary of The Invention

In a first aspect, provided herein is a compound of formula VIII:

wherein R is¹Is unsubstituted or (by R)⁶) Substituted aryl or heteroaryl; r²Selected from H, (C)₁-C₃) Alkyl, halogen, -CN, -O- (C)₁-C₃) Alkyl, -O- (CH)₂)_nX、-N(R⁷)(R⁸)、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX and-SO₂(C₁-C₃) An alkyl group; r³And R⁴Each independently is H, (C)₁-C₆) Alkyl or CN; r⁵Is- (C)₁-C₃) Alkyl or- (C) substituted by 1-3 fluorine₁-C₃) An alkyl group; r⁶Is H, OH, NH₂、(C₁-C₃) Alkyl, halogen, -CN, -O (C)₁-C₃) Alkyl, -O (CH)₂)_nX、-N(R⁷)(R⁸)、-CONH₂、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX or-SO₂(C₁-C₃) An alkyl group; x is OR⁹、N(R⁷)(R⁸)；R⁷And R⁸Each independently is hydrogen or (C)₁-C₄) Alkyl or (C)₁-C₄) Alkoxy groups, and may form a ring therebetween; n is 2 or 3; and R is⁹Is H or (C)₁-C₃) An alkyl group. Also provided are salts, isomers, stereoisomers, enantiomers, racemates, solvates, hydrates, polymorphs and prodrugs of formula VIII.

In some embodiments, the compound of formula VIII is selected from: n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1); 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) -N- (5- (tert-butyl) isoxazol-3-yl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; and N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide.

In a particular embodiment, the compound of formula VIII is N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1).

In a second broad aspect, provided herein is a method of making a tyrosine kinase inhibitor. The first method comprises reacting a substituted aniline with an activated fluoride compound in a nucleophilic addition reaction to produce an addition product; selectively reducing said addition product to produce a first reduction product; cyclizing the reduced product to produce a cyclized intermediate; coupling the cyclized intermediate with a boronic acid (boronic acid) or tin derivative to produce an ester; and reducing the ester to produce a second reduction product; and ammoniating the second reduction product to produce the tyrosine kinase inhibitor.

In some embodiments, the substituted aniline comprises bromine in the para position. In some embodiments, the selective reduction comprises reduction of NO₂Reduction of the radical to NH₂Without reducing the bromine. In some embodiments, the cyclization comprises activating the reduced product with an acid. In a specific embodiment, the acid comprises pTSA. In some embodiments, the cyclized intermediate comprises 2- (4- (5-bromo-1H-benzo [ d)]Imidazol-1-yl) phenyl) acetic acid ethyl ester.

In some embodiments, the coupling is palladium catalyzed. In some embodiments, bromine is a leaving group in the coupling step. In some embodiments, the coupling step comprises a Suzuki coupling. In some embodiments, the ester is selected from: ethyl 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and ethyl 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate. In some embodiments, the ester consists essentially of ethyl 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

In some embodiments, the second reduction product comprises a compound selected from the group consisting of: 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; lithium 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and lithium 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate. In some embodiments, the second reduction product consists essentially of lithium 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

The second method comprises reacting a bromoborate ester (boronic ester) with a halo-aryl or heteroaryl compound in the presence of a catalyst and a base to produce an intermediate; coupling said intermediate with a boronic acid or tin derivative to produce a tyrosine kinase inhibitor precursor; and reducing and aminating the tyrosine kinase inhibitor precursor to produce the tyrosine kinase inhibitor. In some embodiments, the base comprises potassium acetate.

In some embodiments, the halo-aryl or heteroaryl group comprises a pyrrolyl, pyrazolyl, pyranyl, thiopyranyl, furanyl, imidazolyl, pyridyl, thiazolyl, triazinyl, phthalimidyl, indolyl, purinyl, benzothiazolyl group, or a combination thereof. In some embodiments, the halo-aryl or heteroaryl comprises a heterocyclic residue selected from the group consisting of: oxiranyl, aziridinyl, aziridyl, 1, 2-oxathiacyclopentyl, thienyl, furyl, tetrahydrofuranyl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuryl, benzofuryl, chromenyl, 2H-pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, (S-oxo or S, S-dioxo) -thiomorpholinyl, indolizinyl, isoindolyl, 3H-indolyl, benzimidazolyl, coumaryl, indazolyl, triazolyl, tetrazolyl, Purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothienyl, dibenzothienyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, peridinaphthyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl and chromanyl.

In some embodiments, the intermediate comprises ethyl 2- (4- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

In some embodiments, the tyrosine kinase inhibitor precursor is selected from the group consisting of: ethyl 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and ethyl 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate. In some embodiments, the tyrosine kinase inhibitor precursor consists essentially of ethyl 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

In some embodiments, the tyrosine kinase inhibitor precursor is reduced to a reduction product comprising a compound selected from the group consisting of: 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; lithium 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and lithium 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate. In some embodiments, the tyrosine kinase inhibitor precursor is reduced to a reduction product consisting essentially of 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

In another broad aspect, provided herein are pharmaceutical compositions. The pharmaceutical composition comprises a compound of formula VIII and a pharmaceutically acceptable carrier, diluent or excipient.

Also provided are methods of treating a subject having cancer, inhibiting phosphorylation, and inhibiting proliferation of cells, including thyroid cancer cells. In some embodiments, the method comprises administering to the subject or cell an effective amount of a compound of formula VIII. In some embodiments, the compound comprises N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1). In some embodiments, the thyroid cancer cell comprises MTC.

Also provided are methods of inhibiting tyrosine kinases comprising treating a cell with an effective amount of a compound of formula VIII. In some embodiments, the tyrosine kinase is selected from RET, FLT3, C-Kit, VEGFR, Trk-A, Trk-B, Trk-C, and PDGFR. In some embodiments, the compounds exhibit an IC of less than 1 μ Μ₅₀A kinase domain of value.

Methods of treating pain associated with cancer are also provided. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound of formula VIII and a pharmaceutically acceptable carrier, diluent or excipient.

Kits are also provided. In a first embodiment, a kit for preparing a tyrosine kinase inhibitor comprises a first container holding a substituted aniline and a second container holding an activated fluoride compound. In some embodiments, the kit further comprises one or more reducing agents boric acid or tin derivatives. In a second embodiment, a kit for preparing a tyrosine kinase inhibitor includes a first container holding a borate ester, a second container holding a halo-aryl or heteroaryl compound and a catalyst. In some embodiments, the kit further comprises one or more reducing agents. In a third embodiment, a kit for preparing a pharmaceutical composition comprises a first container containing a compound of formula VIII and a second container containing a pharmaceutically acceptable carrier, diluent, or excipient.

Detailed Description

Various embodiments are described in the compounds, methods of making the compounds, and methods of using the compounds disclosed herein. Those skilled in the art will realize that the following detailed description of the embodiments is illustrative only and is not intended to be in any way limiting. Additional embodiments will readily suggest themselves to those skilled in the art having the benefit of this disclosure. References in the disclosure to "an embodiment," "an aspect," or "an example" indicate that the embodiment of the invention so described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, repeated usage of the term "in one embodiment" does not necessarily refer to the same embodiment, although it may.

Definition of

General chemical terms used herein have their ordinary meaning in the art. For example, the term "C" as used herein₁-C₄Alkyl "alone or in combination denotes a straight-chain or branched C consisting of carbon and hydrogen atoms₁-C₄Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and the like.

The term "halo" or "halogen" as used herein means fluoro, chloro, bromo or iodo. The term "C" as used herein₁-C₆Alkyl "refers to a straight or branched, monovalent, saturated aliphatic chain of 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, and hexyl. The term "C₁-C₆Alkyl "includes within its definition the term" C₁-C₄Alkyl "and" C₁-C₃Alkyl groups ". The term "carboxyl (carboxyl)" or "carboxyl (carboxyl)" refers to a carboxylic acid. The term "carboxamide" means a substituted NH₂A partially substituted carbonyl group. The term "oxo" refers to a carbonyl group.

The term "heteroaryl" as used herein refers to an aryl moiety comprising 1 to 5 heteroatoms selected from O, S and N. Examples of heteroaryl groups include pyrrolyl, pyrazolyl, pyranyl, thiopyranyl, furanyl, imidazolyl, pyridyl, thiazolyl, triazinyl, phthalimidyl, indolyl, purinyl, and benzothiazolyl. Heteroaryl is in particular a heterocyclyl residue selected from the group consisting of oxiranyl, aziridinyl, 1, 2-oxathiacyclopentyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuryl, benzofuryl, chromenyl, 2H-pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, (S-oxo or S, S-dioxo) -thiomorpholinyl, indolizinyl, isoindolyl, 3H-indolyl, benzimidazolyl, coumaryl (cumaroyl), Indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothienyl, dibenzothienyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidine, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochroman, and chromanyl.

Various abbreviations are used herein. The term DCM refers to dichloromethane. The term DIPEA refers to N, N-diisopropylethylamine. The term Pd₂(dba)₃Refers to tris (dibenzylideneacetone) dipalladium. The term dppf refers to 1, 1' -bis (diphenylphosphino) ferrocene. The term DMA refers to N, N-dimethylacetamide. The term DMF refers to N, N-dimethylformamide. The term DMSO refers to dimethylsulfoxide. The term EDC refers to 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. The term EtOAc refers to ethyl acetate. The term EtOH refers to ethanol. The term ES refers to electrospray. The term h refers to hours. The term HOAc refers to acetic acid. The term HOAt refers to 1-hydroxy-7-azabenzotriazole. The term IPA refers to isopropyl alcohol. The term KOAc refers to potassium acetate. The term LC refers to liquid chromatography. The term LiOH refers to lithium hydroxide. The term MgSO₄Refers to magnesium sulfate. The term min refers to minutes. The term mL refers to milliliters. The term mmol refers to millimoles. The term MS refers to mass spectrometry. The term NaHCO₃Refers to sodium bicarbonate. The term pTSA refers to p-toluenesulfonic acid. The term P (CY)₃Refers to tricyclohexylphosphine. The term RT refers to room temperature. The term THF means tetrahydrofuran. The term TLC means thinLayer chromatography. The term TMOF refers to trimethyl orthoformate.

If the plural form is used for a compound, salt, pharmaceutical agent, disease, disorder, etc., it is also intended to refer to a single compound, salt, pharmaceutical agent, disease, etc. If "a" or "an" is used, it refers to the indefinite article or preferably "a".

General description

Provided herein are compounds of formula VIII and salts, isomers, stereoisomers, enantiomers, racemates, solvates, hydrates, polymorphs and prodrugs thereof:

wherein R is¹Is unsubstituted or (by R)⁶) Substituted aryl or heteroaryl; r²Selected from H, (C)₁-C₃) Alkyl, halogen, -CN, -O- (C)₁-C₃) Alkyl, -O- (CH)₂)_nX、-N(R⁷)(R⁸)、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX and-SO₂(C₁-C₃) An alkyl group; r³And R⁴Each independently is H, (C)₁-C₆) Alkyl or CN; r⁵Is- (C)₁-C₃) Alkyl or- (C) substituted by 1-3 fluorine₁-C₃) An alkyl group; r⁶Is H, OH, NH₂、(C₁-C₃) Alkyl, halogen, -CN, -O (C)₁-C₃) Alkyl, -O (CH)₂)_nX、-N(R⁷)(R⁸)、-CONH₂、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX or-SO₂(C₁-C₃) An alkyl group; x is OR⁹,N(R⁷)(R⁸)；R⁷And R⁸Each independently is hydrogen or (C)₁-C₄) Alkyl or (C)₁-C₄) Alkoxy radicalAnd may form a ring therebetween;

n is 2 or 3; and R is⁹Is H or (C)₁-C₃) An alkyl group. Also provided herein are metabolic precursors, such as esters or amides, of the compounds of formula VIII.

As a non-limiting example, compounds of formula VIII specifically include, but are not limited to, the compounds listed below:

1) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

2)2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) -N- (5- (tert-butyl) isoxazol-3-yl) acetamide;

3) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

4) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

5) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

6) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

7) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

8) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

9) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

10) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

11) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

12) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

13) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

14) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

15) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

16) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

17) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

18) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

19) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

20) n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide;

and pharmaceutically acceptable salts thereof. It will be appreciated that the ordinal numbers before the compound names in the above list can be used herein to identify the particular compound to which it corresponds. It should also be understood that the compounds exemplified in the above list are only representative of the invention and are not limiting in any way. Any of the compounds of the present disclosure are useful as tyrosine kinase inhibitors.

In the context of the present specification, an example of a compound of formula VIII may be referred to as compound 1 (i.e. N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide), Pz-1, Pz-01 or any combination of these terms.

One skilled in the art will recognize that any of the compounds described herein can form salts. By way of non-limiting example, the compounds are reacted with any number of organic or inorganic acids to form pharmaceutically acceptable acid addition salts.

Preparation method

In some embodiments, the compounds disclosed herein may be prepared according to the following schemes and examples. It will be appreciated that many additional methods of preparing the compounds are possible. For example, it will be appreciated by those skilled in the art that the introduction of certain substituents may result in asymmetry in the compound of formula VIII. The invention encompasses all enantiomers and mixtures of enantiomers, including racemates. Preferably, the compounds of the invention containing a chiral center are single enantiomers, but this is not required.

The compounds of the present disclosure can be prepared by various methods, some examples of which are in the schemes shown below. Those skilled in the art will recognize that the various steps in the following schemes may be varied to obtain compounds of formula VIII. The particular sequence of steps required to produce the compound of formula VIII depends on the relative lability of the particular compound synthesized, the starting compound, and the substituent group.

In some embodiments, the compound of formula VIII is synthesized by scheme I, shown below.

Scheme I

Scheme IStep a of (a) describes the nucleophilic addition reaction of an appropriately substituted aniline (2) to an activated fluoride compound (1). Para-bromo substitution is important for this reaction; it not only accelerates fluorine NH₂Substitution, but also bromine atom can form a carbon-carbon bond by Suzuki reaction in the obtained intermediate (5). The product may be isolated and purified by techniques well known in the art, such as precipitation, filtration, extraction, evaporation, chromatography, and recrystallization.

In step b of scheme I, some conditions apply to intermediate 3, yielding intermediate 4. Typically, the reaction is carried out in a suitable solvent, such as MeOH, EtOH or acid, to selectively react NO₂Reduction of the radical to NH₂Instead of reducing bromine, but alternative methods are possible. The reaction is carried out below room temperature (e.g. in an ice bath) to ambient temperature for 4-8 h. The product may be isolated and purified by the techniques described above.

Step c of scheme 1 describes the cyclization of intermediate 4 to give optionally substituted intermediate 5. Typically, suitable intermediate 4 is activated with a suitable acid (preferably pTSA) in a suitable solvent or neat TMOF at temperatures of 100 ℃ and above. The product may be isolated and purified by the techniques described above.

Step d of scheme I describes the palladium catalyzed coupling of intermediate 5 with boronic acid or tin derivative to give intermediate 6. Typically, the halide (especially bromine) of intermediate 5 is in a suitable catalyst (preferably Pd)₂(dba)₃) And a suitable base (e.g., potassium acetate) as a leaving group in a boronic acid or tin analog combination, for further synthesis of compounds of formula VIII (Suzuki reaction: see, e.g., Miyaura, N, et al Synth. Commun, 1981, 513-.

In some embodiments, the compound of formula VIII can be synthesized by scheme II shown below.

Scheme II

Step e of scheme II describes the synthesis of the boronate ester of intermediate 9 by methods well known in the art (Li et al, J. org. chem.,2002, 5394-5397). In the same manner as in step d, the halo-aryl or heteroaryl compound may be reacted with intermediate 9 in a suitable catalyst (e.g. Pd)₂(dba)₃) And a suitable base (e.g. potassium acetate) in the presence of a base to further synthesize the compound of formula VIII (Suzuki reaction: see, e.g., Miyaura, N, et al Synth. Commun, 1981, 513-. Compounds 17-20 (whose IUPAC names are specified in the above list) can be synthesized by scheme II, using the same 2 remaining steps as scheme I, steps e and f.

Pharmaceutical composition

The compounds disclosed herein can be incorporated into pharmaceutical compositions for the treatment of various diseases. In some embodiments, the salt of formula VIII is particularly useful in pharmaceutical compositions.

Pharmaceutical compositions may be formulated with any compound of formula VIII which is a tyrosine kinase inhibitor, in association with any conventional excipient, diluent or carrier. The compositions may be compressed into tablets or formulated as elixirs or solutions for convenient administration either orally or by intramuscular or intravenous routes. The compounds may be administered transdermally and may be formulated as sustained release dosage forms and the like.

The compounds, compositions, and formulations herein are useful for treating various diseases in animals (e.g., humans). The methods of treating human patients disclosed herein comprise administering an effective amount of a tyrosine kinase inhibitor or a pharmaceutical composition comprising a tyrosine kinase inhibitor. The tyrosine kinase inhibitors may be formulated as compositions which may be administered by the oral and rectal routes by topical, parenteral (e.g., by injection and by continuous or discontinuous intraarterial infusion) administration in the form of, for example, tablets, troches, sublingual tablets, sachets, cachets, elixirs, gels, suspensions, aerosols, ointments containing, for example, 1-10% by weight of the active compound in a suitable base, soft and hard gelatin capsules, suppositories, injectable solutions and suspensions in a physiologically acceptable medium, and sterile packaged powders for the preparation of the injectable solution adsorbed on a support material. For this purpose, the compositions may advantageously be presented in unit dosage form, preferably containing from about 5 to about 500mg (about 5 to about 50mg in the case of parenteral or inhalation administration; and from about 25 to about 500mg in the case of oral or rectal administration) of the compound per dosage unit. A daily dose of about 0.5 to about 300mg/kg, preferably 0.5 to 20mg/kg of active ingredient may be administered, although it will of course be readily understood that the amount of compound actually administered will be determined by the clinician in accordance with all relevant circumstances, including the condition to be treated, the choice of compound to be administered and the choice of route of administration. Thus, the dosage ranges discussed herein are not intended to limit the scope of the invention in any way.

Formulations for the single administration of a tyrosine kinase inhibitor will generally comprise at least one compound selected from compounds of formula VIII (which may be referred to herein as the active ingredient or substance) admixed with or diluted or encapsulated by a carrier or an ingestible carrier in the form of a capsule, sachet, cachet, paper or other container or encapsulated by a disposable container (e.g., an ampoule). The carrier or diluent may be a solid, semi-solid, or liquid material that serves as a vehicle, excipient, or medium for the active therapeutic substance. Some examples of diluents or carriers which may be used in the pharmaceutical compositions of the invention are lactose, dextrose, sucrose, sorbitol, mannitol, propylene glycol, liquid paraffin, white soft paraffin, kaolin, fumed silica, microcrystalline cellulose, calcium silicate, silicon dioxide, polyvinylpyrrolidone, cetostearyl alcohol, starch, modified starch, gum arabic, calcium phosphate, cocoa butter, ethoxylated esters, theobroma oil, peanut oil, alginate, tragacanth. Gelatin, syrup, methylcellulose, polyoxyethylene lauryl sorbitan, ethyl lactate, methyl and propyl hydroxybenzoates, sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol and propellants, such as trichlorofluoromethane, dichlorodifluoromethane and dichlorotetrafluoroethane. In the case of tablets, lubricants may be incorporated to prevent the powdered ingredient from sticking and binding to the die and punches of the tablet press. For such purposes, for example, aluminum, magnesium or calcium stearate, talc or mineral oil may be used.

In some embodiments, the pharmaceutical compositions of the present disclosure comprise an effective amount of a compound of formula VIII and/or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The term "drug" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or otherwise undesirable response when administered to an animal (e.g., a human). Methods for preparing Pharmaceutical compositions comprising at least one compound or additional active ingredient are known to those skilled in the art in light of this disclosure, exemplified by Remington's Pharmaceutical Sciences,2003, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, and general safety and purity Standards as required by FDA Office of biological Standards.

The compositions disclosed herein may contain different types of carriers depending on whether the administration is in solid, liquid or aerosol form and whether such routes are sterile for administration as an injection. The compositions disclosed herein can be administered intravenously, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosteally, periprosthetically, topically, intramuscularly, subcutaneously, by mucosal, intrauterine, orally, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion bathing target cells directly, by catheter, by lavage, with an emulsion, with a liquid composition (e.g., liposomes), or by other methods, or by a combination of any of the foregoing, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences,2003, incorporated herein by reference).

The actual dosage of the compositions disclosed herein for administration to an animal or human patient may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease to be treated, prior or concurrent therapeutic intervention, patient's characteristics, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and/or the number of administrations of the effective amount will vary depending on the response of the subject. The compounds of the present disclosure are generally effective over a wide dosage range. In any event, the medical personnel responsible for administration can determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

In some embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In further embodiments, for example, the active compound may comprise from about 2% to about 75% or from about 25% to about 60% of the unit weight and any range derived therein. Naturally, the amount of active compound in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in the unit dose specified for any compound. Such factors as solubility, bioavailability, biological half-life, route of administration, product life, and other pharmacological considerations should be of interest to one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens are contemplated.

In additional non-limiting examples, the dose can further comprise about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight per administration, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight to about 1000 mg/kg/body weight or any range derivable therein. In non-limiting examples from the derivable ranges set forth herein, based on the above values, about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, and the like, can be administered.

In some embodiments, the composition and/or additional agent administered by the digestive route may be formulated. The digestive route includes all possible routes of administration, wherein the composition is in direct contact with the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or an ingestable carrier which is meltable, or they may be enclosed in hard or soft gelatin capsules or they may be compressed into tablets or they may be incorporated directly into dietary foods.

In additional embodiments, the compositions described herein may be administered by a parenteral route. The term "parenteral" as used herein includes a route that bypasses the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered, for example, but not limited to, intravenously, transdermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally (U.S. Pat. nos. US6,753,514, US6,613,308, US5,466,468, US5,543,158, US5,641,515, and US5,399,363 each specifically incorporated herein by reference in their entirety).

Solutions of the compositions disclosed herein as free bases or pharmaceutically acceptable salts can be prepared with water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can be prepared with glycerol, liquid polyethylene glycols and mixtures thereof and with oils. Under normal conditions of storage and use, these formulations contain preservatives in order to prevent microbial growth. Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468 is specifically incorporated herein by reference in its entirety). In all cases, the dosage form should be sterile and fluid to the extent that ease of syringability is achieved. It should be stable under the conditions of preparation and storage and should be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like can be used to prevent the action of microorganisms. In many cases, it will be preferable to include isotonic agents, for example, but not limited to, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin.

For parenteral administration with an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, 1 dose is dissolved in 1mL of isotonic NaCl solution and added to 1000mL of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15 th edition, pages 1035- & 1038 & 1570- & 1580). Some variation in dosage will necessarily occur depending on the condition of the subject to be treated. The person responsible for administration can in any case determine the appropriate dosage for the individual subject.

Sterile injectable solutions are prepared by incorporating the composition in the required amount in the appropriate solvent with various additional ingredients enumerated above, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterile compositions into a sterile vehicle which contains a basic dispersion medium and the required additional ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition is combined with a liquid carrier, such as water or saline solution, with or without a stabilizer.

In further embodiments, the compositions are formulated for administration by a variety of different routes, such as topical (i.e., transdermal), mucosal (intranasal, vaginal, etc.), and/or inhalation.

Pharmaceutical compositions for topical administration may include compositions formulated for medical use, such as ointments, pastes, creams or powders. Ointments include all oily absorbent emulsions and compositions for topical application based on water solubility, while creams and lotions are those compositions that include only an emulsion base. Topically applied drugs may contain a penetration enhancer to facilitate absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Possible bases for compositions for topical application include polyethylene glycols, lanolin, cold cream and petrolatum as well as other suitable absorbent emulsion or water-soluble ointment bases. The topical formulation may also include emulsifiers, gelling agents, and antimicrobial preservatives (if necessary) to preserve the composition and provide a homogeneous mixture. Transdermal administration of the composition may also include the use of a "patch". For example, the patch may provide one or more compositions at a predetermined rate and continuously over a fixed period of time.

In some embodiments, eye drops, nasal sprays, inhalants, and/or other aerosol delivery vehicles may be provided to deliver the composition. Methods for delivering compositions directly to the lungs via nasal sprays are described in U.S. Pat. nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the use of intranasal microparticle resins (Takenaga et al, 1998) and lysophosphatidyl glycerol compounds (U.S. Pat. No. 5,5,725,871, specifically incorporated herein by reference in its entirety) for drug delivery is well known in the pharmaceutical arts and may be used to deliver the compositions described herein. Likewise, transmucosal delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety) and can be used to deliver the compositions described herein.

It is also contemplated that the compositions disclosed herein may be delivered by aerosol. The term aerosol refers to a colloidal system of fine solid powder or liquid particles dispersed in a liquefied or pressurized gaseous propellant. A typical aerosol formulation for inhalation consists of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers vary depending on the pressure requirements of the propellant. The aerosol administration may vary according to the age, weight and severity of symptoms and response of the subject.

Preferred pharmaceutical dosage forms of the invention are capsules, tablets, suppositories, injection solutions, creams and ointments, particularly preferred are preparations for inhalation, such as aerosols; formulations for injection and for oral ingestion.

Medicine box

It is also contemplated that a compound or composition disclosed herein may be packaged in the form of a kit comprising a plurality of containers. Many embodiments of such kits are possible. In some embodiments, the kit comprises a plurality of components for use in a method of making a tyrosine kinase inhibitor compound. In particular embodiments, the kit comprises a substituted aniline, an activated fluoride compound, one or more reducing agents, and a boronic acid or tin derivative. In another embodiment, the kit comprises a borate ester, a halo-aryl or heteroaryl compound, a catalyst, and optionally one or more reducing agents. In additional embodiments, the kit for preparing a pharmaceutical composition comprises a tyrosine kinase inhibitor (e.g., a compound of formula VIII) and a pharmaceutically acceptable carrier, diluent, or excipient. Many additional variations and embodiments of the kit are contemplated.

The kits typically also include instructions for using the components of the kit to practice the subject methods, but such instructions need not be included. The instructions for carrying out the subject methods are generally recorded on a suitable recording medium. For example. The instructions may be present in the kit as a package insert or on a label for the container of the kit or a component thereof. In further embodiments, the instructions reside as an electronic stored data file, which may be presented on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, or flash drive. In other embodiments, the exact instructions are not present in the kit, but rather provide a means for obtaining the instructions from a remote source (e.g., via the internet). An example of this embodiment is a kit comprising a website where the instructions may be visual and/or downloaded therefrom. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

Examples

Example 1 preparation of formula III

Ethyl 4-aminophenylacetate (3.67g,20.45mmol) was charged to a 20mL microwave vial along with 4-bromo-1-fluoro-2-nitrobenzene (3.00g,13.64mmol) and DMA (10 mL). The reaction mixture was sealed and placed in microwave irradiation at 160 ℃ for 30 minutes. Water was added to the crude reaction mixture and the product was extracted with EtOAc. The organic extracts were washed with brine 1x, acidified water (pH 4)2x, and brine 2 x. Collecting the organic layer, then using MgSO₄And (5) drying. The concentrated crude product was purified using flash chromatography with hexanes/EtOAc to give ethyl 2- (4- ((4-bromo-2-nitrophenyl) amino) phenyl) acetate 3 as a red-blood oil (4.2g, 81%). ESMS M/z379(M + H)⁺The structure of ethyl 2- (4- ((4-bromo-2-nitrophenyl) amino) phenyl) acetate (formula III) is shown below:

example 2 preparation of formula IV.

Ethyl 2- (4- ((4-bromo-2-nitrophenyl) amino) phenyl) acetate (2.026g,5.34mmol) was placed in a 100mL round bottom flask. Adding E into a flasktOH (20mL) and zinc (3.49g,53.4mmol), and the reaction mixture was then cooled with an ice bath. Acetic acid (2.246g,37.4mmol) was diluted with EtOH (10mL) and the reaction mixture was added dropwise over 1 h. The reaction mixture was stirred at 0 ℃ for 5h, then filtered and EtOH evaporated. With NaHCO₃The reaction mixture was basified with aqueous solution and the product was extracted with ether. With NaHCO₃The reaction mixture was washed 3 times with an aqueous solution, and an organic layer was collected. With MgSO₄The organic layer was dried and the solvent was evaporated to give ethyl 2- (4- ((2-amino-4-bromophenyl) amino) phenyl) acetate 4 as a pale purple solid (1.834g, 98%). ESMS M/z 349(M + H)⁺The structure of ethyl 2- (4- ((2-amino-4-bromophenyl) amino) phenyl) acetate (formula IV) is shown below:

example 3 preparation of 3-preparation of formula V.

Ethyl 2- (4- ((2-amino-4-bromophenyl) amino) phenyl) acetate (2g,5.73mmol), prepared as described above, was placed in a 50mL round bottom flask. TMOF (15mL) was added to the flask followed by pTSA (0.109g,0.573 mmol). The reaction mixture was stirred at RT for 3 h. Evaporating excess solvent and using NaHCO₃The reaction mixture was washed with an aqueous solution. The product was extracted with diethyl ether and NaHCO₃The organic layer was washed with aqueous 2x and 1x brine. Collecting organic layer, drying, and concentrating to obtain 2- (4- (5-bromo-1H-benzo [ d)]Imidazol-1-yl) phenyl) acetic acid ethyl ester 5 as a brown solid (2.04g, 99%). ESMSm/z 359(M + H)⁺2- (4- (5-bromo-1H-benzo [ d ]]Imidazol-1-yl) phenyl) ethyl acetate (formula V) has the following structure:

example 4 preparation of formula IX.

Reacting 2- (4- (5-bromo-1H-benzo [ d ]]Imidazol-1-yl) phenyl) acetic acid ethyl ester (4g,11.14mmol), bis (pinacolato) diboron (8.48g,33.4mmol) and KOAc (3.28g,33.4mmol) were dissolved in dioxane (100 mL). With N₂The reaction mixture was degassed for 10min and Pd was added₂(dba)₃(0.102g,0.111mmol) and P (Cy)₃(0.094g,334 mmol). Sealing the test tube at positive N₂Heat to 85 ℃ under pressure for 12h or until all starting material is consumed as determined by TLC and LC-MS. The solvent was evaporated and the product was adsorbed onto silica gel. Purifying the compound by flash chromatography to obtain 2- (4- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-benzo [ d [ -D)]Imidazol-1-yl) phenyl) acetic acid ethyl ester 9(4.09g, 86%). ESMS M/z 407(M + H)⁺¹.2- (4- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-benzo [ d ]]Imidazol-1-yl) phenyl) ethyl acetate the structure (formula IX) is shown below:

example 5 preparation of 5-preparation of formula VI.

Ethyl 2- (4- ((2-amino-4-bromophenyl) amino) phenyl) acetate (800mg,2.227mmol) was mixed with 4: 1 DMF/water (20mL) was placed together in a 30mL CEM microwave vial. A vial was charged with 1-methylpyrazole-4-boronic acid pinacol ester (556mg,2.67mmol) and Na₂CO₃(1,168mg,11.14 mmol). With N₂The reaction vessel was degassed for 10min, then Pd (dppf) Cl was added₂(91mg,0.111 mmol). The reaction mixture was heated at 130 ℃ for 20min with a microwave reactor and the solvent was evaporated. The reaction residue was adsorbed on silica gel and the product was purified by flash chromatography to give 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d]Imidazol-1-yl) phenyl) acetic acid ethyl ester 6(365mg, 45.5%). ESMS M/z 361(M + H)⁺¹.2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d)]Imidazol-1-yl) phenyl) ethyl acetate having the structure (formula VI) as followsThe following steps:

using the Suzuki coupling procedure as described in preparation 5 above, the intermediates described in table 1 below were obtained:

table 1.

Example 6 preparation of formula VII.

LiOH (72.5mg,3.03mmol) was reacted with H₂O (4mL) was added together to a 30mL CEM microwave vial and 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ]]Imidazol-1-yl) phenyl) acetic acid ethyl ester (365.3mg,1.014mmol) was dissolved in THF (4mL) and transferred to a microwave vial. The reaction mixture was heated in a microwave at 100 ℃ for 10min, the hydrolysis was verified by TLC and LC-MS to be complete, and all solvent was evaporated. By H₂The reaction mixture was redissolved and washed 1 time with DCM. Then concentrating the aqueous layer to obtainTo the compound 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d]Imidazol-1-yl) phenyl) lithium acetate 7(302mg, 90%). ESMS M/z 331(M-Li)^-1.2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d)]Imidazol-1-yl) phenyl) lithium acetate has the following structure (formula VII):

using a hydrolysis procedure as described in preparation 6 above, the intermediates listed in table 2 below were obtained:

TABLE 2

Example 7 preparation of formula VIII.

To 100mLAdding 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] into a round-bottom flask]Imidazol-1-yl) phenyl) lithium acetate (600mg,1.805mmol), EDC (700mg,4.51mmol), HOAt (246mg,1.805mmol), DIPEA (0.377mL,2.166mmol) and 5- (tert-butyl) isoxazol-3-amine (380mg,2.71 mmol). DMF (15mL) was then added to the reaction mixture and the flask was sealed under positive nitrogen pressure. Stirring was carried out at RT (. about.20-25 ℃ C.) for 12 h. At the 12h time point, the reaction mixture was purified using flash chromatography by TLC and LC-MS to verify that all starting material had been consumed. Collecting the product peak, and condensing to obtain N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d]Imidazol-1-yl) phenyl) acetamide 8(589.2mg, 71.8%). ESMS M/z 455(M + H)⁺¹. N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d]Imidazol-1-yl) phenyl) acetamide (compound 1: compounds of formula VIII) are shown below:

using the condensation procedure as described in example 1 above, the intermediates listed in table 3 below were obtained:

TABLE 3

Example 8-discovery of inhibitors of bidirectional RET and VEGFR2 kinases.

RET and VEGFR2 computer binding model creation

The RET proto-oncogene tyrosine kinase domain was crystallized with pyrazolopyrimidine PP1 and 4-anilinoquinazoline ZD6474 (vantanib). These compounds are type I inhibitors of protein kinases and have been shown to bind to RET kinase in intra-DFG conformations. The scaffold was determined to bind RET kinase (type II) in the DFG outer conformation by kinetic means based on incubation studies. A model for RET kinase outside the DFG was generated by removing residues SER 774-GLN 781 from the back of the allosteric pocket of the crystal structure of ZD 6474/RET. This refined DFG external RET kinase model was used for molecular docking. Discovery studio3.5visualizer, AutoDock Vina and AutoDock Tools are used for molecular modeling of potential small molecule inhibitors. The DFG external model was shown to accurately predict the binding pattern of type II inhibitors.

Drug discovery and development

Suitable screening assays for RET and VEGFR2 small molecule kinase inhibitors (from Kinase Directed Fragment (KDF) libraries) were established. RET kinase assay EZ Reader Electrophoresis Mobility chip instrument (Caliper Life Science) was used. In this assay, 2nM recombinant RET enzyme (Invitrogen) was incubated for 30min with small molecule inhibitors or control buffer toAllowing the type II kinase inhibitor to capture the conformation to which the inhibitor binds outside the DFG. Such inhibitors compete indirectly with ATP and bind slowly with kinetics (low k)_d) The inhibitor can be made to exhibit substantially non-competitive and substantially pseudo-irreversibility. Because of the noncompetitiveness of these inhibitors, a more biologically relevant concentration of ATP substrate (200 μ M) was incorporated in this assay. After 30min preincubation, a solution containing 200. mu.M ATP, 20mM MgCl was added₂And 1.5. mu.M of a fluorescently labeled RET substrate peptide (Caliper LS, peptide 22; Perkin Elmer, USA). The level of conversion of RET substrate peptide was then determined using an EZ Reader instrument. Similarly, VEGFR2 and RET/V804M kinase assays were also established using the same peptide substrates.

Because of the high homology of ATP active sites of RET and VEGFR2, screening and optimization was mainly focused on RET. VEGFR2 activity was assayed with only potent RET inhibitors in order to determine selectivity, and imidazole analogs were identified by fragment (and/or specially treated structures, molecular weight: less than 300) screening using ATP concentrations of 200 μ M to avoid non-specific binding and false positives. Homology modeling of RET kinases with the VEGFR-2 crystal structure known to determine the DFG pocket of RET kinases facilitates the addition of lipophilic groups (e.g., meta-trifluoromethylphenyl groups). The combination of structure-activity correlation (SAR) information with further optimization of the DFG pocket and substitution at position 5 of benzimidazole yielded the clinical candidate Pz-1(N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d [ -d ] ] -]Imidazol-1-yl) phenyl) acetamide, which is active in biochemical assays and IC of RET wild type, gatekeeper mutant RETV804M, and VEGFR2₅₀Less than 1 nM. The actual potency of Pz-1 exceeds the measurable limits of biochemical assays.

The activity of pRET and pVEGFR2 was determined in a cell-based assay, and Pz-1 inhibited both phosphate biomarkers equivalently. The inhibitory activity of auto-RET phosphorylation (in TT cell line) and auto-VEGFR 2 phosphorylation (in HEK293 cell line) as shown in figure 3 and figure 4, respectively, showed strong correlation with the biochemical assays of RET and VEGFR 2. The clinical candidate Pz-1 inhibited 90% RET and VEGFR2 autophosphorylation at nanomolar concentrations in all cell lines and mutations tested (fig. 1-4).

Mechanism of action and overall kinase selectivity

Pz-1 was particularly effective at less than 1nM for RET and all tested RET mutations, including gatekeeper mutant RET/V804M (FIGS. 1-3). The RET biochemical assay described herein is not suitable for determining IC based on less than 1nM₅₀Because the concentration of RET enzyme in this assay is 2 nM. Thus, the intermediate compounds were used for kinetic studies. Extension of incubation time from 5min to 20min or 60min resulted in IC₅₀A 3-fold increase (100nM to 30nM), indicating a conformational change from inside to outside of DFG, a standard feature of type II kinase inhibitors. Screening for 91 kinases in KINOMEscan with Pz-1 at 50nM concentration represented the diversity of each Kinome cluster. The kinase assay in kinosscan is more sensitive than the biochemical assay described above. Although there was-90% inhibition of 5 kinases (TRKB, TRKC, GKA, FYN and SRC), Pz-1 was still significantly selective for these kinases (strongly inhibited pRET at1 nM) based on the available cellular data, indicating good overall Kinomie selectivity.

Inhibition of pRET and pVEGFR2 activity by Pz-1 in cell-based assays

Inhibition of RET autophosphorylation by Pz-1 was determined in several different cell lines, including NIH3T3 fibroblasts expressing RET/C634Y or RET/M918T, TT expressing RET/C634W (MTC), nth-ory-3-1 (thyroid, untransformed with RET; negative control), HEK293/RET/PTC 1(CCDC6-RET which can be found in PTC & lung cancer), HEK293/RET-KIF5B (which can be found in lung cancer), HEK293/RET/PTC3(nc 4-RET, which can be found in PTC & lung cancer) and HEK293 transfected with RET/C634R-V804M (fig. 1-3), HEK293 cells were transiently transfected with expression vectors containing the desired mutants. Cells were serum starved for 12hrs post transfection at 36hrs, and then treated with Pz-1 at the indicated concentration for 2hrs, and total cell lysates were subjected to immunoblotting for testing anti-phospho-Y1062 (α p1062) and anti-phospho-Y905 (α p905) RET antibodies. Blots were normalized using anti-RET (α RET) antibodies. Pz-1 strongly inhibited pRET at 1nM in all cell lines (FIGS. 1-3), including cells expressing RET/C634R-V804M. Pz-1 also strongly inhibited VEGFR2 autophosphorylation in HEK293 cells transiently transfected with VEGFR2 and stimulated with VEGF at 1nM (FIG. 4). pMAPK is a downstream marker of the signal that many kinases pass through the RAS/MEK/ERK pathway. In TT cells containing RET mutants (fig. 3), pMAPK and pRET were similarly strongly inhibited at1 nM. In contrast, pMAPK was inhibited only at 100nM in the thyroid cell line (Nthy-ory-3-1, untransformed with the oncogene RET) (FIG. 3), confirming the overall kineme selectivity for Pz-1.

Antiproliferative effects of Pz-1 in RET-driven cancer cell lines

Nthy-ory-3-1 and TT cells were seeded on 60-mm dishes. Cells were maintained in either 2% (Nthy-ori-3-1) or 10% (TT) fetal calf serum. Different concentrations of Pz-1 or vehicle were added to the medium 1 day after plating and changed every 2-3 days. Cells were counted every 2-3 days. Ba/F3 cell proliferation is dependent on IL3 and this dependence is lost upon expression of constitutively active kinases such as RET. Ba/F3 expressing the parent and RET/PTC was maintained in 10% fetal calf serum in 6-well plates and counted every other day for 4 consecutive days, with media changed every 2 days. To compare cell growth, unpaired student's t-test was performed using the Instat Software program (Graphpad Software Inc). All p values are double-sided. IC calculation by Curve fitting analysis, PRISM Software program (Graphpad Software Inc)₅₀And (4) dosage. Pz-1 inhibits RET-driven cancer cell proliferation, wherein IC on TT cells₅₀1.86nM (FIG. 5), 907pM for LC-2/ad cells (FIG. 5) and 340pM for RET/PTC3 transfected Ba/F3 cells (FIG. 6). The selectivity was confirmed by not significantly reducing the growth of parental NIH3T3 fibroblasts until 1000nM Pz-1 when used for 3 days.

In Vivo Target Inhibition (IVTI) assay and potency model for pRET and pVEGFR2

Mixing TT cells (7.5x 10)⁶Mice) were inoculated subcutaneously into the dorsal part (bilateral) of 39 SCID mice (Jackson Laboratories, Bar Harbor, Maine).After 5 weeks, at least one tumor was present in each mouse; tumor-bearing mice were randomly grouped to receive Pz-1(0.3, 1.0 or 3.0mg/kg daily) (29 mice, 52 tumors) or vehicle (10 mice, 18 tumors) by oral gavage. Treatment was applied for 28 consecutive days. Tumor diameter was measured weekly using calipers. Tumor volume (V) was calculated by the rotational ellipsoid formula: v is AxB²And/2 (a ═ shaft diameter; B ═ rotation diameter) and reported as mean volume ± standard deviation. Pz-1 strongly inhibited tumor growth at all doses tested (FIG. 7). The anti-tumorigenic activity of Pz-1 was also evaluated in nude mice implanted with RET/C634Y or HRAS (G12V) transformed NIH3T3 fibroblasts. In this case, NIH3T3RET/C634Y or NIH3T3HRAS/G12V cells were inoculated subcutaneously into the dorsal part (bilateral) of BALB/C nu/nu mice (n.31 mice/cell line) (Jackson Laboratories, Bar Harbor, Maine). After 4 days, animals were randomized to receive Pz-1(1.0, 3.0, or 10mg/kg daily) by oral gavage (23 mice/cell line: 8 mice/group, 1.0 and 3.0mg/kg daily dose, and 7 mice 10dose mg/kg daily) or vehicle control group (8 mice) before tumor emergence. Tumor diameters were measured every 1-2 days using calipers. Tumor volume (V) was calculated by the rotational ellipsoid formula: v is AxB²And/2 (a ═ shaft diameter; B ═ rotation diameter) and reported as mean volume ± standard deviation. Although the treatment prevented RET cell-induced tumor formation overall, it only reduced, rather than eradicated, HRAS oncogene-driven tumor formation (fig. 8).

At the end of the tumor growth experiment reported in figure 8, some of the vehicle-treated tumors were tested with different doses of Pz-1(1.0 or 3.0mg/kg daily) for 48hr or remained untreated. At the end of the treatment, protein lysates were immunoblotted with anti-phospho-Y1062 (α p1062) and anti-phospho-905 (α p905) RET antibodies, anti-phospho-MAPK (α pMAPK, T302/Y304), anti-phospho-SHC (α pSHC, Y317), anti-phospho-p 70S6K (α pp70S6K, T389), anti-phospho-S6 RP (α pS6RP, S235/S236) and anti-phospho-VEGFR 2(α pvfr 2, pY1175) antibodies. Pz-1 treatment inhibited pvgfr 2 in RET/C634Y-and HRAS/G12V-induced tumors, and inhibited RET phosphorylation and intracellular signaling (SHC, MAPK, p70S6K and S6RP) only in RET/C634Y-induced tumors (fig. 9).

Preclinical formulation, PK, PD and toxicology of Pz-1

To perform PK studies of Pz-1 in animals, solution formulations in 500 μ L polysorbate 80, 500 μ L EtOH and 1.0mL pH 2 buffer were generated for oral administration. For toxicity studies, suspension formulations in tween 20 (20%) and xanthan gum (0.125%) were generated.

Pz-1 has an AUC (solution formulation) of 6.5. mu.g/hr/mL and an elimination half-life of 3.8h at 2mg/kg in mice. In an oral PK study in rats, Pz-1 showed a high bioavailability of 97% at 10mg/kg (oral) with an AUC of 19. mu.g/hr/mL and an AUC of 1.9. mu.g/hr/mL at 1mg/kg (IV). The elimination half-life in the oral study was 4.2h, clearance was 9mL/min/kg, and the volume distribution was 2.02L/kg. T in rats_1/2(3-4hrs) with T in mice_1/2Consistent (4.2 hrs).

In vitro, Pz-1 is in a cell comprising RET^C634WIs antiproliferative in MTC TT cell lines, IC₅₀1.86nM and antiproliferative effect on Ba/F3 cells transfected with oncogene RET (RET/PTC3), IC₅₀It was 0.34 nM. Pz-1 has minimal activity in the patch clamp hERG assay, where IC of hERG assay₅₀At 10. mu.M (GenScript). The selectivity for RET was 10,000-fold over hERG, indicating clear cardiovascular toxicity profiles. Pz-1 vs CYP2D6 (IC)₅₀13.8. mu.M) and CYP3A4 (IC)₅₀7.4 μ M) had minimal activity, indicating that there is no drug-drug interaction problem with the two major CYP isoforms.

In a 1 week-long toxicology study in mice using daily dose escalation of 10 mg/kg-100 mg/kg suspension formulations, there was no significant toxicity. Organ pathology analysis in heart, kidney and liver showed no adverse reactions at the 100mg/kg dose. Similarly, toxicity markers were unaffected at any dose, including phosphorus, creatinine, total bilirubin, GGT, ALP, glucose, total protein, albumin, globulin, calcium, cholesterol, and BUN. The hepatotoxicity marker ALT increased proportionally from 22U/L to 51U/L at 30mg/kg and 96U/L at 100mg/kg in serum (blood serum) at 10 mg/kg. At the highest dose, ALT concentrations were slightly above normal 80U/L ALT levels, but below the toxicity threshold of 200U/L. ALT levels are easy to monitor and can be used as a reversible toxicity marker in a clinical setting.

Example 9-method.

RET biochemical assay

RET kinase assay uses a microfluidics based instrument for direct kinase activity readout (Caliper life science). In this assay, 1-2nM recombinant RET enzyme (Invitrogen) was preincubated for 30min with small molecule inhibitors or control buffer to allow the inhibitors to capture the DFG outer conformation. Such inhibitors compete indirectly with ATP and have slow binding kinetics (very slow k)_off) The inhibitor can be made to exhibit substantially non-competitive and substantially pseudo-irreversibility. Because of the noncompetitiveness of these inhibitors, a more biologically relevant concentration of ATP substrate was incorporated in this assay. After 30min pre-incubation, a substrate mixture comprising 180-200. mu.M ATP and 1.0-1.5. mu.M fluorescently labeled RET substrate peptide (Caliper LS, peptide 22; Perkin Elmer, USA) was added. The level of conversion of RET substrate peptide was then determined using an EZ Reader instrument. The final test concentrations of the buffer components were as follows: 50mM HEPES, 0.075% (v/v) Brij-35, 0.10% (v/v) polysorbate 20, 0.02% (w/v) NaN₃，10mM MgCl₂And 2mM DDT. All exemplary compounds were shown to have IC₅₀Value of<1 μ M RET kinase domain.

VEGFR2 Biochemical assays

The VEGFR-2 assay was identical to the RET assay, but the RET was replaced with 1-2nM recombinant VEGFR-2. All exemplary compounds were shown to have IC₅₀Value of<VEGFR2 kinase domain at 1. mu.M value.

FLT3 Biochemical assay

The FLT3 assay was identical to the RET assay, but replacing RET with 1-2nM recombinant FLT 3. In addition, peptide 22 was replaced with 1.0-1.5. mu.M fluorescently labeled FLT3 substrate peptide (Caliper LS, peptide 2; Perkin Elmer, USA). All exemplary compounds were shown to have IC₅₀Value of<FLT3 kinase domain at 1. mu.M value.

Cell culture assay

RET oncogene-transformed fibroblasts were cultured in DMEM medium (GIBCO, Paisley, Pa.) with 5% calf serum (NIH3T3) or 10% fetal bovine serum (RAT1), 2mM L-glutamine and 100 units/ml penicillin-streptomycin. HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2mM L-glutamine and 100 units/ml penicillin-streptomycin (GIBCO, Paisley, Pa.). Transient transfections were performed with pcDNA-RET/C634R-V804M, pBABE-RET/PTC1(CCDC6-RET), -RET/PTC3(NCOA4-RET) or-KIF 5B/RET vectors using lipofectamine reagent according to the manufacturer's instructions (GIBCO). The RET constructs used for transfection of fibroblasts and HEK293 encode a short isoform of the RET protein (RET-9). Parental Ba/F3 and Ba/F3 cells stably expressing RET/PTC3 were cultured in RPMI (GIBCO, Paisley, Pa.) supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 100 units/ml penicillin-streptomycin. Ba/F3 cells stably expressing RET/PTC3 protein were generated by transfecting the long isoform RET/PTC3(RET-51) with electroporation. Parental cells were grown in the presence of 10ng/mL IL 3. Nthy-ORI-3-1(NTHY-ORI) cell lines derived from normal thyroid follicular tissue and immortalized with SV40Large T were grown in RPMI supplemented with 10% fetal bovine serum, 2mM L-glutamine and 100 units/ml penicillin-streptomycin. TT cell lines derived from human MTC (Carlomagno,1995) containing the RET/C634W mutation were cultured in RPMI (GIBCO, Paisley, Pa.) with 20% calf serum, 2mM L-glutamine and 100 units/ml penicillin-streptomycin.

Immunoblotting and growth Curve assays

Protein lysates were prepared according to standard methods. Briefly, cells were lysed with a buffer containing 50mM N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES; pH 7.5), 1% (vol/vol) Triton X-100, 150mM NaCl, 5mM EGTA, 50mM NaF, 20mM sodium pyrophosphate, 1mM sodium orthovanadate, 2mM phenylmethylsulfonyl fluoride (PMSF) and 1. mu.g/mL aprotinin. Lysates were purified by centrifugation at 10,000Xg for 15 min. Lysates containing comparable amounts of protein, evaluated by a modified Bradford assay (Bio-Rad, Munich, Germany), were subjected to direct Western blotting. The immune complexes were detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfort, UK). anti-phospho-Shc (# Y317), which recognizes the SHC protein when phosphorylated on Y317, is from Upstate Biotechnology Inc. (Lake Placid, NY). anti-Shc (H-108) was from Santa Cruz Biotechnology (Santa Cruz, Calif.). anti-MAPK (#9101) and anti-phospho-MAPK (#9102) specific for p44/42MAPK (ERK1/2) phosphorylated on Thr202/Tyr204 were from Cell Signaling Technologies (Danvers, MA, USA). anti-phospho-VEGFR-2/KDR (#2479) and anti-VEGFR-2/KDR (#2478) with specificity for VEGFR2/KDR phosphorylated on Tyr1175 are from Cell Signaling Technologies (Danvers, MA, USA). anti-phospho-S6 ribosomal protein (#2211), anti-S6 ribosomal protein (#2217), anti-p 70S6 kinase (p70S6K) (#2708), and anti-phospho p70S6 kinase (T389) (#9234) specific for the S6 ribosomal protein phosphorylated at Ser235-236 are from Cell Signaling technologies (Danvers, MA, USA). anti-RET is a polyclonal antibody directed against the tyrosine kinase protein fragment of human RET (Santoro, 1995). Anti-phospho 905 is a phospho-specific polyclonal antibody that recognizes RET protein phosphorylated on Y905. Anti-phospho 1062 is a phospho-specific polyclonal antibody that recognizes the RET protein phosphorylated on Y1062. Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology.

IC₅₀Value of

For these examples, dose response curves and ICs were fitted using curve fitting PRISM Software (GraphPad Software)₅₀And (6) dose charting. Unpaired student-t test (inst program, GraphPad software) was used to compare tumor growth. P value is in P<Was statistically significant at time 05.

Some embodiments of the compounds and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating specific embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various uses and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the true scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.

Claims (52)

1. A compound of formula VIII and salts, isomers, stereoisomers, enantiomers, racemates, solvates, hydrates, polymorphs and prodrugs thereof,

wherein:

R¹is unsubstituted or (by R)⁶) Substituted aryl or heteroaryl;

R²selected from H,(C₁-C₃) Alkyl, halogen, -CN, -O- (C)₁-C₃) Alkyl, -O- (CH)₂)_nX、-N(R⁷)(R⁸)、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX and-SO₂(C₁-C₃) An alkyl group;

R³and R⁴Each independently is H, (C)₁-C₆) Alkyl or CN;

R⁵is- (C)₁-C₃) Alkyl or- (C) substituted by 1-3 fluorine₁-C₃) An alkyl group;

R⁶is H, OH, NH₂、(C₁-C₃) Alkyl, halogen, -CN, -O (C)₁-C₃) Alkyl, -O (CH)₂)nX、-N(R⁷)(R⁸)、-CONH₂、-CONH(CH₂)_nX、-SO₂NH(CH₂)_nX or-SO₂(C₁-C₃) An alkyl group; x is OR⁹,N(R⁷)(R⁸)；

R⁷And R⁸Each independently is hydrogen or (C)₁-C₄) Alkyl or (C)₁-C₄) Alkoxy groups, and may form a ring therebetween; n is 2 or 3; and is

R⁹Is H or (C)₁-C₃) An alkyl group.

2. The compound of claim 1, wherein the compound is selected from the group consisting of: n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1); 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) -N- (5- (tert-butyl) isoxazol-3-yl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; and N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide.

3. The compound of claim 1, wherein the compound consists essentially of N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1).

4. A method of making a tyrosine kinase inhibitor comprising:

reacting a substituted aniline with an activated fluoride compound in a nucleophilic addition reaction to produce an addition product;

selectively reducing said addition product to produce a first reduction product;

cyclizing the reduced product to produce a cyclized intermediate;

coupling the cyclized intermediate with a boronic acid or tin derivative to produce an ester; and

reducing the ester to produce a second reduction product; and

ammoniating the second reduction product to produce the tyrosine kinase inhibitor.

5. The method of claim 4, wherein the substituted aniline comprises bromine in the para position.

6. The process of claim 4, wherein the addition product comprises ethyl 2- (4- ((4-bromo-2-nitrophenyl) amino) phenyl) acetate.

7. The method of claim 4, wherein selectively reducing comprises reducing NO₂Reduction of the radical to NH₂Groups, but not bromine.

8. The process of claim 4, wherein the first reduction product comprises 2- (4- ((2-amino-4-bromophenyl) amino) phenyl) acetate.

9. The process of claim 4, wherein said cyclizing comprises activating the first reduction product using an acid.

10. The method of claim 9, wherein the acid comprises pTSA.

11. The process of claim 4 wherein said cyclized intermediate comprises ethyl 2- (4- (5-bromo-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

12. The method of claim 4, wherein the coupling is palladium catalyzed.

13. The process of claim 4, wherein bromine is a leaving group in the coupling step.

14. The method of claim 4, wherein the coupling step comprises a Suzuki coupling.

15. The method of claim 4, wherein the ester is selected from the group consisting of: ethyl 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and ethyl 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

16. The method of claim 4, wherein the ester consists essentially of ethyl 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

17. The method of claim 4, wherein the second reduction product comprises a compound selected from the group consisting of: 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; lithium 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and lithium 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

18. The process of claim 4, wherein the second reduction product consists essentially of lithium 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

19. The method of claim 4, wherein the tyrosine kinase inhibitor is selected from the group consisting of: n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1); 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) -N- (5- (tert-butyl) isoxazol-3-yl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; and N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide.

20. The method of claim 4 wherein the tyrosine kinase inhibitor consists essentially of N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1).

21. A method of making a tyrosine kinase inhibitor, the method comprising:

reacting a boronic ester with a halo-aryl or heteroaryl in the presence of a catalyst and a base to produce an intermediate;

coupling said intermediate with a boronic acid or tin derivative to produce a tyrosine kinase inhibitor precursor; and

reducing and aminating the tyrosine kinase inhibitor precursor to produce the tyrosine kinase inhibitor.

22. The method of claim 21, wherein the base comprises potassium acetate.

23. The method of claim 21, wherein the halo-aryl or heteroaryl comprises pyrrolyl, pyrazolyl, pyranyl, thiopyranyl, furanyl, imidazolyl, pyridyl, thiazolyl, triazinyl, phthalimidyl, indolyl, purinyl, benzothiazolyl, or a combination thereof.

24. The method of claim 21, wherein said halo-aryl or heteroaryl comprises a heterocyclyl residue selected from the group consisting of oxiranyl, aziridinyl, 1, 2-oxathiacyclopentyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuryl, benzofuryl, chromenyl, 2H-pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, (S-oxo or S, S-dioxo) -thiomorpholinyl, indolizinyl, isoindolyl, 3H-indolyl, Indolyl, benzimidazolyl, coumarinyl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothienyl, dibenzothienyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, β -carbolinyl, phenanthridinyl, acridinyl, peridinaphthenyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochroman, and chromanyl.

25. The process of claim 21 wherein said intermediate comprises ethyl 2- (4- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

26. The method of claim 21, wherein the tyrosine kinase inhibitor precursor is selected from the group consisting of: ethyl 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; ethyl 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and ethyl 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

27. The method of claim 21, wherein the tyrosine kinase inhibitor precursor consists essentially of ethyl 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

28. The method of claim 21, wherein the tyrosine kinase inhibitor precursor is reduced to a reduction product comprising a compound selected from the group consisting of: 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; lithium 2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; lithium 2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetic acid lithium; 2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; 2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate; and lithium 2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

29. The method of claim 21, wherein the tyrosine kinase inhibitor precursor is reduced to a reduction product consisting essentially of lithium 2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetate.

30. The method of claim 21, wherein the tyrosine kinase inhibitor is selected from the group consisting of: n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1); 2- (4- (5- (1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) -N- (5- (tert-butyl) isoxazol-3-yl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 3-dimethylphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dimethoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (thiophen-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-difluorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2, 4-dichlorophenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylthio) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxyphenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-fluoropyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (2-methoxypyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-morpholinopyridin-3-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (4- (methylsulfonyl) phenyl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyrimidin-5-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (pyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; n- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (6-methylpyridin-2-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide; and N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5-phenyl-1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide.

31. The method of claim 21, wherein the tyrosine kinase inhibitor consists essentially of N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1).

32. The product of the process of any one of claims 4-31.

33. A pharmaceutical composition comprising:

a compound of claim 1; and

a pharmaceutically acceptable carrier, diluent or excipient.

34. The pharmaceutical composition of claim 33, wherein said compound consists essentially of N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1), or a pharmaceutically acceptable salt thereof.

35. A method of treating a subject having cancer, the method comprising administering to a subject in need thereof an effective dose of the pharmaceutical composition of claim 33.

36. The method of claim 35, wherein the pharmaceutical composition is administered in combination with any another anticancer drug.

37. The method of claim 35, wherein the cancer is selected from thyroid cancer, lung cancer or breast cancer.

38. A method of inhibiting RET phosphorylation comprising treating cells expressing RET gene with an effective amount of a compound of claim 1.

39. The method of claim 38, wherein the compound comprises N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H-benzo [ d ] imidazol-1-yl) phenyl) acetamide (Pz-1).

40. A method of inhibiting phosphorylation of VEGFR2/KDR, comprising treating a cell expressing a VEGFR protein with an effective amount of the compound of claim 1.

41. A method of inhibiting proliferation of thyroid cancer cells comprising treating thyroid cancer cells with an effective amount of a compound of claim 1.

42. The method of claim 41, wherein the thyroid cancer cell comprises MTC.

43. A method of inhibiting tyrosine kinase activity comprising treating a cell with an effective amount of a compound of claim 1.

44. The method of claim 43, wherein said tyrosine kinase is selected from the group consisting of RET, Trk-A, Trk-B, Trk-C, FLT3-ITD, c-Kit, VEGFR, and PDGFR.

45. The method of claim 43, wherein said compound exhibits an IC₅₀Value of<Inhibitory activity against the kinase domain at 1. mu.M.

46. A method of treating pain associated with cancer comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 33.

47. A kit for preparing a tyrosine kinase inhibitor comprising:

a first vessel comprising a substituted aniline; and

a second vessel containing an activated fluoride compound.

48. The kit of claim 47, further comprising one or more reducing agents.

49. The kit of claim 47, further comprising one or more of: boric acid or tin derivatives.

50. A kit for preparing a tyrosine kinase inhibitor comprising:

a first container comprising a borate ester;

a second container comprising a halo-aryl or heteroaryl compound; and

a catalyst.

51. The kit of claim 50, further comprising one or more reducing agents.

52. A kit for preparing a pharmaceutical composition comprising:

a first container comprising the compound of claim 1; and

a second container comprising a pharmaceutically acceptable carrier, diluent or excipient.