HK40005718A

HK40005718A - Compounds for treatment of cancer

Info

Publication number: HK40005718A
Application number: HK19129078.2A
Authority: HK
Inventors: J·王; 陈建军; D·D.·米勒; 李伟
Original assignee: 田纳西大学研究基金会
Priority date: 2013-03-05
Filing date: 2019-09-03
Publication date: 2020-05-15

Description

Compounds for the treatment of cancer

This application is a divisional application of chinese patent application 201480025267.4 entitled "compound for treating cancer" filed 3/5/2014.

Technical Field

The present invention relates to pharmaceutical compositions comprising BRAF inhibitors (e.g. vemurafenib) and/or MEK inhibitors (e.g. trametinib, RO5068760) and the anti-tubulin compounds of the invention or other known tubulin inhibitors for the treatment of cancer and the use of such compositions for the treatment of cancers such as melanoma, drug resistant cancers and cancer metastasis.

Background

Cancer is the second most common cause of death in the united states, second only to heart disease. Cancer accounts for one-fourth of the causes of death in the united states. The 5-year relative survival rate for all patients diagnosed with Cancer was 66% between 1996 and 2003, and increased over 50% between 1975 and 1977 (Cancer Facts & Figures American Cancer Society: Atlanta, GA (2008)). The new cancer case rate drops on average by 0.6% per year in men and remains unchanged in women from 2000 to 2009. From 2000 to 2009, the overall mortality rate for all cancers decreased by an average of 1.8% per year for men and 1.4% per year for women. This improvement in survival reflects advances in early diagnosis and improvement in treatment. The discovery of highly potent and low-toxic anticancer agents is a major goal of cancer research.

Microtubules are cytoskeletal filaments composed of αβ -tubulin heterodimers and are involved in a wide range of cellular functions including shape maintenance, vesicle transport, cell motility and division tubulin is the major structural component of microtubules and is a well-documented target for a variety of very successful anticancer drugs.

Unfortunately, there are two major problems in common with the clinically used microtubule-interacting anticancer drugs: drug resistance and neurotoxicity.

Malignant melanoma is the most dangerous form of skin cancer, accounting for approximately 75% of deaths from skin cancer. The incidence of melanoma continues to increase in the population in western countries. The number of cases doubled in the last 20 years. Approximately 160,000 new melanoma cases are diagnosed worldwide each year and are more common in men and caucasians. According to WHO reports, about 48,000 melanoma-associated deaths occur worldwide each year.

Currently, there is no effective method for treating advanced/metastatic melanoma. It is very resistant to current chemotherapy, radiotherapy and immunotherapy. The prognosis for advanced/metastatic melanoma is poor, with a median survival rate of 6 months and a 5-year survival rate of less than 5%.

Various centers use a variety of chemotherapeutic agents, including dacarbazine (also known as DTIC), immunotherapy (with interleukin-2 (IL-2) or Interferon (IFN)), and local perfusion. The overall success of metastatic melanoma is very limited. In 20 years, IL-2 (aldesleukin) was the first approved new therapy for the treatment of metastatic melanoma. However, patients with complete remission are only less than 5%. In recent years, great efforts have been made to try to combat advanced melanoma. Whether DTIC or otherwiseNeither combination of chemotherapeutic drugs (e.g., cisplatin, vinblastine, and carmustine) nor addition of interferon- α 2b to DTIC demonstrated a survival benefit over treatment with DTIC aloneIs a drug that uses the immune system of the patient to combat melanoma. The use of an monopigma xylostella antibody treats advanced melanoma that has spread beyond its original location. Targeted therapies use drugs designed to target specific weaknesses in cancer cells.

BRAF mutations are found in about 60% of melanoma patients, and FDA-approved BRAF inhibitors (BRAFi; e.g., Verofenib and Dalafinib (GSK2118436)) and MEK inhibitors (MEKi; e.g., trametinib (GSK1120212), RO5068760) have been found in BRAF^V600A dramatic clinical response was shown in the treatment of mutant melanoma. The prior use of the BRAFi + MEKi combination was very effective during initial therapy, but due to tumor heterogeneity and activation of the alternative pathway, drug resistance developed within about 9 months, leading to patient disease relapse and death.

VerofiniIs a targeted therapy approved for the treatment of advanced melanoma that cannot be treated surgically or melanoma that has spread throughout the body. In the case of melanoma, vemurafenib only treats patients with certain genetic mutations (BRAF)^V600) The tumor of (2). Likewise, vemurafenib and other BRAF inhibitors may be effective in a variety of BRAF mutant cancers. Examples where B-RAF is mutated at a high frequency include melanoma (30-60%), thyroid cancer (30-50%), colorectal cancer (5-20%), ovarian cancer (about 30%) and other cancers (1-3%) (Wellbrock C, Karasarides M, Marais R. "The RafProtein Takes Centre Stage". Nat. Rev. (2004) 5: 875-885).

Virofenib inSuffering from BRAF^V600The sustained clinical activity in patients with mutant melanomas is limited by the rapid progression of acquired resistance (Lee JT, Li L, Brafford PA et al, "PLX 4032, a patent inhibitor of organic phase, B-Raf V600E oncogene, selective inhibitors of V600E-porous melanemes." Pigmentcell Melanoma Res. (2010) 23: 820. tans H, Higgins B, Kolinsky K et al "RG 7204(PLX4032), a selective BRAFV600E inhibitor of display inhibitor of organic activity in the representative melanemes.". Cancer modification ". tans. (2010) 70: 5518 5527; Yangh, Higg B, antibody K et al" BRA activity of organic phase, branched antibody of molecular phase 779 ". 3. tumor model of Cancer 779"). The mechanism of drug Resistance progression has been extensively studied (Little AS, Smith PD, Cook SJ., "Mechanisms of acquired Resistance to ERK1/2path inhibition". Oncogene (2013)32 (10): 1207-). 1215,. Bollag G, Hirth P, Tsai J et al, "Clinical efficacy of aAF inhibition novel bound target block in BRAF-mutant membrane". Nature (2010) 467: 596-599; Flaherty KT., "Targeting strategy pathway". Annu Re2012 (63: 171-). 183. WD. waiting Q et al, "Resistance pathway modification". 97: flow 968). A number of different mechanisms have been proposed in the literature, including intrinsic resistance to BRAFi, amplification of BRAF Cancer groups (Shi H, Moriceau G, Kong X et al, "Melanoma white-isomer sequence identifiers (V600E) B-RAF amplification-mediated acquisition B-RAF inhibition response," Nat. Commun. (2012) 3: 724), upregulation or activation mutations of downstream MEK kinases, upregulation of CRAF expression (Montagut C, Sharma SV, Shioda T et al, "expressed F as a particulate protein kinase a structural inhibition of acquired inhibition response to F inhibition in cell inhibition in tumor". Cancer Res. (68: 4853), upregulation of NRR inhibition genes (S4853) S "" (S11: S-S11: 11), activation of NRF inhibition genes (R11: S-S.) (R7: M-P11) and the pathways of RNA inhibition of Cancer genes (R-S11. RTK-P + 7. RTK-M-P-M-11. RTK-P-3. RTK-P-11. RTK-P-K-11. RTK-11, Sanchez-Laoden B et al"Cancer disorders" (2013)3(2) of the neighbor EFG receiver or SRC family kinase signaling and signaling in melanem: 158-: 35ra 41; balzano D, Santaguida S, Musacchio a, Villa f. "a genetic frame for inhibition of resistance in protein kinases." chem.biol. (2011) 18: 966-975; sierra JR, ceero V, Giordano s. "Molecular mechanisms of acquisition and to type of kinase target therapy." mol.cancer (2010) 9: 75) growth factor receptors (e.g., insulin-like growth factor 1 receptor) (IFG1R) (Villanueva J, Vultur A, Lee JT et al. "Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch enzyme and IGF-1R/PI 3K". Cancer Cell (2010) 18: 683695) or platelet-derived growth factor receptor (PDGFR), as well as several other resistance mechanisms (Wilson TR, fridland J, Yan Y et al, "wide pore for growth-factor-drive resistance to antibody kinase inhibitors". Nature (2012) 487: 505 — 509; straussman R, Morikawa T, Shee K et al, "Tumour micro-environmental aspects to RAF inhibitors through HGF session". Nature (2012) 487: 500-504). Several methods have been described for maintaining the levels of phosphorylated extracellular signal-associated kinases 1 and 2(p-ERK1/2) in the presence of BRAF inhibitor drugs, including ERK kinase 1(MEK1) mutations, recruitment of other alternative MEK1/2 activators, RAS mutations, or upregulation of Receptor Tyrosine Kinases (RTKs). Thus, in many cases, Verafenib resistant cells have cross-resistance to MEK inhibitors (Little AS, Smith PD, Cook SJ. "Mechanisms of acquired resistance to ERK1/2pathway inhibition". Oncogene (2013)32 (20110): 1207) 1215; Atefi M, von Euw E, Attar N et al, "Reversing melano cross-resistance to MEK BRAF and inhibition by mTOR One (2011) 6: E28973; Poulikofos PI, saudy, Janakiraman et al., RAF inhibition resistance and inhibition resistance mechanism inhibition of MEKrandly specialized BRAF (V600E) "Nature (2011) 480: 387-390). Since one of the major acquired vemurafenib resistance mechanisms is persistent downstream MEK/ERK activation, BRAFi + MEKi combinations targeting elements within the RAF-MEK-ERK pathway attract the most attention, leading to approval of the darafinil + trametinib combination by the FDA in 2013. However, due to tumor heterogeneity and activation of the alternative pathway in melanoma, resistance to this combination therapy developed on average within 9.4 months, and the combination had very low clinical activity after resistance developed.

The use of drug combinations of agents with different anti-cancer mechanisms can enhance tumor response and patient survival, particularly in the treatment of patients with advanced cancer (Carrick S, Parker S, Wilken N et al, "Single agent versatemporal chemotherapy for metastic breakdown cancer". C. Database Syst. Rev. 2005: CD 003372; Fassnarht M, Terzolo M, Allolio B et al, "combinatorial chemotherapy in advanced regenerative medicine Carcinoma". N.Engl. J.Med. (2012) 366: 2189-. Although virofibrine has been extensively studied in combination with agents targeting the same mitogen-activated protein kinase (MAPK) pathway, such as MEK or ERK inhibitors, and has demonstrated clinical efficacy (Greger JG, Eastman SD, Zhang V et al, "Combinations of BRAF, MEK, and PI3K/mTOR inhibitors over complex response to the BRAFinhibitor GSK2118436 databrafenib, media by NRAS muscles" mol. Cancer ther 367 (11: 909. 920; Patel SP, Lazar AJ, Papodosullos NE et al, Median stresses into compositions (AZD 44; ARR-142886) -transformed genes NE, 20111. J., "clinical responses to metals 62119, J. branched protein, J.," coding ". 11. 12. J.," coding ". 3. 12. J.," coding ". 31. 12. 9. blend J.," coding ". 31. the coding gene, 2. 11. the coding gene, J. 11. the coding gene, 2. 7. the invention, 2.₀/G₁Cells in phase (c). Such a combination strategy is not possibleDrug resistant cells that can escape from this cell cycle arrest are effectively treated.

Long-term selection of Verofinib-resistant human melanoma cells (e.g., A375RF21) cannot be arrested at G375 by Verofinib at a concentration effective for the sensitive parental cell line (i.e., A375)₀/G₁And vemurafenib resistant cells readily progress to G₂the/M phase (FIG. 2A). Therefore, vemurafenib strongly induces subsequent G₂Combinations of compounds retarded by phase/M should be successfully captured from G₀/G₁It is expected that escaping vemurafenib-resistant cells will be blocked, thereby creating a strong synergistic effect.

Recently, a new class of antimitotic agents has been found, represented by the skeleton of 2-aryl-4-benzoylimidazole (ABI) (Chen J, Li CM, Wang J et al, "Synthesis and anti-catalytic activity of novel 2-aryl-4-phenyl-organic derivative targeting molecules". Bioorg.Med.Chem. (2011) 19: 4782-, lu Y, Chen J et al, "organic bioavailable tubulin antagonists for paclitaxel-defractoracer". pharm.res. (2012) 29: 3053-3063). These compounds exhibit antiproliferative IC in the low nanomolar (nM) range in several human and mouse melanoma cell lines₅₀The value is obtained. They bind to tubulin at the colchicine binding site. Compared to many existing tubulin inhibitors (e.g., paclitaxel and vinblastine), ABI compounds can effectively circumvent several clinically relevant multidrug resistance mechanisms, including resistance mediated by P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), and Breast Cancer Resistance Protein (BCRP). In vivo studies showed that they significantly inhibited melanoma B16-F10 cell lung metastasis in mice (Wang Z, Chen J, Wang)J et al, "Novel tubulin polymerization inhibitors over communicating responses and reduction melanises to the lung". pharm.Res. (2012) 29: 3040-3052).

With the rapid rise in the incidence of cancer, particularly melanoma, and the high resistance to current therapeutic agents, identifying more effective drug combinations targeting the alternative pathway to overcome the BRAFi resistance of melanoma would provide significant benefits to patients. In addition, since BRAF mutations are also common in many other types of cancer, including ovarian, colorectal, and papillary thyroid cancers, the development of new combination strategies can have a broader impact on these types of cancer where little clinical activity is exhibited using existing BRAFi + MEKi combinations and is urgently needed.

Disclosure of Invention

In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor; and a pharmaceutically acceptable carrier.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound represented by the structure of formula II:

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

R₁is H, C₁-C₆Linear or branched alkyl, aryl, phenyl, benzyl, haloalkyl, aminoalkyl, -OCH₂Ph、SO₂Aryl, SO₂-phenyl, - (C ═ O) -aryl, - (C ═ O) -phenyl or OH;

R₄and R₅Each independently is hydrogen, C₁-C₆Linear or branched alkyl, C₁-C₆Linear or branched haloalkyl, C₁-C₆Linear or branched alkoxy, C₁-C₆Linear or branched haloalkoxy, F, Cl, Br, I, CF₃、CN、-CH₂CN、NH₂、OH、-OC(O)CF₃Alkylamino, aminoalkyl, -OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, -C (O) NH₂Or NO₂(ii) a And is

n is an integer from 1 to 4;

or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof; and

at least one of a BRAF inhibitor or a MEK inhibitor; and a pharmaceutically acceptable carrier.

In one embodiment, the present invention relates to a method of treating, suppressing, (i) a BRAF mutant cancer, (ii) a BRAF inhibitor-resistant cancer, (iii) a melanoma, (iv) a drug-resistant melanoma, (v) cancer metastasis, reducing the severity, reducing the risk, or inhibiting thereof in a subject; or (vi) a method of delaying or preventing a BRAF inhibitor-resistant cancer in a subject, the method comprising administering to a subject having a BRAF mutant cancer a composition comprising: at least one of a BRAF inhibitor or a MEK inhibitor; and a compound represented by the structure of formula II:

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

n is an integer from 1 to 4;

or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer or isomer thereof.

In one embodiment, the invention relates to a method of treating, suppressing, reducing, inhibiting, eliminating, delaying or preventing secondary cancer resistance to a taxane drug in a subject having cancer who was previously treated with the taxane drug, the method comprising administering to the subject a composition comprising: at least one of a BRAF inhibitor or a MEK inhibitor and a compound represented by the structure of formula II:

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

n is an integer from 1 to 4;

In one embodiment, the present invention relates to: (i) methods of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer; (ii) a method of suppressing acquired BRAF inhibitor resistance; (iii) a method of delaying or preventing the progression of BRAF inhibitor resistance; or (iv) a method of treating, suppressing, inhibiting, eliminating, reducing, delaying or preventing cancer metastasis, the method comprising administering to a subject having a drug-resistant cancer a composition comprising at least one of a BRAF inhibitor and a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer.

In another embodiment, the compound of the invention is compound 12 da. In another embodiment, the compound of the present invention is compound 17 ya.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a tubulin inhibitor, a BRAF inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the tubulin inhibitor is a compound of the present invention.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound represented by the structure:

and a BRAF inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the BRAF inhibitor is vemurafenib.

and a MEK inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the MEK inhibitor is RO 5068760.

In one embodiment, the present invention relates to: (a) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a BRAF mutant cancer in a subject; (b) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a BRAF inhibitor-resistant cancer; (c) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting melanoma; (d) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting drug resistant melanoma; (e) methods of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer; (f) a method of overcoming resistance to treatment with a BRAF inhibitor in an individual with resistant cancer; or (g) a method of preventing, eliminating, reducing, or delaying resistance to cancer treatment in a subject having cancer, the method comprising administering a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the cancer is melanoma, thyroid cancer, colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the cancer is a drug-resistant cancer. In another embodiment, the melanoma is drug resistant melanoma. In another embodiment, the compound of the invention is compound 12 da. In another embodiment, the compound of the present invention is compound 17 ya.

In one embodiment, the invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer, comprising administering to a subject having a drug-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the cancer is melanoma, thyroid cancer, colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma.

Drawings

The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

figure 1 shows establishment of vemurafenib resistant a375 melanoma cell line (a375RF21) from a parent a375 cell line with increasing concentrations of vemurafenib using long term selection over 3 months. MTS assay showed IC for proliferation inhibition in parental A375 melanoma₅₀Values (0.57 ± 0.03 μ M) increased by more than 50-fold when tested in vemurafenib resistant a375RF21 cells (28.9 ± 0.6 μ M). In contrast, IC of Compound 12da₅₀Values were not significantly affected (10.7 ± 1.5nM in a375 parental cell line and 13.6 ± 4.4nM in a375RF21, respectively). The structures of compound 12da and vemurafenib are shown in the figure.

Fig. 2 shows a cell cycle analysis (n-4). A, a375 or a375RF21 cells treated with 1 μ M vemurafenib for 24h and compared to DMSO controls. 1 μ M Verofinib efficiently arrested A375 cells at G₀/G₁Stage, but failed to arrest drug-resistant a375RF21 cells. B, 24h a375RF21 cells were treated with DMSO, 30 μ M vemurafenib, 20nM compound 12da, 20nM docetaxel, and a combination. Compound 12da and docetaxel induced G in A375RF21 cells₂M block, and their combination with vemurafenib blocks cells at G₁/G₂And a/M period.

Figure 3 shows that the combination of tubulin inhibitor and vemurafenib synergistically increases the proportion of apoptosis or death of drug resistant a375RF21 cells. A, representative quadrant plots show cell distribution in Q1 (early apoptosis), Q2 (apoptosis), Q3 (live), and Q4 (dead). Cell clusters with high SSC (side scatter)/low FSC (forward scatter) cell morphology stained black. There was no back-gating (back-gating) difference between the gray and black populations. B, the apoptosis score was calculated by adding together the distribution percentages in Q1, Q2, and Q4. The proportion of apoptosis induced by the drug combination group was significantly higher compared to the simple sum of the fraction of apoptosis in the two single agent treated groups (. P < 0.05).

Figure 4 shows the effect of single agent and combination therapy on purified protein-based tubulin polymerization assays (n-3). Vemurafenib at 20 μ M did not significantly affect tubulin polymerization compared to DMSO control. The tubulin polymerization inhibitory effect in the combination treatment group was contributed only by compound 12 da.

FIG. 5 shows Western blot analysis of lysates of A375RF21(A), MDA-MB-435 and WM164 cells (B) after 48h treatment with the indicated antibodies. GAPDH was used as loading control. A, although the indicated combination treatments resulted in only moderately reduced p-ERK levels, they were greatly inhibitedAKT phosphorylates and increases the level of apoptotic markers including cleaved PARP and cleaved caspase-3. B, Compound 12da is also on the other two BRAFs^V600EAKT knock-out effect was shown in mutant human melanoma cell lines MDA-MB-435 and WM 164.

Figure 6 shows the in vivo combination of vemurafenib and compound 12da in the drug resistant a375RF21 xenograft model (n ═ 7). A, pictures of isolated tumor tissue. B, tumor volume growth curve. C, mouse body weight versus time curve. D, representative immunohistochemical images of H & E, Ki67, pAKT, pERK, and S100 staining of tumor tissue sections three weeks after treatment with single agents or combinations. The blue scale bar in each image represents 100 μm.

Figure 7 shows the in vivo combination of high dose vemurafenib (30mg/kg) and compound 12da (15mg/kg) in an a375RF21 xenograft model (n-5). A, pictures of isolated tumor tissue. B, tumor volume growth curve. C, mouse body weight versus time curve. The combination of compound 12da and vemurafenib at this dose achieved 44.9% tumor regression.

Figure 8 shows a synthetic route for the preparation of aryl-benzoyl-imidazole (ABI) compounds of the invention. Reagents and conditions: (a) t-BuOH, I₂Ethylenediamine, K₂CO₃Refluxing; (b) PhI (OAc)₂，K₂CO₃，DMSO；(c)DBU，CBrCl₃，DMF；(d)NaH，PhSO₂Cl, THF, 0 ℃ to RT; (e) t-BuLi, substituted benzoyl chloride, THF, -78 ℃; (f) bu₄NF，THF，RT。

Figure 9 shows a synthetic route for the preparation of aryl-benzoyl-imidazole (ABI) compounds of the invention. Reagents and conditions: (a) NH (NH)₄OH, glyoxal, ethanol, RT; (b) NaH, PhSO₂Cl, THF, 0 ℃ to RT; (c) t-BuLi, substituted benzoyl chloride, THF, -78 ℃; (d) bu₄NF，THF，RT；(e)BBr₃，CH₂Cl₂(ii) a (f) c-HCl, AcOH, reflux.

Figure 10 shows a synthetic route for the preparation of aryl-benzoyl-imidazole (ABI) compounds of the invention. Reagents and conditions: (a) NaH, substituted benzoyl chloride, THF.

FIG. 11 shows the synthetic route for compounds 12dc, 12fc, 12daa, 12dab, 12 cba. (a) AlCl₃THF, reflux; (b) NaH, CH for 12dab and 12cba₃I, and BnBr for 12daa, THF, reflux.

FIG. 12 shows the synthetic route for compounds 11gaa, 12 la. (a) NH (NH)₄OH, ethanol, glyoxal, RT; (b) NaH, substituted PhSO₂Cl, THF, 0 ℃ to RT; (c) t-BuLi (1.7M in pentane), substituted benzoyl chloride, THF, -78 ℃; (d) bu₄NF，RT。

Fig. 13 shows a synthetic route to 12 fa. (a) NH (NH)₄OH, glyoxal, ethanol, RT; (b) NaH, PhSO₂Cl, THF, 0 ℃ to RT; (c) t-BuLi, 3, 4, 5-trimethoxybenzoyl chloride, THF, -78 deg.C; (d) bu₄NF，THF，RT。

Fig. 14 shows the synthetic routes of 17ya, 17yab and 17 yac. (a) KOH, ethanol, 2.PhSO₂Cl, acetone, RT; (b) NH (NH)₄OH, glyoxal, ethanol, RT; (c) NaH, PhSO₂Cl, THF, 0 ℃ to RT; (d) t-BuLi (1.7M in pentane), 3, 4, 5-trimethoxybenzoyl chloride, THF, -78 ℃; (e) NaOH, ethanol, H₂O, refluxing; (f) TBAF, THF, RT; (g) NaH, CH₃I，THF。

Fig. 15 shows the cell cycle distribution of PC3 treated with the compounds of the invention (12q, 70a, 70f, and 70m) for 24 hours.

Figure 16 shows dose response curves for 2-aryl-4-benzoyl-imidazole compounds (ABI) against multi-drug resistant melanoma cell lines (MDR cells) and corresponding sensitive parental cell lines (normal melanoma cells) compared to other anti-cancer drugs and compounds. For paclitaxel, vinblastine and colchicine, the large distance between the two curves indicates that they are substrates for the P-glycoprotein (P-gp). The overlap of the two curves for each ABI compound indicates that the ABI compound is not a substrate for P-gp and overcomes multidrug resistance.

Figure 17 shows the effect of ABI compounds on tubulin polymerization in vitro. Tubulin (0.4 mg/assay) was exposed to 10 μ M of the ABI compound (vehicle control, 5% DMSO). Absorbance at 340nm was monitored at 37 ℃ per minute for 15 minutes and the ABI compounds 12da, 12db and 12cb were shown to inhibit tubulin polymerization in vitro.

Figure 18 shows a B16-F1 melanoma colony formation assay in soft agar, which indicates that ABI compounds inhibit colony formation in a concentration-dependent manner. Fig. 18A shows representative pictures of the control and each test compound (12cb, 12da, and 12fb) at 100 nM. The diameter of each hole was 35 mm. Fig. 18B shows a quantitative representation of the assay results for each test compound (12cb, 12da, and 12 fb). P values were calculated by GraphPad Prism software compared to controls using Student's t test. Column, average of triplicate determinations; bar, SD.

Figure 19 shows an in vivo study of ABI compounds. FIG. 19A shows the in vivo activity of 12cb against B16-F1 melanoma tumors in C57/BL mice. FIG. 19B shows the in vivo activity of 12fb on B16-F1 melanoma in C57BL/6 mice and SHO nude mice. The results indicate that 12fb inhibits melanoma tumor growth in a dose-dependent manner. C57BL/6 mice bearing B16-F1 melanoma allografts (n-5 per group). Each mouse received 0.5X 10 of the drug by subcutaneous injection into the flank⁶And (4) cells. When the tumor size reaches about 100mm³At that time, 30 μ L of intraperitoneal therapy per day was started. Figure 19C shows the in vivo activity of 12fb on a375 human melanoma xenografts. SHO nude mice with a375 human melanoma xenografts (n-5 per group). Each mouse received 2.5X 10 injections subcutaneously into the flank⁶And (4) cells. When the tumor size reaches about 150mm³At that time, 30 μ L of intraperitoneal therapy per day was started. Control, vehicle solution only; point, average; bar, SD. DTIC, (5- (3, 3-dimethyl-1-triazenyl) -imidazole-4-carboxamide, dacarbazine.

Fig. 20 shows the effect of 17ya and 55 on tubulin polymerization. Compounds 17ya and 55 bind to the colchicine binding site on tubulin and inhibit tubulin polymerization. Fig. 20A, competitive weight binding. Tubulin (1mg/mL) and colchicine (1.2. mu.M) were incubated with different concentrations of podophyllotoxin, vinblastine, compound 17ya and 55. N is 3; mean. + -. SD. Podophyllotoxin and vinblastine were used as positive and negative controls, respectively. Figure 20B, effect on tubulin polymerization. Tubulin (0.4mg) was exposed to the test compound (5 μ M). Colchicine was used as a positive control. Figures 20C and 20D, 17ya and 55 enhanced the ability of cytoplasmic DNA-histone complex formation (apoptosis) in PC-3(C) and PC-3/txr (D) cells at 24h (N ═ 3); mean. + -. SD. Docetaxel was used as a positive control.

Figure 21 shows in vivo anticancer efficacy. Figure 21A, PC-3 tumor-laden nude mice were treated with docetaxel (i.v., 10 or 20mg/kg) on days 1 and 9. (N-5-6). Rod, SE. Figure 21B, PC-3/TxR tumor-loaded nude mice were treated with docetaxel (i.v., 10 or 20mg/kg) on days 1 and 9, with compound 17ya treatment (p.o., 6.7mg/kg) once daily every five days of the week. (N-4-5). Rod, SE. Figure 21C, PC-3/TxR tumor-loaded nude mice were treated twice daily with compound 17ya (PO, 3.3mg/kg) for four days in the first week, followed by five days per week once daily (N ═ 7) for weeks 2-4, and compound 55 twice daily (p.o., 10 or 30mg/kg) for five days per week for four weeks (N ═ 7). Rod, SE. Figure 21D, nude mice loaded with PC-3/TxR tumors (N ═ 5) were treated with compound 17ya (PO, 10mg/kg) three times a week for four weeks. Rod, SE.

Figure 22 shows the in vivo anti-cancer efficacy of 17ya in HL60 leukemia cell xenografts.

Figure 23 shows anti-phosphorylated histone H3 and PI (propidium iodide) two-variable staining cell cycle analysis of vemurafenib resistant cells. A375RF21 cells (biological replicates n-4) were treated with cell culture medium containing 5% DMSO (vehicle control), single agent or indicated combination for 24H, followed by anti-phospho histone H3-488 antibodies and PI were stained and then analyzed by flow cytometry. A, a representative graph showing cell distribution. The red line was defined manually to show how the phase distribution of the cell cycle was calculated accordingly. B, quantification of phase distribution of the cell cycle (mean ± SD). Comparison: 5 per mill DMSO; vem: vemurafenib 30 μ M; ABI: compound 12da20 nM; vem + ABI: vemurafenib 30 μ M + compound 12da20 nM; and Doc: docetaxel 20 nM; vem + Doc: vemurafenib 30 μ M + docetaxel 20 nM.

Figure 24 shows the in vitro dose response curves (n-5) for each combination in a375 cells and a375RF21 cells. The X-axis of each curve is the IC for drug A or B versus A375 cells or A375RF21 cells in the A + B combination treatment₅₀Dose density of concentration. FIGS. 24A-24C are data from A375 cells, and FIGS. 24D-24F are data from A375RF21 cells.

FIG. 25 shows that the major virofinib resistance mechanisms of A375RF21 cells are overexpression of PDGF β and activation of the PI3K-AKT pathway, both resistance mechanisms have been well established by clinical tumors, indicating that the resistance mechanism of A375RF21 cells may represent clinically relevant resistance mechanisms FIG. A Western blot analysis to compare differential protein levels in sensitive parent A375 cells and virofibrib resistant A375RF21 cells in the presence or absence of 2.5 μ M virofibrib (A375RF21 cell culture maintenance concentration), cells were incubated with control vehicle or 2.5 μ M virofibrib for 24h, phosphorylated PI3K levels were determined after 30 min stimulation with 30 μ M hydrogen peroxide_a0Comparison of values (shown as mean ± SD).

Fig. 26 shows in vitro growth curves for a375 cells and vemurafenib resistant subline a375RF21 cells. 20 μ l of cell growth medium00 cells were seeded into each well (n-6) of a 96-well plate and incubated at 37 ℃ with 5% CO₂The incubation was performed. Total protein amounts were determined accordingly by SRB assay at each indicated time point. The absorbance at 564nm was then plotted against growth time.

Fig. 27 shows a diagrammatic depiction of the study strategy. NSG mice implanted with P2 vemurafenib-sensitive or vemurafenib-resistant PDX tumors were purchased from JAX. The P2 tumor was propagated in additional NSG mice to generate sufficient P3 tumor for study. Genetic maps and histology of harvested P3 tumors were characterized and validated with standards from the initial P0 tumor established in JAX to ensure tumor fidelity. P3 tumor masses were implanted into mice to form P4 tumors for efficacy studies described in targets 1 and 2 (example 11). In addition, a single cell suspension from P3 tumor was injected tail vein into mice in an experimental melanoma lung metastasis model to assess the efficacy of the combination in target 3 on melanoma metastasis.

Figure 28 shows inhibition of melanoma to lung metastasis in mice by ABI 12cb, 12da, and 12 fb. FIG. A: representative photographs of lungs with melanoma nodules (black dots, n ═ 8 per group). Treatment was i.p. injections 5 days a week for 2 weeks. And B: number of melanoma nodules on each lung. Point: the number of each node; long line in the middle: average value; top and bottom stubs: 95% confidence interval^**And is and^##: p is less than 0.01. And (C) figure: change in body weight of mice during the course of the experiment. Point: average value; stick: and (7) SD. Comparison: vehicle solution only.

Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

Detailed Description

In one embodiment, the present invention is directed to a compound represented by the structure of formula I:

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

z is O or S;

R₂、R₃、R₄and R₅Each independently is hydrogen, C₁-C₆Linear or branched alkyl, C₁-C₆Linear or branched haloalkyl, C₁-C₆Linear or branched alkoxy, C₁-C₆Linear or branched haloalkoxy, F, Cl, Br, I, CF₃、CN、-CH₂CN、NH₂、OH、-OC(O)CF₃Alkylamino, aminoalkyl, -OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, -C (O) NH₂Or NO₂(ii) a And is

m and n are each independently an integer from 0 to 4;

In one embodiment, a is aryl. In another embodiment, a is phenyl. In another embodiment, a is indolyl. In another embodiment, A is 3-indolyl. In another embodiment, Z is O.

In one embodiment, R₂Is OMe. In another embodiment, R₃Is H. In another embodiment, m is 3. In another embodiment, R₂Is OMe, R₃Is H, and m is 3.

In one embodiment, R₄Is C₁-C₆Linear or branched alkyl. In another embodiment, R₄Is Me. In another embodiment, R₄Is H. In another embodiment, R₅Is H. In another embodiment, n is 1. In another embodiment, R₄Is Me, R₅Is H, and n is 1. In another embodiment, R₄Is H, R₅Is H, and n is 1.

In another embodiment, R₁Is H. In another embodiment, R₁Is C₁-C₆Linear or branched alkyl. In another embodiment, R₁Is Me.

In one embodiment, the present invention is directed to a compound represented by the structure of formula II:

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

R₄and R₅Each independently is hydrogen, C₁-C₆Linear or branched alkyl, C₁-C₆Linear or branched haloalkyl, C₁-C₆LinearityOr branched alkoxy, C₁-C₆Linear or branched haloalkoxy, F, Cl, Br, I, CF₃、CN、-CH₂CN、NH₂、OH、-OC(O)CF₃Alkylamino, aminoalkyl, -OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, -C (O) NH₂Or NO₂(ii) a And is

n is an integer from 1 to 4;

In one embodiment, a is aryl. In another embodiment, a is phenyl. In another embodiment, a is indolyl. In another embodiment, A is 3-indolyl.

In one embodiment, the present invention is directed to a compound represented by the structure of formula III:

wherein

R₁And R₉Each independently is H, C₁-C₆Linear or branched alkyl, aryl, phenyl, benzyl, haloalkyl, aminoalkyl, -OCH₂Ph、SO₂Aryl, SO₂-phenyl, - (C ═ O) -aryl, - (C ═ O) -phenyl or OH;

R₄and R₅Independently of one another is hydrogen, C₁-C₆Linear or branched alkyl, C₁-C₆Linear or branched haloalkyl, C₁-C₆Linear or branched alkoxy, C₁-C₆Linear or branched haloalkoxy, F, Cl, Br, I, CF₃、CN、-CH₂CN、NH₂、OH、-OC(O)CF₃Alkylamino, aminoalkyl, -OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, -C (O) NH₂Or NO₂(ii) a And is

n is an integer from 1 to 4;

In one embodiment, R₄Is H. In another embodiment, R₅Is H. In another embodiment, n is 1. In another embodiment, R₄Is H, R₅Is H, and n is 1. In another embodiment, R₉Is H. In another embodiment, R₉Is Me.

In another embodiment, the compound of formula III is represented by the structure of compound 17 ya:

in one embodiment, the present invention is directed to a compound represented by the structure of formula IV:

wherein

n is an integer from 1 to 4;

In one embodiment, R₄Is C₁-C₆Linear or branched alkyl. In another embodiment, R₄Is Me. In another embodiment, R₄Is H. In another embodiment, R₅Is H. In another embodiment, n is 1. In another embodiment, R₄Is Me, R₅Is H, and n is 1.

In another embodiment, R₁Is H. In another embodiment, R₁Is C₁-C₆Linear or branched alkyl radicals. In another embodiment, R₁Is Me.

In another embodiment, the compound of formula IV is represented by the structure of compound 12 da:

in one embodiment, a of the compounds of formula I and II is Ph. In another embodiment, a of the compounds of formula I and II is indolyl. In another embodiment, A of the compounds of formula I and II is 2-indolyl. In another embodiment, A of the compounds of formula I and II is 3-indolyl. In another embodiment, A of the compounds of formula I and II is 4-indolyl. In another embodiment, A of the compounds of formula I and II is 5-indolyl. In another embodiment, A of the compounds of formula I and II is 6-indolyl. In another embodiment, A of the compounds of formula I and II is 7-indolyl.

In another embodiment, R₅Is positioned at the contraposition. In another embodiment, R₅Is located at a meta position. In another embodiment, R₅In the ortho position. In another embodiment, R₅Is 4-Me. In another embodiment, R₅Is H. In another embodiment, R₅Is 4-F.

In another embodiment, R₄Is H.

In another embodiment, n is 1.

In one embodiment, the a groups of formulas I and II are furyl, benzofuryl, benzothienyl, indolyl, pyridyl, phenyl, biphenyl, triphenyl, diphenylmethane, adamantyl, fluorenyl, and other heterocyclic analogs, such as, for example, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, pyrrolizinyl, indolyl, isoquinolyl, quinolinyl, isoquinolinyl, benzimidazolyl, indazolyl, quinazo, and the likeAzinyl, cinnolinyl, quinazolinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, furyl, pyrylium, benzodioxolyl, thienylpropyl, thienylbutyl, tetrahydrothienyl, dithiocyclopentyl, tetrahydrothiopyranyl, thienyl, thiaquinoxalinyl, quinoxalinyl, oxatyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, furyl, pyrylium, benzodioxolyl, thiofuranyl, thianyl, thiathienyl, thiaphenyl, thianaphthylA thienyl group, a thioindenyl group, an oxathiolanyl group, a morpholinyl group, a thiazoyl group, a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, an oxazolyl group, an isoxazolyl group, an oxadiazolyl group.

In one embodiment, a is phenyl. In another embodiment, a is indolyl; most preferred are 3-indolyl and 5-indolyl.

In one embodiment, Z of formula I is O. In another embodiment, Z is S.

In one embodiment, R of formulas I, II, III and IV₁Is hydrogen. In another embodiment, R₁Is C₁-C₆Linear or branched alkyl. In another embodiment, R₁Is Me. In another embodiment, R₁Is C₁-C₆Linear or branched haloalkyl. In another embodiment, R₁Is CF₃. In another embodiment, R₁Is phenyl. In another embodiment, R₁Is benzyl. In another embodiment, R₁Is SO₂-an aryl group. In another embodiment, R₁Is (C ═ O) -aryl.

In one embodiment, R of formula I₃Is positioned at the contraposition. In another embodiment, R₃Is located at a meta position. In another embodiment, R₃In the ortho position.

In one embodiment, R of formula I₂And R₃Independent of each otherAnd ground is hydrogen. In another embodiment, R₂And R₃Independently is C₁-C₆Linear or branched alkoxy groups. In another embodiment, R₂And R₃Independently is OCH₃. In another embodiment, R₂And R₃Independently F. In another embodiment, R₂And R₃Independently 4-F. In another embodiment, R₂And R₃Independently Cl. In another embodiment, R₂And R₃Independently is Br. In another embodiment, R₂And R₃Independently is I. In another embodiment, R₂And R₃Independently is C₁-C₆Linear or branched haloalkyl. In another embodiment, R₂And R₃Independently of one another is CF₃. In another embodiment, R₂And R₃Independently CN. In another embodiment, R₂And R₃Independently is NH₂. In another embodiment, R₂And R₃Independently is OH. In another embodiment, R₂And R₃Independently is C₁-C₆Linear or branched alkyl. In another embodiment, R₂And R₃Independently is CH₃. In another embodiment, R₂And R₃Independently is NO₂. In another embodiment, R₂And R₃Independently an alkylamino group. In another embodiment, R₂And R₃Independently is 4-N (Me)₂。

In one embodiment, m of formula I is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

In one embodiment, R of formulas I, II, III and IV₅Is positioned at the contraposition. In another embodiment, R₅Is located at a meta position. In another embodiment, R₅In the ortho position.

In a fruitIn embodiments, R of formulas I, II, III and IV₄And R₅Independently hydrogen. In another embodiment, R₄And R₅Independently is C₁-C₆Linear or branched alkoxy groups. In another embodiment, R₄And R₅Independently OMe. In another embodiment, R₄And R₅Independently F. In another embodiment, R₄And R₅Independently Cl. In another embodiment, R₄And R₅Independently is Br. In another embodiment, R₄And R₅Independently is I. In another embodiment, R₄And R₅Independently is C₁-C₆Linear or branched haloalkyl. In another embodiment, R₄And R₅Independently of one another is CF₃. In another embodiment, R₄And R₅Independently CN. In another embodiment, R₄And R₅Independently is NH₂. In another embodiment, R₄And R₅Independently is OH. In another embodiment, R₄And R₅Independently is C₁-C₆Linear or branched alkyl. In another embodiment, R₄And R₅Independently is NO₂. In another embodiment, R₄And R₅Independently an alkylamino group.

In one embodiment, n of formulae I, II, III and IV is 0. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4.

It is understood that for heterocycles, n is limited to the number of positions that can be substituted, i.e., the number of CH groups minus 1. Thus, if the a ring is, for example, furyl, thienyl or pyrrolyl, n is 0-2; and n is 0 or 1 if the a ring is, for example, oxazolyl, imidazolyl, or thiazolyl; and n is 0 if the a ring is, for example, oxadiazolyl or thiadiazolyl.

In one embodiment, R of formula III₉Is hydrogen. In another embodiment, R₉Is C₁-C₆Linear or branched alkyl. In another embodiment, R₉Is CH₃. In another embodiment, R₉Is C₁-C₆Linear or branched haloalkyl. In another embodiment, R₉Is CF₃. In another embodiment, R₉Is phenyl. In another embodiment, R₉is-CH₂Ph. In another embodiment, R₉Is SO₂-an aryl group. In another embodiment, R₉Is (C ═ O) -aryl. In another embodiment, R₉Is (SO)₂) Ph. In another embodiment, R₉Is (SO)₂)-Ph-OCH₃。

As used herein, a "monocyclic or fused aromatic or heteroaromatic ring system" can be any such ring, including, but not limited to, phenyl, indolyl, 1H-indole, isoindolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, tetrazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, pyrrolyl, furanyl, thienyl, isoquinolyl, naphthyl, anthracyl, benzimidazolyl, indazolyl, 2H-indazolyl, 4, 5, 6, 7-tetrahydro-2H-indazolyl, 3H-indol-3-one, purinyl, benzoxazolyl, 1, 3-benzoxazolyl, benzisoxazolyl, benzothiazolyl, 1, 3-benzothiazole, 4, 5, 6, 7-tetrahydro-1, 3-benzothiazole, 4H-indole, 3-thiazole, and mixtures thereof, Quinazolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, quinolinyl, isoquinolinyl, acridinyl, benzofuranyl, 1-benzofuran, isobenzofuranyl, benzothienyl, benzo [ c ] thienyl, benzodioxolyl, thiadiazolyl, [1, 3] oxazolo [4, 5-b ] pyridine, oxadiazolyl, imidazo [2, 1-b ] [1, 3] thiazole, 4H, 5H, 6H-cyclopenta [ d ] [1, 3] thiazole, 5H, 6H, 7H, 8H-imidazo [1, 2-a ] pyridine, 7-oxo-6H, 7H- [1, 3] thiazolo [4, 5-d ] pyrimidine, [1, 3] thiazolo [5, 4-b ] pyridine, 2H, 3H-imidazo [2, 1-b ] [1, 3] thiazoles, thieno [3, 2-d ] pyrimidin-4 (3H) -ones, 4-oxo-4H-thieno [3, 2-d ] [1, 3] thiazine, imidazo [1, 2-a ] pyridine, pyrazolo [1, 5-a ] pyridine, imidazo [1, 2-a ] pyrazine, imidazo [1, 2-a ] pyrimidine, 1H-pyrrolo [2, 3-b ] pyridine, pyrido [2, 3-b ] pyrazine-3 (4H) -ones, 4H-thieno [3, 2-b ] pyrrole, quinoxaline-2 (1H) -ones, 1H-pyrrolo [3, 2-b ] pyridine, 7H-pyrrolo [2, 3-d ] pyrimidine, Oxazolo [5, 4-b ] pyridine, thiazolo [5, 4-b ] pyridine, and the like.

As used herein, "heterocyclic system" refers to saturated or unsaturated N-heterocycles, including but not limited to aza-and diaza-cycloalkyls, such as aziridinyl, azetidinyl, diazatidinyl, pyrrolidinyl, piperidinyl, piperazinyl, and azoctyl (azocanyl), pyrrolyl, pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, indazolyl, quinolizinyl, cinnolinyl, quinolonyl (quinoxalinyl), phthalazinyl, naphthyridinyl, quinoxalinyl, and the like; saturated or unsaturated O-heterocycles including, but not limited to, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, furanyl, pyrylium, benzofuranyl, benzodioxolyl, and the like; saturated or unsaturated S-heterocycles, including but not limited to, thienylpropyl, thienylbutyl, tetrahydrothienyl, dithiolyl, tetrahydrothiopyranyl, thienyl, benzothienyl, thiaPhenyl, thioindenyl, and the like; a saturated or unsaturated mixed heterocycle which may be any heterocycle containing two or more S-, N-or O-heteroatoms including, but not limited to, oxathiolanyl, morpholinyl, thiaxalkyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, and the like.

The term "alkyl" as used herein, unless otherwise indicated, can be any straight or branched chain alkyl group containing up to about 30 carbons. In another embodiment, the alkyl group includes C₁-C₆Carbon. In another embodiment, the alkyl group includes C₁-C₈Carbon. In another embodiment, the alkyl group includes C₁-C₁₀Carbon. In another embodiment, alkyl is C₁-C₁₂Carbon. In another embodiment, alkyl is C₁-C₂₀Carbon. In another embodiment, the cycloalkyl group has 3 to 8 carbons. In another embodiment, the branched alkyl is an alkyl substituted with an alkyl side chain of 1 to 5 carbons. In one embodiment, the alkyl group may be substituted. In another embodiment, the alkyl group may be substituted with halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H. Amino, alkylamino, dialkylamino, carboxyl, thio and/or thioalkyl substituted.

The alkyl group may be a single substituent or it may be a component of a larger substituent, for example in alkoxy, haloalkyl, arylalkyl, alkylamino, dialkylamino, alkylamido, alkylurea and the like. Preferred alkyl groups are methyl, ethyl and propyl, and thus halomethyl, dihalomethyl, trihalomethyl, haloethyl, dihaloethyl, trihaloethyl, halopropyl, dihalopropyl, trihalopropyl, methoxy, ethoxy, propoxy, arylmethyl, arylethyl, arylpropyl, methylamino, ethylamino, propylamino, dimethylamino, diethylamino, methylamido, acetylamino, propylamido, halomethylamido, haloethylamido, halopropylamido, methylurea, ethylurea, propylurea, and the like.

The term "aryl" as used herein refers to any aromatic ring directly bonded to another group. The aryl group may be a single substituent or the aryl group may be a more substituted component, for example in arylalkyl, arylamino, arylamido, and the like. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, xylyl, furyl, naphthyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, thiazolyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thienyl, pyrrolyl, phenylmethyl, phenylethyl, phenylAmino, phenylamido, and the like. Substitutions include, but are not limited to: F. cl, Br, I, C₁-C₅Linear or branched alkyl, C₁-C₅Linear or branched haloalkyl, C₁-C₅Linear or branched alkoxy, C₁-C₅Linear or branched haloalkoxy, CF₃、CN、NO₂、-CH₂CN、NH₂NH-alkyl, N (alkyl)₂Hydroxy, -OC (O) CF₃、-OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, or-C (O) NH₂。

The term "alkoxy" as used herein refers to an ether group substituted with an alkyl group as defined above. Alkoxy refers to both linear and branched alkoxy groups. Non-limiting examples of alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy.

The term "aminoalkyl" as used herein refers to an amine group substituted with an alkyl group as defined above. Aminoalkyl refers to monoalkylamine, dialkylamine, or trialkylamine. Non-limiting examples of aminoalkyl are-N (Me)₂、-NHMe、-NH₃。

In another embodiment, "haloalkyl" refers to an alkyl group as defined above substituted with one or more halogen atoms (e.g., F, Cl, Br, or I). A non-limiting example of a haloalkyl is CF₃、CF₂CF₃、CH₂CF₃。

In another embodiment, "alkoxyalkyl" refers to an alkyl group as defined above substituted with an alkoxy group (e.g., methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, etc.) as defined above. A non-limiting example of an alkoxyalkyl group is-CH₂-O-CH₃、-CH₂-O-CH(CH₃)₂、-CH₂-O-C(CH₃)₃、-CH₂-CH₂-O-CH₃、-CH₂-CH₂-O-CH(CH₃)₂、-CH₂-CH₂-O-C(CH₃)₃。

In one embodiment, a "cycloalkyl" or "carbocyclic" group refers to a ring structure that includes a carbon atom as a ring atom, which may be saturated or unsaturated, substituted or unsubstituted. In another embodiment, the cycloalkyl group is a 3-12 membered ring. In another embodiment, the cycloalkyl is a 6 membered ring. In another embodiment, the cycloalkyl group is a 5-7 membered ring. In another embodiment, the cycloalkyl is a 3-8 membered ring. In another embodiment, the cycloalkyl group may be unsubstituted or substituted with halogen, alkyl, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H. Amino, alkylamino, dialkylamino, carboxyl, thio and/or thioalkyl substituted. In another embodiment, the cycloalkyl ring may be fused to another saturated or unsaturated cycloalkyl or heterocyclic 3-8 membered ring. In another embodiment, the cycloalkyl ring is a saturated ring. In another embodiment, the cycloalkyl ring is an unsaturated ring. Non-limiting examples of cycloalkyl groups include cyclohexyl, cyclohexenyl, cyclopropyl, cyclopropenyl, cyclopentyl, cyclopentenyl, cyclobutyl, cyclobutenyl, cyclooctyl, Cyclooctadienyl (COD), Cyclooctene (COE), and the like.

In one embodiment, "heterocycle" or "heterocyclyl" refers to a ring structure that contains sulfur, oxygen, nitrogen, or any combination thereof as part of the ring in addition to carbon atoms. In another embodiment, the heterocycle is a 3-12 membered ring. In another embodiment, the heterocycle is a 6 membered ring. In another embodiment, the heterocycle is a 5-7 membered ring. In another embodiment, the heterocycle is a 3-8 membered ring. In another embodiment, the heterocyclyl group may be unsubstituted or substituted with halo, alkyl, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H. Amino, alkylamino, dialkylamino, carboxyl, thio and/or thioalkyl substituted. In another embodiment, the heterocyclic ring may be fused to another saturated or unsaturated cycloalkyl or heterocyclic 3-8 membered ring. In another embodiment, the heterocyclic ring is a saturated ring. In another embodimentThe heterocyclic ring is an unsaturated ring. Non-limiting examples of heterocycles include pyridine, piperidine, morpholine, piperazine, thiophene, pyrrole, benzodioxole, or indole.

In one embodiment, the present invention provides a compound of the present invention or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, hydrate, N-oxide, polymorph or crystal thereof, or a combination thereof. In one embodiment, the invention provides isomers of the compounds of the invention. In another embodiment, the invention provides a metabolite of a compound of the invention. In another embodiment, the invention provides a pharmaceutically acceptable salt of a compound of the invention. In another embodiment, the invention provides a pharmaceutical product of a compound of the invention. In another embodiment, the invention provides tautomers of the compounds of the invention. In another embodiment, the present invention provides a hydrate of the compound of the present invention. In another embodiment, the invention provides an N-oxide of a compound of the invention. In another embodiment, the present invention provides a polymorph of a compound of the present invention. In another embodiment, the invention provides crystals of a compound of the invention. In another embodiment, the present invention provides a composition comprising a compound of the present invention as described herein, or in another embodiment, a combination of isomers, metabolites, pharmaceutically acceptable salts, pharmaceutical products, tautomers, hydrates, N-oxides, polymorphs, or crystals of a compound of the present invention.

In one embodiment, the term "isomer" includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like. In another embodiment, the isomers are optical isomers.

In one embodiment, the compounds of the invention are pure (E) -isomers. In another embodiment, the compounds of the present invention are pure (Z) -isomers. In another embodiment, the compounds of the present invention are mixtures of (E) isomer and (Z) isomer. In one embodiment, the compounds of the present invention are pure (R) -isomers. In another embodiment, the compounds of the present invention are pure (S) -isomers. In another embodiment, the compounds of the present invention are mixtures of (R) isomers and (S) isomers.

The compounds of the invention may also exist as racemic mixtures containing substantially equal amounts of the stereoisomers. In another embodiment, the compounds of the present invention may be prepared or isolated using known methods to obtain stereoisomers substantially free of their corresponding stereoisomers (i.e., substantially pure). By substantially pure is meant that the stereoisomer is at least about 95% pure, more preferably at least about 98% pure, and most preferably at least about 99% pure.

The compounds of the present invention may also be in the form of hydrates, which means that the compounds also contain stoichiometric or non-stoichiometric amounts of water bound by non-covalent intermolecular forces.

The compounds of the invention may exist in the form of one or more possible tautomers, and depending on the particular conditions, some or all tautomers may be separated into single and different entities. It is to be understood that all possible tautomers are encompassed herein, including all additional enol-and keto-tautomers and/or isomers. For example, including but not limited to the following tautomers.

Tautomerization of imidazole rings

The tautomers of the present invention are free tautomers, not unresolved mixtures. The imidazoles and other ring systems of the present invention are tautomeric. All tautomers are considered to be part of the present invention.

It is well understood that in structures presented in the present invention in which the nitrogen atom has fewer than 3 bonds, an H atom is present to complete the valence of the nitrogen.

The present invention includes "pharmaceutically acceptable salts" of the compounds of the present invention, which may be prepared by reacting a compound of the present invention with an acid or base. Certain compounds, particularly those having acidic or basic groups, may also be in the form of salts, preferably pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts that retain the biological effects and properties of the free base or free acid, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, ferrihydric acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like. Other salts are known to those skilled in the art and can be readily adapted for use in accordance with the present invention.

Suitable pharmaceutically acceptable salts of amines of the compounds of the present invention may be prepared from inorganic or organic acids. In one embodiment, examples of inorganic salts of amines are bisulfate, borate, bromide, chloride, hemisulfate, hydrobromide, hydrochloride, 2-hydroxyethylsulfonate (hydroxyethanesulfonate), iodate, iodide, isothionate (isothionate), nitrate, persulfate, phosphate, sulfate, sulfamate, sulfa, sulfonic acid (alkylsulfonate, arylsulfonate, halogen-substituted alkylsulfonate, halogen-substituted arylsulfonate), sulfonate, and thiocyanate salts.

In one embodiment, examples of organic salts of amines may be selected from aliphatic acids, cycloaliphatic acids, aromatic acids, araliphatic acids, heterocyclic acids, carboxylic acids and sulfonic organic acids, examples of which are acetate, arginine, aspartate, ascorbate, adipate, anthranilate, alginate, alkane carboxylate, substituted alkane carboxylate, alginate, benzenesulfonate, benzoate, bisulfate, butyrate, bicarbonate, bitartrate, citrate, camphorate, camphorsulfonate, cyclohexylsulfamate, cyclopentanepropionate, calcium edetate, camphorsulfonate, carbonate, clavulanate, cinnamate, dicarboxylate, digluconate, dodecylsulfonate, dihydrochloride, caprate, heptanoate (enanthurate), ethanesulfonate, edetate, ethanedisulfonate, etonate, ethanesulfonate, fumarate, formate, fluoride, galacturonate, gluconate, glutamate, glycolate, glucarate (glucoronate), glucoheptonate, glycerophosphate, glucoheptonate, glycollate, p-acetylsalicylate (acetylsalicylate, mandelate, maleate, mandelate, maleate, mandelate, maleate, and sulfonate, mandelate.

In one embodiment, examples of inorganic salts of carboxylic acids or hydroxyl groups may be selected from ammonium; alkali metals including lithium, sodium, potassium, cesium; alkaline earth metals including calcium, magnesium, aluminum; zinc; barium; choline; quaternary ammonium.

In another embodiment, examples of organic salts of carboxylic acids or hydroxyl groups may be selected from arginine; organic amines including aliphatic organic amines, alicyclic organic amines, aromatic organic amines; benzathine penicillin; tert-butylamine; benzphetamine (N-benzylphenethylamine); dicyclohexylamine; dimethylamine; diethanolamine; ethanolamine; ethylene diamine; hydrabamine; imidazole; lysine; a methylamine; melamine (meglamine); N-methyl-D-glucamine; n, N' -dibenzylethylenediamine; nicotinamide; an organic amine; ornithine; pyridine; picoline (picoliy); piperazine; procaine; tris (hydroxymethyl) methylamine; triethylamine; triethanolamine; trimethylamine; tromethamine and urea.

In one embodiment, the salt may be formed by conventional methods, for example by reacting the free base or free acid form of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble or in a solvent such as water (which is removed in vacuo or by lyophilization), or by exchanging an ion of an existing salt for another ion, or a suitable ion exchange resin.

Pharmaceutical composition

Another aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one compound according to an aspect of the invention. The pharmaceutical compositions may comprise one or more of the above-described compounds of the invention. Typically, the pharmaceutical compositions of the invention will comprise a compound of the invention, e.g., a compound of formula I, II, III or IV or 17ya or 12da or a pharmaceutically acceptable salt thereof, and at least one of a BRAF inhibitor or a MEK inhibitor; and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention may also comprise at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor; and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to any suitable adjuvant, carrier, excipient or stabilizer, and may be in solid or liquid form, such as a tablet, capsule, powder, solution, suspension or emulsion. In some embodiments, the pharmaceutical composition comprises a compound of the invention, e.g., a compound of formula I, II, III, or IV or a combination of 17ya or 12da, or a pharmaceutically acceptable salt thereof, and a BRAF inhibitor, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of the invention, e.g., a compound of formula I, II, III, or IV or a combination of 17ya or 12da, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a compound of the present invention, e.g., a compound of formula I, II, III, or IV or 17ya or 12da or a pharmaceutically acceptable salt thereof, in combination with a BRAF inhibitor and a MEK inhibitor, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a combination of a tubulin inhibitor and a BRAF inhibitor, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a tubulin inhibitor in combination with a MEK inhibitor, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a tubulin inhibitor in combination with a BRAF inhibitor, a MEK inhibitor, and a pharmaceutically acceptable carrier.

The term "BRAF" as used herein refers to a human gene that produces a protein called B-Raf. The B-Raf protein is involved in signaling intracellular signals that are involved in directing cell growth. In 2002, it was pointed out that it is mutated in human cancers. Drugs have been developed to treat cancers caused by BRAF. Vemurafenib is a BRAF inhibitor drug approved by the FDA for the treatment of advanced melanoma. Other specific inhibitors of mutant B-raf proteins (the "BRAF inhibitors" as used herein) are being developed for anti-cancer use. These include: GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib and LGX 818.

The term "MEK" as used herein refers to the mitogen-activated protein kinase kinases MEK1 and/or MEK 2. MEK is a kinase that phosphorylates mitogen-activated protein kinases. MEK is a member of the MAPK signaling cascade that is activated in melanoma. When MEK is inhibited, cell proliferation is prevented and apoptosis (controlled cell death) is induced.

The term "MEK inhibitor" refers to a chemical or drug that inhibits the mitogen-activated protein kinase kinases MEK1 and/or MEK 2. They can be used to affect the MAPK/ERK pathway, which is normally overactive in some cancers. Thus, MEK inhibitors have potential in the treatment of some cancers, particularly BRAF mutant melanoma and KRAS/BRAF mutant colorectal cancer. MEK inhibitors include, but are not limited to: trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, cobinetinib or XL518 and CI-1040 or PD 035901.

In one embodiment, the present invention relates to a pharmaceutical composition comprising therapeutically effective amounts of two compounds having anti-cancer activity and a pharmaceutically acceptable carrier. In another embodiment, the composition comprises a BRAF inhibitor and a compound of the invention, e.g., a compound of formula I, II, III, or IV or 17ya or 12 da. In another embodiment, the composition comprises a MEK inhibitor and a compound of the present invention, for example a compound of formula I, II, III or IV or 17ya or 12 da. In one embodiment, the present invention relates to a pharmaceutical composition comprising therapeutically effective amounts of three compounds having anti-cancer activity and a pharmaceutically acceptable carrier. In another embodiment, the composition comprises a BRAF inhibitor, a MEK inhibitor and a compound of the invention, for example a compound of formula I, II, III or IV or 17ya or 12 da. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, or XL518, CI-1040, or PD035901, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the compound of the invention is a compound of formula I, II, III or IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da. In another embodiment, the compound is in the form of a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor; and a pharmaceutically acceptable carrier. In one embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a combination of a BRAF inhibitor and a tubulin inhibitor and a pharmaceutically acceptable carrier. In one embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a combination of a MEK inhibitor and a tubulin inhibitor, in combination with a pharmaceutically acceptable carrier. In one embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a combination of a BRAF inhibitor, a MEK inhibitor, a tubulin inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, or XL518, CI-1040, or PD035901, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib or RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine (vinfluine), dolastatin (dolastatin), halichondrin (halichondrin), hemiasterlin (hemiasterlin), cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel.

Generally, the compositions will contain from about 0.01% to about 99%, preferably from about 20% to about 75%, of the active compound, together with adjuvants, carriers and/or excipients. Although individual requirements may vary, the determination of an optimal range for effective amounts of each component is within the skill of the art. Typical dosages contain from about 0.01 to about 100mg/kg body weight. Preferred doses comprise from about 0.1 to about 100mg/kg body weight. The most preferred dosage comprises from about 1 to about 100mg/kg body weight. One of ordinary skill in the art can also readily determine the treatment regimen for administering the compounds of the present invention. That is, the frequency of administration and the size of the dose may be determined by routine optimization methods, preferably while minimizing any side effects.

In one embodiment, the methods of the invention may comprise administering the compounds of formulae I-IV of the invention in various doses. In one embodiment, the compounds of formulae I-IV are administered at a dose of 0.1-200 mg/kg. In one embodiment, the compounds of formulae I-IV are administered at a dose of 0.01-1 mg/kg. In one embodiment, the compound of formula I-IV is administered at a dose of 0.1 to 10mg/kg or in another embodiment, 0.1 to 25mg/kg or in another embodiment, 10 to 50mg/kg or in another embodiment, 10 to 25mg/kg or in another embodiment, 0.3 to 30mg/kg or in another embodiment, 0.5 to 25mg/kg or in another embodiment, 0.5 to 50mg/kg or in another embodiment, 0.75 to 15mg/kg or in another embodiment, 0.75 to 60mg/kg or in another embodiment, 1 to 5mg/kg or in another embodiment, 1 to 20mg/kg or in another embodiment, 3 to 15mg/kg or in another embodiment, 30-50mg/kg or, in another embodiment, 30-75mg/kg or, in another embodiment, 100-2000 mg/kg. In another embodiment, the compounds of formulas 1-IV are administered at a dose of 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, or 35 mg/kg. In another embodiment, the compounds of formulas I-IV are administered at a dose of 10 mg/kg. In another embodiment, the compounds of formulas I-IV are administered at a dose of 15 mg/kg. In another embodiment, the compounds of formulas I-IV are administered at a dose of 25 mg/kg.

In one embodiment, the compounds of formulae I-IV are administered at a dose of 10 mg. In one embodiment, the compounds of formulae I-IV are administered at a dose of 15 mg. In one embodiment, the compounds of formulae I-IV are administered at a dose of 25 mg. In another embodiment, a compound of formula I-IV is administered at a dose of 0.01mg, 0.03mg, 0.1mg, 0.3mg, 0.75mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, or 100 mg.

In one embodiment, the methods of the invention may comprise administering a BRAF inhibitor of the invention in various doses. In one embodiment, the BRAF inhibitor is administered at a dose of 0.1 to 200 mg/kg. In one embodiment, the BRAF inhibitor is administered at a dose of 0.01-1 mg/kg. In one embodiment, the BRAF inhibitor is administered at a dose of 0.1 to 10mg/kg or in another embodiment, 0.1 to 25mg/kg or in another embodiment, 10 to 50mg/kg or in another embodiment, 10 to 25mg/kg or in another embodiment, 0.3 to 30mg/kg or in another embodiment, 0.5 to 25mg/kg or in another embodiment, 0.5 to 50mg/kg or in another embodiment, 0.75 to 15mg/kg or in another embodiment, 0.75 to 60mg/kg or in another embodiment, 1 to 5mg/kg or in another embodiment, 1 to 20mg/kg or in another embodiment, 3 to 15mg/kg or in another embodiment, 30 to 50mg/kg or in another embodiment, 30-75mg/kg or, in another embodiment, 100-2000 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, or 50 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 10 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 20 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 30 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 40 mg/kg. In another embodiment, the BRAF inhibitor is administered at a dose of 45 mg/kg. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib.

In one embodiment, the BRAF inhibitor is administered at a dose of 10 mg. In one embodiment, the BRAF inhibitor is administered at a dose of 15 mg. In one embodiment, the BRAF inhibitor is administered at a dose of 25 mg. In one embodiment, the BRAF inhibitor is administered at a dose of 45 mg. In another embodiment, the BRAF inhibitor is administered at a dose of 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, or 50 mg.

In one embodiment, the methods of the invention may comprise administering the MEK inhibitors of the present invention in various doses. In one embodiment, the MEK inhibitor is administered in a dose of 0.1-200 mg/kg. In one embodiment, the MEK inhibitor is administered at a dose of 0.01-1 mg/kg. In one embodiment, the MEK inhibitor is administered at a dose of 0.1 to 1mg/kg or in another embodiment, 0.1 to 25mg/kg or in another embodiment, 10 to 50mg/kg or in another embodiment, 10 to 25mg/kg or in another embodiment, 0.3 to 0.5mg/kg or in another embodiment, 0.5 to 25mg/kg or in another embodiment, 0.5 to 50mg/kg or in another embodiment, 0.75 to 15mg/kg or in another embodiment, 0.75 to 60mg/kg or in another embodiment, 1 to 5mg/kg or in another embodiment, 1 to 20mg/kg or in another embodiment, 3 to 15mg/kg or in another embodiment, 30 to 50mg/kg or in another embodiment, 30-75mg/kg or, in another embodiment, 100-2000 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, or 1 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 0.1 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 0.3 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 0.5 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 0.7 mg/kg. In another embodiment, the MEK inhibitor is administered at a dose of 1 mg/kg. In another embodiment, the MEK inhibitor is trametinib or RO 5068760.

The solid unit dosage form may be of conventional type. The solid dosage form may be a capsule or the like, for example of the conventional gelatin type comprising a compound of the invention and a carrier, for example a lubricant and an inert filler such as lactose, sucrose or corn starch. In another embodiment, these compounds are tableted with conventional tablet bases (e.g., lactose, sucrose or corn starch) and binders (e.g., acacia, corn starch or gelatin), disintegrants (e.g., corn starch, potato starch or alginic acid) and lubricants (e.g., stearic acid or magnesium stearate).

Tablets, capsules and the like may also contain binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. In addition to the active ingredients, syrups may contain sucrose as a sweetening agent, methylparaben and propylparaben as preservatives, dyes and flavors such as cherry or orange flavor.

For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and formulations should contain at least 0.1% of the active compound. The percentage of the compound in these compositions may, of course, vary and may conveniently be from about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions will be such that a suitable dosage is obtained. Preferred compositions of the invention are prepared such that an oral dosage unit contains from about 1mg to 800mg of the active compound.

The active compounds of the present invention may be administered orally, for example with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft gelatin capsules, or they may be compressed into tablets, or they may be admixed directly with the food of the diet.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that it can be easily injected. It should be stable under the conditions of manufacture and storage and should be protected from 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 (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

The compounds or pharmaceutical compositions of the present invention may also be administered in injectable doses by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical adjuvant, carrier or excipient. Such adjuvants, carriers or/excipients include, but are not limited to, sterile liquids, such as water and oils, with or without the addition of surfactants and other pharmaceutically and physiologically acceptable components. Exemplary oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions and glycols (e.g., propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions.

These active compounds can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersants may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Exemplary oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions and glycols (e.g., propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

For use as an aerosol, a solution or suspension of a compound of the invention may be packaged in a pressurised aerosol container together with a suitable propellant, for example a hydrocarbon propellant such as propane, butane or isobutane, and conventional adjuvants. The materials of the present invention may also be administered in a non-pressurized form, for example in a nebulizer or atomizer.

In one embodiment, the present invention provides a pharmaceutical composition comprising a compound of formulae I-IV as described herein and/or its isomers, pharmaceutically acceptable salts, pharmaceutical products, hydrates, N-oxides, or any combination thereof, alone or in combination with another therapeutic agent, such as an anti-cancer agent, including but not limited to: a tubulin inhibitor, BRAF inhibitor, MEK inhibitor or other agent suitable for use in the applications described herein. In one embodiment, the pharmaceutical composition of the compounds of formulae I-IV as described herein comprises a compound of the present invention in combination with a BRAF inhibitor. In another embodiment, the pharmaceutical composition comprises a compound of the invention and a MEK inhibitor. In another embodiment, the pharmaceutical composition comprises a compound of the invention in combination with a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, or XL518, CI-1040, or PD035901, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib or RO 5068760.

In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor; and a pharmaceutically acceptable carrier. In one embodiment, the present invention relates to a pharmaceutical composition comprising a tubulin inhibitor in combination with a BRAF inhibitor and a pharmaceutically acceptable carrier. In one embodiment, the invention relates to a pharmaceutical composition comprising a tubulin inhibitor in combination with a MEK inhibitor and a pharmaceutically acceptable carrier. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, or XL518, CI-1040, or PD035901, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib or RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel.

In one embodiment, the compounds of the present invention are administered in combination with an anti-cancer agent. In one embodiment, the anti-cancer agent is a BRAF inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In one embodiment, the anti-cancer agent is a MEK inhibitor. In another embodiment, the MEK inhibitor is trametinib (GSK1120212), semetinib, RO5068760, MEK162, PD-325901, or XL518, CI-1040, or PD035901, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib or RO 5068760.

wherein

A is a monocyclic or fused aromatic or heteroaromatic ring system;

n is an integer from 1 to 4;

or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof; and at least one of a BRAF inhibitor or a MEK inhibitor; and a pharmaceutically acceptable carrier. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

Yet another aspect of the invention relates to a method of treating cancer, comprising selecting a subject in need of treatment for cancer, and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II, III, or IV, and at least one of a BRAF inhibitor or a MEK inhibitor; and a pharmaceutically acceptable carrier. In another embodiment, the cancer is a drug-resistant cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is BRAF mutant melanoma. In another embodiment, the cancer is a vemurafenib resistant cancer. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

When the compounds of the present invention are administered, they may be administered systemically or, alternatively, they may be administered directly to the specific site where the cancer cells or precancerous cells are present. Thus, administration can be accomplished in any manner effective to deliver the compound or the pharmaceutical composition to the cancer cells or precancerous cells. Exemplary modes of administration include, but are not limited to, administration of the compound or composition by oral administration, topical administration, transdermal administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, intranasal instillation administration, intracavity or intravesical instillation administration, intraocular administration, intraarterial administration, intralesional administration, or by administration to mucous membranes (e.g., mucous membranes of the nose, throat, and bronchi).

In some embodiments, in any form or embodiment as described herein, any of the compositions of the present invention comprise a compound of formulae I-IV or a tubulin inhibitor, and at least one of a BRAF inhibitor or a MEK inhibitor. In some embodiments, in any form or embodiment as described herein, any of the compositions of the present invention consist of a compound of formulae I-IV or a tubulin inhibitor, and at least one of a BRAF inhibitor or a MEK inhibitor. In some embodiments, in any form or embodiment as described herein, any of the compositions of the present invention consists essentially of a compound of formulae I-IV or a tubulin inhibitor, and at least one of a BRAF inhibitor or a MEK inhibitor. In some embodiments, the term "comprising" means including the indicated active agent, e.g., a compound of formulas I-IV or tubulin inhibitor, and including other active agents, as well as pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of …" means that the only active ingredient of the composition is the indicated active ingredient, however, other compounds useful for stabilization, preservation, etc. of the formulation, but not directly involved in the therapeutic effect of the indicated active ingredient, may also be included. In some embodiments, the term "consisting essentially of …" may refer to a component that facilitates the release of an active ingredient. In some embodiments, the term "consisting of …" refers to a composition comprising an active ingredient and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the present invention provides a combination formulation. In one embodiment, the term "combined preparation" is defined especially as "kit", in the sense that the combination partners as defined above can be administered separately or by using different fixed combinations with different amounts of the combination partners, i.e. simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit may then be administered, e.g., simultaneously or chronologically staggered, i.e., at different time points and at equal or different time intervals for any part of the kit. In some embodiments, the ratio of the total amounts of the combination parts may be administered in a combined preparation. In one embodiment, the combined preparation may be varied, for example, to address the needs of a patient sub-population to be treated or the needs of an individual patient (different needs may arise from a particular disease, severity of disease, age, sex or body weight which can be readily determined by one skilled in the art).

It is to be understood that the present invention relates to compositions and combination therapies as described herein for any disease, disorder or condition as understood by one of skill in the art as appropriate. Certain applications of such compositions and combination therapies have been described above for particular diseases, disorders, and conditions, which represent embodiments of the invention, and methods of treating such diseases, disorders, and conditions in a subject by administering a compound as described herein (alone or as part of a combination therapy) or using a composition of the invention represent additional embodiments of the invention.

Biological activity

In one embodiment, the present invention provides compounds and compositions (including any of the embodiments described herein) for use in any of the methods of the present invention. In one embodiment, the use of a compound of the invention or a composition comprising the same has utility in inhibiting, suppressing, enhancing or stimulating a desired response in an individual, as understood by one of skill in the art. In another embodiment, the composition may further comprise additional active ingredients, the activity of which may be useful for the particular application for which the compounds of the present invention are to be administered.

In one embodiment, the invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of developing, or inhibiting a cancer, comprising administering to a subject having cancer a compound of the invention under conditions effective to treat the cancer. In another embodiment, the compound is administered in combination with a BRAF inhibitor. In another embodiment, the compound is administered in combination with a MEK inhibitor. In another embodiment, the compound is administered in combination with a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, colorectal cancer, or ovarian cancer.

In one embodiment, the present invention provides: a) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a drug-resistant tumor; b) methods of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting metastatic cancer; c) methods of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer; d) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting melanoma; e) a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer, wherein the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer; f) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting metastatic melanoma; g) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a BRAF mutant cancer; h) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a BRAF inhibitor-resistant cancer; i) methods of treating, suppressing, reducing the severity, reducing the risk, inhibiting, eliminating, delaying or preventing a BRAF inhibitor-resistant cancer; j) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting a BRAF inhibitor-resistant melanoma; k) a method of treating, suppressing, reducing the severity, reducing the risk, or inhibiting cancer in a subject, wherein the subject was previously treated with chemotherapy, radiation therapy, or biological therapy; l) methods of overcoming resistance of an individual to treatment with a BRAF inhibitor; m) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting thyroid cancer; n) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting colorectal cancer; o) methods of treating, suppressing, reducing the severity, reducing the risk, or inhibiting ovarian cancer; p) methods of treating, suppressing, reducing, inhibiting, eliminating, delaying or preventing cancer metastasis in an individual having cancer; or q) a method of treating, suppressing, reducing, inhibiting, eliminating, delaying or preventing secondary cancer resistance to a taxane drug in a subject previously treated with a taxane drug having cancer, the method comprising administering to the subject a compound of the invention and/or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, hydrate, N-oxide, polymorph or crystal of the compound, or any combination thereof, and at least one of a BRAF inhibitor or a MEK inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a compound of the invention and/or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, hydrate, N-oxide, polymorph or crystal of said compound, or any combination thereof, and a BRAF inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a compound of the invention and/or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, hydrate, N-oxide, polymorph or crystal of said compound, or any combination thereof, and a MEK inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a compound of the invention and/or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, hydrate, N-oxide, polymorph or crystal of the compound, or any combination thereof, and a BRAF inhibitor and a MEK inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering at least one of a BRAF inhibitor or a MEK inhibitor, in combination with a tubulin inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a tubulin inhibitor in combination with a BRAF inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a tubulin inhibitor in combination with a MEK inhibitor; or a composition comprising the same. In another embodiment, the method comprises administering a tubulin inhibitor in combination with a BRAF inhibitor and a MEK inhibitor; or a composition comprising the same.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a BRAF mutant cancer in a subject, comprising administering to a subject having a BRAF mutant cancer a composition comprising a BRAF inhibitor, a MEK inhibitor, or a combination thereof, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is a drug-resistant cancer. In another embodiment, the cancer is resistant to a BRAF inhibitor. In another embodiment, the cancer is resistant to a taxane. In another embodiment, the cancer is resistant to docetaxel. In another embodiment, the compounds of the present invention are of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a BRAF inhibitor-resistant cancer in a subject, the method comprising administering to a subject having a BRAF inhibitor-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor under conditions effective to treat the cancer; and a compound of the invention or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer or isomer thereof. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of the compound, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the compounds of the present invention are of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting vemurafenib-resistant cancer in a subject, comprising administering to a subject having a vemurafenib-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor under conditions effective to treat the cancer; and a compound of the invention or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer or isomer thereof. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the cancer has secondary resistance to a taxane. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting melanoma in a subject, comprising administering to a subject having melanoma a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the melanoma. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the melanoma is drug resistant. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the melanoma is metastatic melanoma. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting thyroid cancer in a subject, comprising administering to a subject having thyroid cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the thyroid cancer. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the thyroid cancer is drug resistant. In another embodiment, the thyroid cancer is a metastatic cancer. In another embodiment, the compounds of the present invention are of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting ovarian cancer in a subject, comprising administering to a subject having ovarian cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the ovarian cancer. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the ovarian cancer is drug resistant. In another embodiment, the ovarian cancer is a metastatic cancer. In another embodiment, the compounds of the present invention are of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting colorectal cancer in a subject, the method comprising administering to a subject having colorectal cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat colorectal cancer. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the colorectal cancer is drug resistant. In another embodiment, the colorectal cancer is a metastatic cancer. In another embodiment, the compounds of the present invention are of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting drug-resistant melanoma in a subject, comprising administering to a subject having drug-resistant melanoma a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the melanoma. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the melanoma is metastatic melanoma. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer in a subject, comprising administering to a subject having a drug-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof, under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is V600E positive melanoma. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of overcoming resistance to treatment with a BRAF inhibitor in a subject having a drug-resistant cancer, the method comprising administering to the subject having a drug-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is V600E positive melanoma. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the invention relates to a method of preventing, eliminating, reducing, or delaying resistance to cancer treatment in a subject having cancer, the method comprising administering to a subject having resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a compound of the present invention, or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof. In another embodiment, the combination consists essentially of a compound of the invention and a BRAF inhibitor. In another embodiment, the combination consists essentially of a compound of the invention and a MEK inhibitor. In another embodiment, the combination consists essentially of a compound of the invention, a BRAF inhibitor, and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is metastatic. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is V600E positive melanoma. In another embodiment, the compound is a compound of formulas I-IV. In another embodiment, the compound of the present invention is compound 17 ya. In another embodiment, the compound of the invention is compound 12 da.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a BRAF mutant cancer in a subject, the method comprising administering to a subject having a BRAF mutant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the cancer is a drug-resistant cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is resistant to a BRAF inhibitor.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a BRAF inhibitor-resistant cancer in a subject, the method comprising administering to a subject having a BRAF inhibitor-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma.

In one embodiment, the invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting vemurafenib-resistant cancer in a subject, comprising administering to a subject having vemurafenib-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the melanoma is V600E positive melanoma.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting melanoma in a subject, the method comprising administering to a subject having melanoma a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the melanoma. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the melanoma is drug resistant. In another embodiment, the melanoma is metastatic melanoma. In another embodiment, the melanoma is V600E positive melanoma.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting thyroid cancer in a subject, comprising administering to a subject having thyroid cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the thyroid cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the thyroid cancer is drug resistant. In another embodiment, the thyroid cancer is metastatic.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting colorectal cancer in a subject, the method comprising administering to a subject having colorectal cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat colorectal cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the colorectal cancer is drug resistant. In another embodiment, the colorectal cancer is metastatic.

In one embodiment, the invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting ovarian cancer in a subject, the method comprising administering to a subject having ovarian cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the ovarian cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the ovarian cancer is drug resistant. In another embodiment, the ovarian cancer is a metastatic cancer.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting drug-resistant melanoma in a subject, the method comprising administering to a subject having drug-resistant melanoma a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the melanoma. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the melanoma is V600E positive melanoma. In another embodiment, the melanoma is metastatic.

In one embodiment, the present invention relates to a method of treating, suppressing, reducing the severity of, reducing the risk of, or inhibiting a drug-resistant cancer in a subject, the method comprising administering to a subject having a drug-resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor under conditions effective to treat the cancer. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is V600E positive melanoma.

In one embodiment, the invention relates to a method of overcoming resistance in a subject to treatment with a BRAF inhibitor, the method comprising administering to a subject having resistant cancer a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and a tubulin inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a BRAF inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor and a MEK inhibitor. In another embodiment, the combination consists essentially of a tubulin inhibitor, a BRAF inhibitor and a MEK inhibitor. In another embodiment, the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof. In another embodiment, the BRAF inhibitor is vemurafenib. In another embodiment, the BRAF inhibitor is dabrafenib. In another embodiment, the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, pemitinib, CI-1040, or any combination thereof. In another embodiment, the MEK inhibitor is trametinib. In another embodiment, the MEK inhibitor is RO 5068760. In another embodiment, the tubulin inhibitor is paclitaxel, epothilone, docetaxel, discodermolide, colchicine, combretastatin, 2-methoxyestradiol, methoxybenzenesulfonamide (E7010), vinblastine, vincristine, vinorelbine, vinflunine, dolastatin, halichondrin, hamiltrin, cryptophysin 52, taxol, or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel, colchicine, vinblastine, taxol or any combination thereof. In another embodiment, the tubulin inhibitor is docetaxel. In another embodiment, the cancer is melanoma, thyroid cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), colorectal cancer, or ovarian cancer. In another embodiment, the cancer is a metastatic cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is V600E positive melanoma.

The compounds of the invention are useful for treating, reducing the severity of, reducing the risk of, or inhibiting cancer, metastatic cancer, drug-resistant tumors, drug-resistant cancer, and various forms of cancer. In a preferred embodiment, the cancer is a skin cancer (e.g., melanoma), thyroid cancer, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, lung cancer, colon cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), leukemia, lymphoma, head and neck cancer, pancreatic cancer, esophageal cancer, renal cancer, or CNS cancer (e.g., glioma, glioblastoma). The embodiments herein provide support for the treatment of these various cancers. Furthermore, based on their recognized mode of action as tubulin inhibitors, it is believed that administration of the compounds or compositions of the present invention to a patient will also treat or prevent other forms of cancer. Preferred compounds of the invention are selectively destructive to cancer cells, which results in ablation of cancer cells, but preferably does not result in ablation of normal cells. It is important that damage to normal cells is minimized because cancer cells are susceptible to destruction at very low concentrations of the compounds of the invention.

In some embodiments, the present invention provides the use of a compound described herein, or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, polymorph, crystal, N-oxide, hydrate, or any combination thereof, in combination with at least one of a BRAF inhibitor or a MEK inhibitor, to treat, suppress, reduce the severity of, reduce the risk of, or inhibit cancer in a subject. In another embodiment, the cancer is a skin cancer (e.g., melanoma), thyroid cancer, colorectal cancer, ovarian cancer, adrenocortical cancer, anal cancer, bladder cancer, brain tumor, brain stem tumor, breast cancer, glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, pineal tumor, hypothalamic glioma, carcinoid tumor, carcinoma, cervical cancer, colon cancer, Central Nervous System (CNS) cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing's family tumor (Pnet), extracranial blastoma, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer, blastoma, extragonadal tumor, gestational trophoblastic tumor, head and neck cancer, hypopharynx cancer, islet cell cancer, larynx cancer, leukemia, acute lymphocytic leukemia, oral cancer, liver cancer, adrenal gland cancer, prostate cancer, Lung cancer, non-small cell lung cancer, lymphoma, AIDS-related lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, Merkel cell carcinoma, metastatic squamous cell carcinoma, multiple myeloma, plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative diseases, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal carcinoma, osteosarcoma, epithelial ovarian carcinoma, ovarian germ cell tumor, ovarian low grade malignant tumor, pancreatic cancer, exocrine pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile carcinoma, pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, kidney cancer, renal cell carcinoma, salivary gland carcinoma, Sezary syndrome, cutaneous T-cell lymphoma, human immunodeficiency virus, Skin cancer, kaposi's sarcoma, skin cancer, melanoma, small intestine cancer, soft tissue sarcoma, testicular cancer, thymoma, malignancy, urinary tract cancer, uterine cancer, sarcoma, unusual cancer in children, vaginal cancer, vulvar cancer, nephroblastoma, or any combination thereof. In another embodiment, the subject has been previously treated with chemotherapy, radiation therapy, or biological therapy.

In some embodiments, the present invention provides the use of a compound described herein, or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, polymorph, crystal, N-oxide, hydrate, or any combination thereof, in combination with at least one of a BRAF inhibitor or a MEK inhibitor, to treat, suppress, reduce the severity of, reduce the risk of, or inhibit metastatic cancer in a subject. In another embodiment, the cancer is a skin cancer (e.g., melanoma), thyroid cancer, colorectal cancer, ovarian cancer, adrenocortical cancer, anal cancer, bladder cancer, brain tumor, brain stem tumor, breast cancer, glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, pineal tumor, hypothalamic glioma, carcinoid tumor, carcinoma, cervical cancer, colon cancer, Central Nervous System (CNS) cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing's family tumor (Pnet), extracranial blastoma, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer, blastoma, extragonadal tumor, gestational trophoblastic tumor, head and neck cancer, hypopharynx cancer, islet cell cancer, larynx cancer, leukemia, acute lymphocytic leukemia, oral cancer, liver cancer, adrenal gland cancer, prostate cancer, Lung cancer, non-small cell lung cancer, lymphoma, AIDS-related lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, Merkel cell carcinoma, metastatic squamous cell carcinoma, multiple myeloma, plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative diseases, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal carcinoma, osteosarcoma, epithelial ovarian carcinoma, reproductive cell tumor of the ovary, low grade ovarian malignancy, pancreatic cancer, exocrine pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, kidney cancer, renal cell carcinoma, salivary gland carcinoma, Sezary syndrome, skin cancer, cutaneous T-cell lymphoma, lymphomas, and other cancers, Skin cancer, kaposi's sarcoma, melanoma, small bowel cancer, soft tissue sarcoma, testicular cancer, thymoma, malignancy, cancer of the urethra, cancer of the uterus, sarcoma, unusual cancer in children, cancer of the vagina, cancer of the vulva, nephroblastoma, or any combination thereof.

In some embodiments, the present invention provides the use of a compound described herein, or an isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, tautomer, polymorph, crystal, N-oxide, hydrate thereof, or any combination thereof, in combination with at least one of a BRAF inhibitor or a MEK inhibitor, to treat, suppress, reduce the severity of, reduce the risk of, or inhibit a drug-resistant or resistant cancer in a subject. In another embodiment, the cancer is a skin cancer (e.g., melanoma), thyroid cancer, colorectal cancer, ovarian cancer, adrenocortical cancer, anal cancer, bladder cancer, brain tumor, brain stem tumor, breast cancer, glioma, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, pineal tumor, hypothalamic glioma, carcinoid tumor, carcinoma, cervical cancer, colon cancer, Central Nervous System (CNS) cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing's family tumor (Pnet), extracranial blastoma, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer, blastoma, extragonadal tumor, gestational trophoblastic tumor, head and neck cancer, hypopharynx cancer, islet cell cancer, larynx cancer, leukemia, acute lymphocytic leukemia, oral cancer, liver cancer, adrenal gland cancer, prostate cancer, Lung cancer, non-small cell lung cancer, lymphoma, AIDS-related lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous cell carcinoma, multiple myeloma, plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disease, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal carcinoma, osteosarcoma, epithelial ovarian carcinoma, ovarian germ cell tumor, ovarian low grade malignancy, pancreatic cancer, exocrine pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid gland cancer, penile cancer, pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma, salivary gland carcinoma, sezary syndrome, skin cancer, lymphoma, chronic lymphocytic leukemia, lymphomatosis, myelodysplasia, and sarcoidosis, Cutaneous T-cell lymphoma, kaposi's sarcoma, skin cancer, melanoma, small intestine cancer, soft tissue sarcoma, testicular cancer, thymoma, malignancy, urinary tract cancer, uterine cancer, sarcoma, childhood unusual cancer, vaginal cancer, vulvar cancer, nephroblastoma, or any combination thereof.

In one embodiment, "metastatic cancer" refers to cancer that spreads (metastasizes) from its initial site to another area of the body. Almost all cancers have the potential to spread. Whether metastasis will occur depends on a complex interaction between a number of tumor cell factors, including the type of cancer, the degree of maturation (differentiation) of the tumor cells, the location and how long the cancer has been present, and other factors that are not fully understood. There are three ways in which metastasis spreads-local spread from the tumor to surrounding tissue, through the bloodstream to distant sites, or through the lymphatic system to adjacent or distant lymph nodes. Each cancer may have a representative route of spread. Tumors are named according to primary site (e.g., breast cancer that has spread to the brain is referred to as metastatic breast cancer that metastasizes to the brain).

In one embodiment, "drug-resistant cancer" refers to cancer cells that acquire resistance to chemotherapy. Cancer cells can acquire resistance to chemotherapy through a range of mechanisms, including mutation or overexpression of drug targets, inactivation of drugs, or elimination of drugs from the cells. Tumors that recur after an initial response to chemotherapy may develop resistance to multiple drugs (they are multidrug resistant). The conventional view of resistance is that one or several cells in a tumor population acquire genetic changes that confer resistance. Therefore, the cause of drug resistance is, among others: a) some cells that are not killed by chemotherapy mutate (change) and become resistant to the drug. Once they proliferate, there may be more resistant cells than cells that are sensitive to chemotherapy; b) and (4) gene amplification. Cancer cells can produce hundreds of copies of a particular gene. This gene triggers the overproduction of proteins that render anticancer drugs ineffective; c) cancer cells can use molecules called p-glycoproteins to pump drugs out of the cell at as fast a rate as they enter; d) cancer cells may stop absorbing drugs due to the failure of proteins that transport the drug across the cell wall; e) cancer cells may learn how to repair DNA breaks caused by some anticancer drugs; f) cancer cells may develop mechanisms that inactivate drugs. One major cause of multidrug resistance is the overexpression of P-glycoprotein (P-gp) or other drug efflux pumps (OAT, OCT, BCRP, etc.). This protein is a clinically important transporter protein belonging to the cell membrane transport ATP-binding cassette family. It can pump substrates, including anticancer drugs, out of tumor cells through ATP-dependent mechanisms. Therefore, resistance to anticancer agents used in chemotherapy is a major cause of therapeutic failure in malignant disorders, resulting in tumors becoming resistant. Drug resistance is a major cause of cancer chemotherapy failure.

In one embodiment, "drug resistant cancer" refers to a drug resistant cancer as described above. In another embodiment, "drug resistant cancer" refers to a cancer cell that has acquired resistance to any treatment (e.g., chemotherapy, radiation therapy, or biological therapy).

In one embodiment, the present invention relates to treating, suppressing, reducing the severity, reducing the risk of or inhibiting cancer in a subject, wherein the subject has been previously treated with chemotherapy, radiation therapy or biological therapy.

In one embodiment, "chemotherapy" refers to the chemotherapy of cancer with, for example, drugs that kill cancer cells directly. Such drugs are known as "anti-cancer" drugs or "anti-tumor drugs". Current therapies use more than 100 drugs to treat cancer. Can be used for treating specific cancers. Chemotherapy is used to control the growth of tumors when they are unlikely to be cured; shrinking tumors prior to surgery or radiation therapy; relief of symptoms (e.g., pain); and destruction of microscopic cancer cells that may be present after removal of a known tumor by surgery (known as adjuvant therapy). Adjuvant therapy is given to prevent possible cancer recurrence.

In one embodiment, "radiotherapy" refers to high energy X-rays and similar rays (e.g., electrons) that treat a disease. Many cancer patients will undergo radiation therapy as part of their treatment. This may be administered in the form of external radiotherapy performed in vitro using X-rays or in vivo radiotherapy performed in vivo. Radiation therapy works by destroying cancer cells in the treatment site. Although normal cells may also be damaged by radiation therapy, they can often repair themselves. Radiation therapy can cure some cancers and also reduce the chances of cancer recurrence after surgery. It can be used for alleviating symptoms of cancer.

In one embodiment, "biotherapy" refers to a substance that destroys cancer cells that occurs naturally in the body. There are several types of treatments, including: monoclonal antibodies, cancer growth inhibitors, vaccines and gene therapy. Biological therapy is also known as immunotherapy.

Yet another aspect of the invention relates to a method of treating or preventing a cancerous condition, the method comprising: compositions comprising a compound of the invention, e.g., a compound of formula I, II, III or IV or 17ya or 12da, and at least one of a BRAF inhibitor or a MEK inhibitor are provided, followed by administering to the patient an effective amount of the compositions in a manner effective to treat or prevent the cancerous condition.

According to one embodiment, the patient to be treated is characterized by the presence of a precancerous condition, and administration of the composition is effective to prevent progression of the precancerous condition to a cancerous condition. This can be done by destroying the precancerous cells before or while they have further progressed to a cancerous state.

According to another embodiment, the patient to be treated is characterized by the presence of a cancerous condition, and administration of the composition is effective to cause regression of the cancerous condition or to inhibit growth of the cancerous condition, i.e., to stop its growth altogether or to reduce its growth rate. This is preferably done by destroying the cancer cells, regardless of their location within the patient. I.e., whether the cancer cells are located at the site of a primary tumor or whether the cancer cells have metastasized and produced a secondary tumor in the patient.

As used herein, an individual or patient refers to any mammalian patient, including but not limited to humans and other primates, dogs, cats, horses, cows, sheep, pigs, rats, mice, and other rodents. In one embodiment, the individual is male. In another embodiment, the individual is female. In some embodiments, the methods described herein can be used to treat both males and females.

When a compound or pharmaceutical composition of the invention is administered to treat, suppress, reduce the severity of, reduce the risk of, or inhibit a cancerous condition, the pharmaceutical composition may also contain, or be administered in conjunction with, other therapeutic agents or regimens now known or later developed for the treatment of, various types of cancer. Examples of other therapeutic agents or treatment regimens include, but are not limited to, radiation therapy, immunotherapy, chemotherapy, surgical intervention, and combinations thereof.

The following examples are presented in order to more fully illustrate preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

Examples

The following examples are set forth for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1

Use for treating BRAF mutant blacksCompound 12da or 17ya and veova for melanoma and vemurafenib resistant cancers Combination of rofenib

Vemurafenib is a novel anti-melanoma drug approved for the V600E mutant, but develops resistance over about 9 months. Several tubulin inhibitors, including compounds 12da and 17ya, were screened to evaluate their antiproliferative combined effect with vemurafenib on parental a375 and MDA-MB-435 cells, both BRAF V600E mutant cell lines. These combinations may help to overcome resistance.

In a group of BRAFs^V600EThe hypothesis of synergistic cell cycle arrest of combinations of vemurafenib with 12da or docetaxel was tested in mutant parent melanoma cell lines and long-term selected vemurafenib-resistant a375RF21 sublines (Su F, BradleyWD, Wang Q et al, "Resistance to selective BRAF inhibition of medium recent upper stream pathway activation." Cancer Res. (2012) 72: 969-. Established vemurafenib resistant a375RF21 cells were used in vitro and in vivo as disease relapse models to test whether the proposed synergistic drug combinations have potential therapeutic benefit in relation to clinical vemurafenib resistance.

Materials and methods

Reagents and cell lines

Vemurafenib (also known as PLX4032, RG7204 or RO5185426), trametinib, sunitinib (malate) and docetaxel were purchased from LC Laboratories (Woburn, MA). Compound 12da was synthesized internally as described below. Compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, st. louis, MO) to prepare 10mM stock solutions. Human melanoma a375 cell line was obtained from ATCC (Manassas, VA). WM164 and MDA-MB-435 cells were obtained from Dr.Meenhard Herlyn (Wistar Institute, Philadelphia, Pa.) and Dr.Robert Clarke (Georgetown University, Washington, DC), respectively. All cell lines were validated before use in the study. Cells were cultured in DMEM medium (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA), 1% antibiotic/antimycotic mixture (Sigma-Aldrich, st. louis, MO) and 5 μ g/mL bovine insulin (Sigma-Aldrich, st. louis, MO).

In vitro acquired vemurafenib resistance

Melanoma cell lines with acquired Verofinib Resistance were long-term selected by culturing parent A375 cells in increasing concentrations of Verofinib for at least three months according to the reported method ("Resistance to selective BRAF inhibition can conditioned by modified upstream pathway activation." (2012) 72: 969-978 by Sun F, Bradley WD, Wang Q et al. IC of the isolated drug-resistant A375RF21 cell line on Verofinib₅₀Stably increased by more than 50-fold (28.9. + -. 0.6. mu.M for A375RF21 cells, FIG. 1, compared to 0.57. + -. 0.03. mu.M for the parental A375 cell line, as determined by the MTS assay). Drug-resistant a375RF21 cell lines were maintained in complete growth medium containing 2.5 μ M vemurafenib.

Cell proliferation and in vitro combinatorial assays

Cell proliferation viability was investigated using MTS or SRB assays as described previously. An in vitro study of the combination of vemurafenib and tubulin inhibitors was designed and performed in five replicates per group treatment using CalcuSyn software (Biosoft, Ferguson, MO). IC based on each test drug from pilot study₅₀The value is selected to select the drug concentration. Synergy, additivity or antagonism was determined by the Chou-Talalay method (Chou TC. "Drug combination study and the same synergy quantification using the Chou-Talalay method," Cancer Res. (2010) 70: 440-.

Cell cycle analysis

Flow cytometry analysis was as previously described (Wang Z, Chen J, Wang J et al, "Novel tubulin polymerization inhibitors over communication multiplex resistance and reductionmelatomalkung metastasis, "pharm. res. (2012) 29: 3040 and 3052). To determine G₂And cell cycle distribution in M phase (FIG. 23), harvesting of cells with trypsin using anti-phospho histone H3-The 488 antibody was stained on ice in the dark for 1 hour, then stained with PI/RNase solution at room temperature in the dark for 30 minutes according to the manufacturer's instructions (# FCCH025103, EMD Millipore Corp., Ballerica, MA). The data were further processed and charted using the Modfit 2.0 program (Verity Software House, Topsham, ME).

Tubulin polymerization assay

HTS-tubulin polymerization assays were performed using commercially available kits as described previously according to the manufacturer's instructions (# BK004P, cytosketon, Denver, CO). Bovine brain tubulin (0.4mg) was mixed with 5. mu.M 12da, 20. mu.M vemurafenib or a combination of the two agents and mixed in 110. mu.L of universal tubulin buffer (80mM PIPES, 2.0mM MgCl)₂0.5mM EDTA and 1mM GTP) at pH 6.9. Absorbance at 340nm was recorded dynamically every 1min at 37 ℃ for 45min by an SYNERGY HT microplate reader (Bio-Tek instruments, Winooski, VT). Data from single or combination treatments were compared to a positive control group of 10 μ M colchicine.

Western blot analysis

At the indicated treatment times, human melanoma A375RF21, MDA-MB 435 or WM164 cells were harvested for investigation of relevant cascade proteins or apoptotic markers by Western blotting. Total protein was extracted by lysis of cells with RIPA buffer (Sigma-Aldrich, st.louis, MO) containing phosphatase-protease inhibitor cocktail (Sigma-Aldrich, st.louis, MO). Then, the general protein concentration was determined by the BCA method using a kit (Sigma-Aldrich, st. Cell lysates were diluted to equal general protein concentrations using Laemmli loading buffer (Bio-Rad, Hercules, Calif.) andboiling for 5 minutes to denature the protein then loading the group containing 10. mu.g of the general protein into wells of a 4-15% Tris-HCl preformed polyacrylamide gel (Bio-Rad, Hercules, CA) for electrophoresis followed by transfer to a 0.2. mu.m nitrocellulose membrane (Bio-Rad, Hercules, CA) after blocking with 5% bovine serum albumin in 1 XTST at room temperature for 1 hour, the membrane was further incubated with the following primary rabbit antibody (Cell Signaling technology, Danvers, MA) alone at 4 ℃ overnight anti-phospho-ERK 1/2(Thr202/Tyr 204;. 9101), anti-p 44/42MAPK (ERK 1/2;. Danvers 9102), anti-phospho-AKT (Ser 473;. 929271), anti-AKT #9272), anti-cyclin D5 (G3938964), anti-caspase # Asp #9433), anti-caspase # Asp # 4133, anti-caspase # Asp #31, anti-caspase # Asp # 4178, anti-caspase # Asp #9433, anti-caspase # Asp # 3633, anti-Asp #9433, anti-Asp # 48, anti-Asp #33, anti-Asp #33, Asp #3, and Asp #9, and Asp #The reagents (Cell Signaling, #7003) were incubated for 1 minute for detection and exposed to X-ray film. The films were scanned in gray scale and lane intensities quantified using ImageJ software (NIH, Bethesda, MD, USA).

Apoptosis detection

A375RF21 cells were seeded in 6-well plates (1X 10 per well)⁶Individual) and treated with growth medium containing 5% DMSO, vemurafenib, 12da, docetaxel, or the indicated combination. After 48 hours of incubation, apoptosis assays were performed using Annexin V-FITC apoptosis detection kit (Abcam, Cambridge, MA) according to the manufacturer's instructions and by BDLSR-II flow cytometer (BD Biosciences, San Jose, Calif.).

Tumor xenografts and treatments

7-8 week old male nude mice were obtained from Charles River Laboratories International publicPurchased from department (Wilmington, MA). A375RF21 cells were suspended in ice-cold phenol red-free and FBS-free DMEM medium and mixed with high concentrations of Matrigel (BD Biosciences, San Joes, Calif.) at a ratio of 1: 1 prior to use. 100 μ L of the extract containing 3X 10⁶This mixture of individual cells was injected subcutaneously (s.c.) into the left dorsal flank of each mouse. One week after vaccination, mice were randomized into 4 groups (n-7 for the initial low dose and n-5 for the subsequent high dose drug combination) and treatment was initiated. Compound 12da or vemurafenib was diluted in PEG300(Sigma-Aldrich, st.louis, MO) and administered once daily by intraperitoneal (i.p.) injection five days a week for three consecutive weeks. Vehicle control groups were i.p. injected with the same volume (100 μ Ι _) of PEG300 at the same dosing frequency. At the end of the experiment, mice were euthanized and tumor tissue was isolated, weighed, and then fixed in 10% buffered formalin phosphate solution.

Tumor volume and body weight of each mouse were evaluated three times per week. Using the formula a x b²X 0.5 tumor volume was calculated, where a and b represent the larger and smaller tumor diameters. Data are shown as mean ± SD for each group and plotted as a function of time. Tumor Growth Inhibition (TGI) was calculated as 100 × [ (T-T)₀)/(C-C₀)]And tumor regression was calculated as (T-T)₀)/T₀X 100, wherein T, T₀C and C₀Mean tumor volume for a particular group on the last day of treatment, mean tumor volume for the same group on the first day of treatment, mean tumor volume for the vehicle control group on the last day of treatment, and mean tumor volume for the vehicle control group on the first day of treatment, respectively.

Pathology and immunohistochemistry analysis

Tumor tissue fixed in formalin buffer for more than one week was stained with hematoxylin and eosin. For Immunohistochemical (IHC) analysis, the following primary antibodies were used: rabbit anti-Ki 67, anti-phospho-AKT (Ser473) and anti-phospho-ERK 1/2(Thr202/Tyr204) (# 9027; # 4060; # 4376; Cell signalling Technology, Danvers, MA). anti-S100 primary antibodies were purchased from Abcam (# ab868, Abcam corporation, Cambridge, MA). The analysis was performed according to the manufacturer's protocol.

Statistical analysis

Data were analyzed using Prism Software 5.0(GraphPad Software, San Diego, Calif.). For the in vitro apoptosis assay and xenograft studies, statistical significance (P < 0.05) was assessed by Mann-Whitney rank test, nonparametric t-test, and one-way ANOVA. Accordingly, the treatment group was compared to the vehicle group, and the combination treatment group was compared to the group receiving single agent treatment.

Results

Combination of vemurafenib with tubulin inhibitors 17ya and 12da in parental and vemurafenib resistant melanin A strong synergy was shown in both tumor cell lines.

Several tubulin inhibitors, including compounds 12da and 17ya, were screened to evaluate their antiproliferative combined effect with vemurafenib on parent a375 and MDA-MB-435 cells, both BRAF^V600EA mutant cell line. Two well-known tubulin inhibitors, docetaxel and colchicine, were included for comparison (table 1 and figure 24). It was found that for the combination of 12da and Vemurafenib, at its ED₅₀Next, the CI values calculated were as low as 0.32 (in the A375 cell line) and 0.10 (in the MDA-MB-435 cell line). Furthermore, MEKi (trametinib) and general receptor tyrosine kinase inhibitors (RTKi; sunitinib) showed only additive CI values (Table 1).

Based on the following results, these combinations can help to overcome the resistance seen in patients treated with vemurafenib.

Both 17ya and 12da showed synergistic effects when combined with vemurafenib or docetaxel. This work began with 17ya, but for in vivo studies, transferred to 12 da. The concept of Combination Index (CI) was introduced. Essentially, a CI value of < 1.0 indicates synergy, a value of 1.0 indicates additive effect, and a value of > 1.0 indicates less than additive or antagonistic effect.

Table 1. combination of vemurafenib with tubulin inhibitors showed synergistic effect in parental and vemurafenib resistant melanoma cell lines. The combination of vemurafenib and tubulin inhibitors maintained a strong synergistic effect in this resistant cell line (CI < 0.9), but Vem + MEKi or Vem + RTKi was only additive. Combination Index (CI) values were calculated based on the results of the cell viability MTS assay (n-5). CI < 0.9 indicates synergy; CI is more than or equal to 0.9 and less than or equal to 1.1, which represents additive effect; CI > 1.1 indicates antagonism between the two drugs tested. From parental A375 cells (IC)₅₀0.57 μ M) stable high verrofenib resistance a375RF21 subline (IC)₅₀＝28.9μM)。

ND-not determined

In order to achieve this goal, parental a375 human melanoma cells were used to establish vemurafenib-resistant a375RF21 subfamily by long-term selection according to literature-reported methods (Su F, Bradley WD, Wang Q et al, "Resistance to selective BRAF inhibition copy proximal activity. (2012) 72: 969-978) in order to establish vemurafenib-resistant a375RF21 subfamily using a375 and a375RF21 cells for western blot analysis to determine that differential protein activation known to cause vemurafenib Resistance was observed in 2.5 μ M vemurafenib (maintenance concentration of its culture medium) cells with no change in PDGF receptor.

As shown in table 1, the drug combination studies repeated using a375RF21 cells all yielded calculated CI values of less than 0.9 (range: 0.53-0.70) for compound 12da combined with vemurafenib, indicating a strong synergy at all concentrations tested. At ED₅₀Next, all three tubulin inhibitors act in a synergistic manner with vemurafenib. The CI values of the docetaxel or colchicine groups increased relatively rapidly with increasing drug concentration. At ED₉₀At the dose of (a), the combination of docetaxel and vemurafenib was almost additive (CI value of 0.90), whereas the combination effect of colchicine and vemurafenib had reversed to antagonistic (CI value of 1.36). Compound 12da, when combined with vemurafenib, showed a stronger synergy in drug resistant a375RF21 cells compared to the other two tubulin inhibitors.

It has surprisingly been found that in EC₅₀Next, the calculated CI values for the combination of 12da and vemurafenib were as low as 0.1 and 0.3. Since clinical tumor regression progressed extensively only 3-6 months after receiving vemurafenib chemotherapy, studies continued as to whether the synergistic effects mainly observed for combination therapy could be consistent for the vemurafenib resistant a375RF21 cell line.

Within the entire chemotherapeutic group tested, the B-RAF mutation inhibitor, vemurafenib, showed particularly synergistic cytotoxicity in MDA-MB-435 cells, which were originally considered breast cancer cells but are currently known as melanoma cells (Table 2), showed a CI value of 0.10 for vemurafenib +17ya, and at EC₇₅And EC₉₀The following also showed strong but lower synergy (table 5). This activity can be rationalized by the fact that the B-RAF inhibitor and the anti-tubulin agent (17ya) arrest the cell in different parts of the cell cycle, G, respectively₁And G₂(as discussed herein). Thus, dual targeting of mitosis should more completely drive mitotic cells towards cell death. Synergistic CI results are also shown in table 3, where a375 melanoma cells were used not only for 17ya, but also for other anti-tubulin agents such as colchicine, vinblastine and taxol. Limited synergy was observed with AKT inhibitors and no synergy was observed with MEK inhibitors (table 4).

TABLE 2 in EC₅₀Lower, CI value for 17ya in combination with multiple drugs.

CI: combination index

The additive effect is as follows: CI-1 synergy: CI < 1 antagonism: CI > 1

TABLE 3 CI values for A375 cell line.

TABLE 4 CI values for A375 cell line.

GSK 1120212: MEK inhibitors at IC₅₀The CI values for vemurafenib and GSK2126458(PI3K inhibitor) were 0.45 ± 0.13.

MK 2206: inhibitors of AKT in EC₉₀The CI values for Vemurafenib and PLX4032 are 0.384.

TABLE 5 CI values for MDA-MB-435 cell line.

12da and Verofini combination produced synergistic cell cycle arrest in A375RF21 cells (Table 1)

As a tubulin inhibitor binding to the colchicine site, Compound 12da effectively blocked the parent A375 cell line at G in a dose-dependent manner₂And a/M period. To determine whether the combination of compound 12da and vemurafenib arrested vemurafenib-resistant cells at different replication stages, cell cycle analysis was performed in a375RF21 cells. The data in figure 2B clearly demonstrate synergistic cell cycle arrest after 24 hours of exposure to a compound solution at the indicated concentrations. For the DMSO control, 50% of a375RF21 cells were distributed at G₀/G₁And is in S or G₂The percentage of cells in the/M phase was 12% or 32%, respectively. For compound 12da single treatment group at 20nM concentration, distribution at G₂The percentage of cells in the/M phase accumulated up to 70%. Production of similar G in drug-resistant A375RF21 cell line using vemurafenib as single agent₀/G₁Cell cycle arrest, the concentration of vemurafenib must be increased to 30 μ M or higher, compared to less than 1 μ M in the parent a375 cells. As expected, the combination of vemurafenib and compound 12da strongly arrested A375RF21 cells at G₀/G₁Period (48%) and G₂stage/M (43%). In addition, the combination treatment produced more cell debris, indicating an increase in cancer cell apoptosis. Treatment with a combination of vemurafenib and docetaxel produced similar synergistic effects.

Combination therapy induces significantly increased apoptotic cell death in vemurafenib resistant cells

To more clearly understand the possible apoptosis-inducing effects of combination therapy, annexin V and propidium iodide co-staining flow cytometry was used to distinguish live and apoptotic cells in a375RF 21. As expected, single agent treatment produced only moderate effects on inducing apoptosis at the tested concentrations; in contrast, the combined treatment group significantly enhanced apoptosis (fig. 3A). As shown in figure 3B (figure 3B quantifies the sum of the percentages of cells distributed in Q1 (early apoptosis), Q2 (apoptosis), and Q4 (dead cells)), the combination of compound 12da and vemurafenib resulted in a count of 50 ± 7.6% apoptosis or death, which is much higher than the simple sum of the apoptosis percentages of the two single-agent treatment groups (11.8 ± 3.0% for compound 12da, and 11.9 ± 3.5% for vemurafenib). Similar synergistic apoptosis-inducing effects were also observed in the docetaxel-containing combination-treated group (6.4 ± 3.0% for docetaxel group, 38.1 ± 2.6% for combination).

The combination moderates acquired vemurafenib resistance by downregulating pAKT or total AKT and activating the apoptotic cascade.

It has been determined that compound 12da targets tubulin polymerization (Chen J, Li CM, Wang J et al, "Synthesis and anti-catalytic activity of novel 2-aryl-4-benzoyl-imidazole derivatives targeting tubulin polymerization." bioorg. Med. chem. (2011) 19: 4782-^V600E. As a first approach to understand the responsible molecular mechanisms leading to this strong synergistic combination, it was investigated whether the synergy was mediated by the enhancement of the direct target of compound 12da or vemurafenib. As shown in fig. 4, vemurafenib itself has no effect on tubulin polymerization even at a high concentration of 20 μ M. Incorporation of vemurafenib into compound 12da polymerized tubulin compared to the single agent compound 12daThe inhibition of (a) is at most slightly increased. Inhibition of tubulin polymerization by combination therapy was achieved only with compound 12da, suggesting that synergistic combinations are not mediated by an enhancement of direct target inhibition by compound 12 da. Next, the combination pair pERK (Verofini inhibitory BRAF) was determined using Western blotting^V600EFlag of) has any effect. Fig. 5A shows that compound 12da or combination treatment had no significant effect on pERK or total ERK levels after 48 hours of incubation with a375RF21 resistant cells. Replacement of compound 12da with another tubulin inhibitor docetaxel gave similar results (fig. 5A). Thus, the synergistic combination is unlikely to pass BRAF^V600EEnhanced achievement of the suppression of (2).

Recently, Fei Su et al reported that pAKT levels were increased in A375 Verofenib resistant clones compared to their parent Verofenib sensitive cells (Su F, Bradley WD, Wang Q et al. "Resistance to selective BRAF inhibition can be used as a medium by test upstream pathwayation." Cancer Res. (2012) 72: 969-. Since a375RF21 cells also had strong pAKT activation (fig. 25A), it was speculated that combination therapy might produce its strong synergistic effect by down-regulating the activity of the AKT pathway in vemurafenib resistant a375RF21 cells. As shown in figure 5A, after 48 hours of incubation, pAKT and total AKT (takt) were both greatly reduced in the single-agent compound 12da or combination treatment group thereof, suggesting that synergistic antiproliferation may be mediated by targeting both ERK and AKT phosphorylation simultaneously. Docetaxel also reduced the expression of pAKT and tAKT and had similar effects in its combination therapy with vemurafenib. For example, in addition to a significant reduction in tAKT levels, the combination of compound 12da and vemurafenib also reduced pAKT levels to 61% (calculated from quantified relative multiples of lane density: 0.08/0.13X 100%) relative to tAKT, whereas single agent treatment only reduced pAKT levels to 77% (12da, 0.6/0.77X 10%) and 70% (vemurafenib, 0.34/0.48X 100%) relative to the corresponding tAKT levels, respectively. Of Compound 12daStrong dose-dependent pAKT/tAKT inhibitory effects on the other two BRAFs^V600EThe mutant cell lines WM164 and MDA-MB-435 were further confirmed (FIG. 5B). Decreased cyclin D1 levels in vemurafenib-resistant cells (fig. 5A), vemurafenib and the combination treatment group indicate G₀/G₁Cell cycle progression is inhibited. The apoptosis markers (cleaved PARP and cleaved caspase-3) are highly induced by tubulin inhibitors, whereas vemurafenib slightly increases their expression. The results are consistent with observations in apoptosis detection experiments.

Combination of vemurafenib and compound 12da to suppress vemurafenib-resistant tumor growth in a synergistic manner in vivo

To assess whether the strong synergy observed in vitro against a375RF21 cells could be transferred into vemurafenib resistant tumors in vivo, the effect of the combined efficacy on tumor growth was compared to the effect of single agent treatment. It has been previously determined that compound 12da effectively suppresses parent A375 melanoma tumor growth in vivo at a dose of 25mg/kg ("Novel tubulin polymerization inhibition compositions resistance and reduction tumor lysis". pharm. Res.29 (11): 3040-3052). Lead studies showed that vemurafenib resistant a375RF21 cells had similar growth kinetics to their parent a375 cells without drug treatment (fig. 26). To avoid any potential undesirable toxicity due to its combination with vemurafenib, the dose of compound 12da was reduced to 10 mg/kg.

Table 6 Tumor Growth Inhibition (TGI) and tumor weight comparison of the in vivo combination of vemurafenib and compound 12da in the drug resistant a375RF21 xenograft model (n ═ 7). The combination of compound 12da at 10mg/kg and vemurafenib at 20mg/kg achieves a higher antitumor activity (TGI) (. P < 0.05) compared to the simple addition of TGI for the two single agent treatment groups. The synergistic tumor inhibition continued after another week without further treatment (. P < 0.05).

As shown in figure 6 and table 6, vemurafenib (20mg/kg) monotherapy achieved only a very low (22.65%) TGI in this vemurafenib-resistant tumor model, and compound 12da alone (10mg/kg) achieved a slightly better TGI of 38.12%; in contrast, its combination treatment significantly enhanced tumor suppression to 88.56% after 3 weeks of treatment (fig. 6A, 6B and 6D, table 6). Three of the seven mice receiving combination therapy were maintained for an additional 7 days without further treatment, showing no significant (P ═ 0.2857) tumor recurrence and maintained 81.27% tumor suppression. None of the mice lost more than 10% of body weight in the four groups throughout the experiment (fig. 6C), indicating that these treatments were not severely toxic. When the mice were euthanized, major organs including brain, heart, kidney, liver, spleen and lung were isolated and subjected to pathological analysis, respectively. No abnormalities of these organs were observed. Taken together, these results strongly suggest that this combination therapy effectively helps to overcome acquired resistance to vemurafenib in the a375RF21 melanoma model and further demonstrate the synergistic antiproliferative effects observed in vitro.

To determine whether the down-regulation of AKT signalling observed in vitro by combination therapy also worked in vivo, immunohistochemical analysis was performed on tumor sections from all experimental groups. The activity of the ERK pathway was also determined and the level of proliferation indicated by the cellular marker Ki-67 in tumor sections was assessed. As demonstrated in fig. 6D, improved pathway and proliferation inhibition in the combination treatment group corresponded well to overall tumor response TGI results. ERK and AKT phosphorylation and Ki67 expression levels in the nucleus or cytoplasm were greatly reduced in the combination treatment group. Furthermore, the broad area of background powder stained by Matrigel in the H & E staining of tumor sections in the combination treatment group indicated that few, if any, tumor cells remained after the combination treatment. The significant reduction of melanoma cells in the combination treatment group was further confirmed by the reduction in density in the S100 immunostaining (fig. 6D).

Higher dose combinations of vemurafenib with compound 12da resulted in significant vemurafenib-resistant tumor regression Without observable toxicity

Since the results shown in figure 6 and table 6 were promising but did not appear to result in tumor regression, the experiment was repeated by increasing the dose of both compound 12da and vemurafenib by 50%. The results are shown in fig. 7 and table 7.

Table 7 Tumor Growth Inhibition (TGI) and tumor weight comparison of in vivo combinations of vemurafenib (30mg/kg) and compound 12da (15mg/kg) in the drug resistant a375RF21 xenograft model (n ═ 5).

Treatment group	TGI(％)	Tumor weight (g)
			Media	-	1.60±0.22
Virofenib 30mg/kg	28.10±4.81	1.05±0.21
			12da 15mg/kg	72.72±8.29	0.51±0.12
Virofenib 30mg/kg +12da 15mg/kg	103.38±1.42	0.08±0.03

The efficacy of vemurafenib was slightly increased (22.65% TGI at 20mg/kg versus 28.10% TGI at 30mg/kg), and the efficacy of compound 12da was greatly increased (38.12% TGI at 10mg/kg versus 72.72% TGI at 15 mg/kg). As shown in figure 7B, moderate tumor regression (44.9%) was evident in the combination treatment group at the increased drug dose, while no regression was seen at the lower dose. Taken together, these data provide the first convincing evidence: for patients with BRAF^V600EMutant melanoma patients, a combination of a novel tubulin inhibitor (e.g., compound 12da) with vemurafenib have the potential to overcome acquired resistance to vemurafenib.

Taken together, these studies strongly suggest that the combination of a BRAF inhibitor (e.g. vemurafenib) and a novel tubulin inhibitor (e.g. compound 12da) effectively overcomes acquired BRAF inhibitor resistance in BRAF mutant melanoma by several possible mechanisms including synergistic cell cycle arrest, enhanced apoptosis and strong inhibition of the AKT pathway. Such a combination appears to be more effective than vemurafenib in combination with a MEK inhibitor or an AKT inhibitor or an existing tubulin inhibitor, at least in vitro. Since the BRAFi + MEKi combination lacks sustained efficacy for melanoma, the development of such a combination strategy targeting the alternative pathway may have high impact in this area.

Discussion of the related Art

Although approved first for use with having a BRAF^V600EThe drug verafenib of mutated melanoma patients shows a significant response in initial treatment, but almost all patients taking this drug develop verafenib resistance within a few months (bolag G, Tsai J, Zhang J et al, "Vemurafenib: the first drug approved for BRAF-mutated cancer." nat. rev. drug Discov. (2012) 11: 873- > 886). Understanding the mechanisms of primary or acquired resistance and developing appropriate combinatorial strategiesA more effective method of overcoming such resistance may be provided. Preclinical studies and Clinical trials seeking effective Combinations of vemurafenib with other agents to eliminate or reduce melanoma tumor resistance to BRAF or MEK1/2 inhibitors are abundant (Greger JG, Eastman, Zhang V et al. "Combinations of BRAF, MEK, and PI3K/mTOR inhibitors over finding resistance to the BRAF inhibitor GSK2118436 barrier, modified by n r m e.s. mektation. mol. Cancer Ther (r) 3/mTOR inhibitors over which 11: 909. 920; Patel SP, Lazar AJ, papado NE et al. medium k. knot of" copolymers of selection "(AZD 6244; ARRY-142886) -basal strand NE et al." methyl ketone reaction of which 1. n. wo 9. k. weed, k. 1983. k. and k. 1. weed, n. k. repair. k. 2. repair. k. repair. n. wo. 11. wo. 1. k. repair. k. 2. the same. repair. the same strain of interest (r. wo; n. wo. 11. the same No. 11. 7. the same was found in et al. "biological reaction of (r. K. 2. the same." repair. the same. "K. 2. K. 2. the same, 2. K. 2. the same was found in et al." experiment of interest; 2. the same, 2. K.A. the same, 2. Attar N et al, "combining therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with discrete drives" (2011) 9: 76; pariiso KH, HaarbergHE, Wood E et al, "The HSP90inhibitor XL888 overrides BRAF overridor restristinanced blocked through reversed mechanisms" clin. 2502-2514; "BRAF inhibitor or antibody activity of adaptive cell immunology" Cancer Res (2012) 72: 3928-3937). Inhibitors of the RAF/MEK/ERK pathway primarily cause G₁Cell cycle arrest rather than melanoma tumor cell death. Thus, combinations of agents targeting different components in the same pathway (e.g., a combination of vemurafenib and a MEK1/2 inhibitor) while initially effective (Little AS, Smith PD, Cook SJ. "Mechanisms of acquired resistance to ERK1/2pathway inhibition," Oncogene (2012)32 (10): 1207-1215), pairs can be derived from these G₁Drug-resistant cells that escape from cell cycle arrest may not be able to maintain long-lasting synergy. Since one of the main hallmarks of tubulin inhibitors is their ability to strongly arrest cells at G₂phase/M, therefore, the combination of vemurafenib and tubulin inhibitors is believed to beSynergistically arresting melanoma cells, thereby achieving enhanced apoptosis and overcoming acquired drug resistance. In this study, the novel tubulin inhibitor compound 12da was selected to study its combination with vemurafenib against melanoma tumors. Vemurafenib resistant human melanoma cell line a375RF21 was generated and the combination of compound 12da and vemurafenib has been shown to have strong synergy in vitro. It was demonstrated that this synergy could not be achieved by an increase in the inhibition of tubulin polymerization or a decrease in p-ERK activation. In contrast, the results of the experiment show that the combination treatment is by synergy G₁And G₂Enhanced induction of apoptosis by/M cell cycle arrest and substantial impairment of survival signaling pathways associated with AKT phosphorylation to overcome acquired vemurafenib resistance. It has been shown that the strong synergy observed in vitro clearly translates into significant in vivo efficacy when tested in the vemurafenib resistant xenograft model. Further immunohistochemical analysis of tissue sections demonstrated strong inhibition of tumor proliferation and reduction of pAKT activity. Activation of The PI3K/AKT/mTOR signaling pathway has been shown to contribute to selective attenuation of ERK1/2 inhibition in human melanoma cell lines (bartholomeuz C, Gonzalez-Angulo AM. "Targeting The PI3K signaling pathway in cancer therapy," Expert opin. targets (2012) 16: 121 acids 130), and several recent studies have shown clearly synergistic combinations of inhibitors Targeting The PI3K/AKT/mTOR pathway and either BRAF inhibitors or MEK inhibitors (Liu R, Liu D, xingming. "The AKT inhibitor MK2206synergizes, but peroxisome agglutinizes, The BRAF (V600 yr 600E) inhibitor PLX 403x 2 and The MEK1/2 inhibitor of mTOR/2 inhibitor azt 34/2 in tissue 6244. nitrine 62182. t of cancer cell strain j 173. n. et al. Several novel classes of compounds have recently been reported AS inhibitors of tubulin polymerization, and they also exhibit strong inhibition of the AKT pathway (Krishnegowda G, Prakasha Gowda AS et al, "Synthesis and biological evaluation of a novel class of organic AS a dual inhibitor of tubulin polymerization and Akt pathway." bioorg.Med.chem. (2011) 19: 6006-6014; Viola G, Borthozzi R, Hamel E et al"MG-2477, a new tubulin inhibitor, antigens autophagythrough inhibition of the Akt/mTOR pathway and delayed apoptosis in A549cells," biochem. Pharmacol (2012) 83: 16-26; zhang C, Yang N, Yang CH et al, "S9, anovel anticipator agent, emerts anti-proliferative activity by interference with booth PI3K-Akt-mTOR signaling and microtubule cytoskeleton," PLoS One (2009) 4: e4881) In that respect In addition, the constitutively active PI3K/AKT pathway has been shown to lead to multidrug resistance to tubulin polymerizing agents (MTPA) targeting microtubules, and inhibition of the PI 3K/AKT-mediated signal transduction pathway has been shown to sensitize cancer cells to MTPA-induced apoptosis (Bhalla KN., "microtube-targeted antibody agents and apoptosis," Oncogene (2003) 22: 9075-9086). These studies indicate a close interaction between tubulin polymerization inhibitors and AKT down-regulation in cancer cells. In addition, it has recently been reported that The MEK inhibitor AZD6244 induces growth arrest and tumor regression of melanoma cells when combined with docetaxel (Haass NK, Sproesser K, Nguyen TK et al. "The mitogen activated protein/extracellular signal-regulated kinase inhibitor AZD6244(ARRY-142886) induced growth arm in tumor cells and tumor regression bound with docetaxel. Clin. cancer Res (2008) 14: 230. 239). Interestingly, the current results are consistent with these studies. The in vivo studies presented in the present invention show effective combination therapy in tumor cells that have been vemurafenib resistant by using the a375RF21 xenograft model. It is envisaged that if the combination is used before the tumour cells become resistant to vemurafenib, tumour regression may be more enhanced and the progression of resistant tumour cells may be significantly delayed or even prevented. This translates at least into a substantially longer progression free time and/or enhanced disease regression for the patient. In summary, the present study provides the first direct evidence and principle for combining potent tubulin inhibitors with inhibitors targeting the RAF/MEK/ERK pathway to greatly improve the treatment of melanoma patients.

Example 2

Synthesis of selected aryl-benzoyl-imidazole compounds

Preparation of 2-aryl-4, 5-dihydro-1H-imidazoles 14b, 14c, 14x (fig. 8).

To a solution of the appropriate benzaldehyde 8(b, c, x) (60mmol) in t-BuOH (300mL) was added ethylenediamine (66mmol) and stirred at RT for 30 min. Potassium carbonate (75mmol) and iodine (180mmol) were added successively to the reaction mixture, followed by stirring at 70 ℃ for 3 h. Sodium sulfite (Na) is added₂SO₃) And the mixture was extracted with chloroform. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (chloroform: methanol 20: 1) to give a white solid. Yield: 50 to 60 percent.

Preparation of 2-aryl-1H-imidazoles (9a-j, p, x; FIGS. 8 and 9).

Method a (only necessary for 9b, 9x, fig. 8): to a solution of 2-aryl-4, 5-dihydro-1H-imidazole 14b, x (35mmol) in DMSO (100mL) was added potassium carbonate (38.5mmol) and diacetoxyiodobenzene (38.5 mmol). The reaction mixture was stirred overnight in the dark. Water was added followed by extraction with dichloromethane. The organic layer was dried over magnesium sulfate and concentrated. The residue was subjected to flash column chromatography (hexane: ethyl acetate 3: 2) to give a white solid. Yield: 30 to 50 percent.

Method B (necessary only for 9 c; FIG. 8): to 2-aryl-4, 5-bisTo a solution of hydrogen-1H-imidazole 14c (50mmol) in DMF (70mL) was added DBU (55mmol) and CBrCl₃(55 mmol). The reaction mixture was stirred overnight and saturated NaHCO was added₃The (aqueous) solution was then extracted with dichloromethane. The organic layer was dried over magnesium sulfate and concentrated. The residue was subjected to flash column chromatography (chloroform: methanol 50: 1) to obtain a white solid. Yield: 7 percent.

Method C (necessary for 9a, 9d-j, 9 p; FIG. 9): to a solution of the appropriate benzaldehyde (8a, 8d-j, 8p) (100mmol) in ethanol (350mL) at 0 deg.C was added 40% aqueous glyoxal (12.8mL, 110mmol) and 29% aqueous ammonium hydroxide (1000mmol, 140 mL). After stirring at RT for 2-3 days, the reaction mixture was concentrated and the residue was subjected to flash column chromatography with dichloromethane as eluent to give the title compound as a yellow powder. Yield: 20 to 40 percent.

Preparation of 2-aryl-1- (phenylsulfonyl) -1H-imidazole (10a-j, p, x; FIGS. 8 and 9).

To a solution of 2-aryl-1H-imidazole 9a-j, p, x (20mmol) in anhydrous THF (200mL) at 0 deg.C was added sodium hydride (60% dispersion in mineral oil, 1.2g, 30mmol) and stirred for 30 min. Benzenesulfonyl chloride (2.82mL, 22mmol) was added and the reaction mixture was stirred overnight. With 100mL saturated NaHCO₃After dilution of the solution (aqueous), the reaction mixture was extracted with ethyl acetate (500 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 2: 1) to give a pale solid. Yield: 50 to 70 percent.

Preparation of aryl (2-aryl-1- (phenylsulfonyl) -1H-imidazol-4-yl) methanones (11aa-ai, ba, ca, cb, da, db, ea, eb, fa, fb, ga, gb, ha, hb, ia, ib, ja, jb, pa; FIGS. 8 and 9).

To a solution of 2-aryl-1- (phenylsulfonyl) -1H-imidazole (6.0mmol)10a-j, p, x in anhydrous THF (30mL) at-78 deg.C was added 1.7M t-butyllithium in pentane (5.3mL, 9.0mmol) and stirred for 10 min. The appropriate substituted benzoyl chloride (7.2mmol) was added at-78 deg.C and stirred overnight. The reaction mixture was taken up with 100mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (200 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 4: 1) to give a white solid. Yield: 15 to 40 percent.

General procedure for the preparation of aryl (2-aryl-1H-imidazol-4-yl) methanones (12aa-ai, ba, ca, cb, da, ab, ea, eb, fa, fb, ga, gb, ha, hb, ia, ib, ja, jb, pa; FIGS. 8 and 9).

To a solution of aryl (2-aryl-1- (phenylsulfonyl) -1H-imidazol-4-yl) methanone (2.0mmol)11aa-ai, ba, ca, cb, da, db, ea, eb, fa, fb, ga, gb, ha, hb, ia, ib, ja, jb, pa in THF (20.0mL) was added 1.0M tetrabutylammonium fluoride (4.0mmol) and stirred overnight. The reaction mixture was taken up with 50mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (100 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 3: 1) or recrystallized from water and methanol to give a white solid. Yield: 80-95 percent.

Preparation of (2- (4-hydroxyphenyl) -1H-imidazol-4-yl) (aryl) methanone (12ka, 12 kb; FIG. 9).

To a solution of (2- (4- (benzyloxy) phenyl) -1H-imidazol-4-yl) (aryl) methanone 12ja or 12jb (1mmol) in AcOH (20mL) was added concentrated HCl (2mL) and refluxed overnight. After removal of the solvent, the residue was recrystallized from dichloromethane to give the title compound as a yellow solid. Yield: 70-85 percent.

Preparation of (2-aryl-1H-imidazol-4-yl) (3, 4, 5-trihydroxyphenyl) methanone 13ea, 13fa, 13ha (FIG. 9).

To aryl (2-aryl-1H-imidazol-4-yl) methanones 12ea, 12fa or 12ha (0.5mmol) in CH₂Cl₂(6.0mL) was added to the solution in CH₂Cl₂1.0M BBr in (1)₃(2mmol) and stirred at RT for 1 h. Water addition to destroy excess BBr₃. The precipitated solid was filtered and recrystallized from MeOH to give a yellow solid. Yield: 60 to 80 percent.

Preparation of aryl (2-aryl-1H-imidazol-4-yl) methanone-HCl salt (12 db-HCl).

To a solution of 12db (0.5mmol) in methanol (20mL) was added a 2M solution of hydrogen chloride (5mmol) in ether and stirred at RT overnight. The reaction mixture was concentrated and the residue was taken up with CH₂Cl₂Washed to obtain the title compound. Yield: 95 percent.

Preparation of aryl (2-phenyl-1H-imidazol-1-yl) methanones (12aba, 12 aaa; FIG. 10).

To a solution of 2-phenyl-1H-imidazole 9a (10mmol) in THF (20mL) at 0 deg.C was added NaH (15mmol) and substituted benzoyl chloride (12 mmol). The reaction mixture was stirred overnight and saturated NaHCO was used₃The solution was diluted and then extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (chloroform) to give a white solid. Yield: 12 to 16 percent.

Preparation of 1-substituted- (2-phenyl-1H-imidazol-1-yl) -aryl-methanones (12dc, 12fc, 12daa, 12dab, 12cba, 11gaa, 12 la; FIGS. 11-12).

The synthesis of 12dc, 12fc and 12daa, 12dab and 12cba is summarized in FIG. 11. Compounds 12da, 12cb and 12fa were synthesized according to the synthesis described above and in figures 8 and 9. Treatment of 12da and 12fa with aluminium chloride provided p-demethylated 12dc, 12fc in which the 3, 5-dimethoxy is intact. Compound 12daa was prepared by benzylation of the N-1 position of 12 da. While methylation of the N-1 position of 12da and 12cb gives compounds 12dab and 12cba, respectively.

Synthesis of 12dc, 12fc, 12daa, 12dab, 12 cba: method d. (for 12dc and 12fc) [ fig. 11 ]:

R₁＝CH₃(12dc)

R_i＝C1(12fc)

to a solution of 12da and 12fa (200mg) in THF (20mL) was added aluminum chloride (10 equivalents). The reaction mixture was stirred overnight. Water was added, followed by extraction with ethyl acetate. The organic layer was dried over magnesium sulfate and concentrated. The residue was subjected to flash column chromatography (hexane: ethyl acetate 1: 1) to give a white-pale yellow solid. Yield: 60 to 80 percent.

Synthesis of 12daa, 12dab, 12cba, method E: [ FIG. 11 ]:

R₁＝Me；R₂＝Bn；R₃＝3，4，5-(OMe)₃(12daa)

R₁＝Me；R₂＝CH₃；R₃＝3，4，5-(OMe)₃(12dab)

R₁＝OMe；R₂＝CH₃；R₃＝F(12cba)

to a solution of 12da and 12cb (100mg) in THF (10mL) in an ice bath was added sodium hydride (1.2 equiv), followed by methyl iodide (for 12dab, 12cba) or benzyl bromide (for 12daa) (2 equiv). The resulting reaction mixture was stirred under reflux for 5 h. With 50mL saturated NaHCO₃After dilution of the solution (aqueous), the reaction mixture was extracted with ethyl acetate (100 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 2: 1) to give a white solid. Yield: 50 to 98 percent. 12 daa: yield: 92.8 percent; mp 135-.¹H NMR(CDCl₃，500MHz)δ7.81(s，1H)，7.80(d，J＝6.5Hz，2H)，7.58(d，J＝8.0Hz，2H)，7.41-7.45(m，3H)，7.31-7.33(m，2H)，7.20(d，J＝7.0Hz，2H)，5.33(s，2H)，3.99(s，3H)，3.98(s，6H)，2.47(s，3H)。MS(ESI)C₂₇H₂₆N₂O₄Calculated value of 442.2, found 443.1[ M + H ]]⁺。HPLC1：t_R4.28min, the purity is more than 99 percent.

Synthesis of 11gaa and 12la (fig. 12):

R₁＝N(Me)₂；R₂＝(4-OMe)PhSO₂(11gaa)

R₁＝Br；R₂＝H(12la)

substituted benzaldehyde compound 8(l, g) was converted to compound 9(l, g) in the presence of ammonium hydroxide and glyoxal to construct the imidazole scaffold. The imidazole ring of compound 9(l, g) was protected with the appropriate phenylsulfonyl followed by coupling with 3, 4, 5-trimethoxybenzoyl chloride to afford compound 11(la, gaa). Treatment of 11la with tetrabutylammonium fluoride removed the protecting group to yield 12 la.

Synthesis of (2- (4-bromophenyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (12la) (FIG. 12)

Synthesis of 9l, 9 g: to a solution of the appropriate benzaldehyde (8l and 8g, 100mmol) in ethanol (400mL) at 0 deg.C was added 40% aqueous glyoxal (oxalaldehyde or glyoxal) (1.1 equiv.) and 29% aqueous ammonium hydroxide (10 equiv.). After stirring at RT for 2-3 days, the reaction mixture was concentrated and the residue was subjected to flash column chromatography with dichloromethane as eluent to give the title compound as a yellow powder. Yield: 10 to 30 percent.

Synthesis of 10la and 10 gb: to a solution of imidazole (9l, 9g) (10mmol) in anhydrous THF (200mL) at 0 deg.C was added sodium hydride (60% dispersion in mineral oil, 1.2 equiv.) and stirred for 20 min. 4-Methoxybenzenesulfonyl chloride (for 10gb) or benzenesulfonyl chloride (for others) (1.2 equivalents) was added and the reaction mixture was stirred overnight. With 200mL saturated NaHCO₃After dilution of the solution (aqueous), the reaction mixture was extracted with ethyl acetate (600 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 2: 1) to give a pale solid. Yield of：40％-95％。

Synthesis of 11la and 11 gaa: to a solution of 2-aryl-1- (phenylsulfonyl) -1H-imidazole (101a, 10gb) (5.0mmol) in anhydrous THF (30mL) at-78 deg.C was added 1.7M t-butyllithium (1.2 equiv.) in pentane and stirred for 10 min. 3, 4, 5-trimethoxybenzoyl chloride (1.2 eq.) was added at-78 ℃ and stirred overnight. The reaction mixture was taken up with 100mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (300 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 3: 1) to give a white solid. Yield: 5 to 45 percent.

Synthesis of 12 la: to a solution of aryl (2-aryl-1- (phenylsulfonyl) -1H-imidazol-4-yl) methanone (11la) (2.0mmol) in THF (25.0mL) was added 1.0M tetrabutylammonium fluoride (2 equiv.) and stirred overnight. The reaction mixture was taken up with 60mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (150 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 4: 1) or recrystallized from water and methanol to give a white solid. Yield: 80 to 98 percent.

Synthesis of (4-fluorophenyl) (2- (4-methoxyphenyl) -1H-imidazol-4-yl) methanone (12cb) (FIG. 8).

To a solution of (4-fluorophenyl) (2- (4-methoxyphenyl) -1- (phenylsulfonyl) -1H-imidazol-4-yl) methanone (11cb, 872mg, 2.0mmol) in THF (20.0mL) was added 1.0M tetrabutylammonium fluoride (4.0mL, 4.0mmol) and stirred overnight. The reaction mixture was taken up with 50mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (100 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was recrystallized from water and methanol to give a white solid. Yield: 90 percent; mp245-247 deg.C.

Synthesis of (2- (p-tolyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (12da) (FIG. 9).

To a solution of (1- (phenylsulfonyl) -2- (p-tolyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (11da, 492mg, 1.0mmol) in THF (15.0mL) was added 1.0M tetrabutylammonium fluoride (2.0mL, 2.0mmol) and stirred overnight. The reaction mixture was taken up with 30mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (80 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was recrystallized from water and methanol to give a white solid. Yield: 88.5 percent.

Synthesis of (2- (4-chlorophenyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (12fa) (FIGS. 9 and 13).

2- (4-chlorophenyl) -1H-imidazole (9 f): to a solution of 4-chlorobenzaldehyde (8f) (100mmol) in ethanol (350mL) at 0 deg.C was added 40% aqueous glyoxal (12.8mL, 110mmol) and 29% aqueous ammonium hydroxide (1000mmol, 140 mL). After stirring at RT for 2-3 days, the reaction mixture was concentrated and the residue was subjected to flash column chromatography with dichloromethane as eluent to give the title compound as a yellow powder. Yield: 19.8 percent.¹H NMR(500MHz，DMSO-d₆)δ13.60(br，1H)，7.94(d，J＝8.5Hz，2H)，7.51(d，J＝8.0Hz，2H)，7.27(s，1H)，7.03(s，1H)。MS(ESI)：C₉H₇ClN₂Calculated value of 178.0, found 178.9[ M + H ]]⁺。

2- (4-chlorophenyl) -1- (phenylsulfonyl) -1H-imidazole (10 f): to a solution of 2- (4-chlorophenyl) -1H-imidazole (9f) (20mmol) in anhydrous THF (200mL) at 0 deg.C was added sodium hydride (60 in mineral oil)% dispersion, 1.2g, 30mmol) and stirred for 30 min. Benzenesulfonyl chloride (2.82mL, 22mmol) was added and the reaction mixture was stirred overnight. With 100mL saturated NaHCO₃After dilution of the solution (aqueous), the reaction mixture was extracted with ethyl acetate (500 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 2: 1) to give a pale solid. Yield: 54.9 percent.¹H NMR(500MHz，CDCl₃)δ7.65(d，J＝2.0Hz，1H)，7.58(t，J＝7.5Hz，1H)，7.43(d，J＝8.5Hz，2H)，7.38(t，J＝8.0Hz，2H)，7.34-7.36(m，4H)，7.12(d，J＝1.5Hz，1H)。MS(ESI)：C₁₅H₁₁ClN₂O₂Calculated value of S is 318.0, found 341.0[ M + Na ]]⁺。

(2- (4-chlorophenyl) -1- (phenylsulfonyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (11 fa): to a solution of 2- (4-chlorophenyl) -1- (phenylsulfonyl) -1H-imidazole (10f) (6.0mmol) in anhydrous THF (30mL) at-78 deg.C was added 1.7M t-butyllithium in pentane (5.3mL, 9.0mmol) and stirred for 10 min. 3, 4, 5-trimethoxybenzoyl chloride (7.2mmol) was added at-78 deg.C and stirred overnight. The reaction mixture was taken up with 100mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (200 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 4: 1) to give a white solid. Yield: 36.8 percent;¹H NMR(500MHz，CDCl₃)δ8.05(d，J＝7.5Hz，2H)，7.77(t，J＝7.5Hz，1H)，7.62(t，J＝8.0Hz，2H)，7.48(s，1H)，7.44(d，J＝9.0Hz，2H)，7.39(d，J＝8.5Hz，2H)，7.37(s，2H)。MS(ESI)：C₂₅H₂₁ClN₂O₆calculated value of S is 512.1, found 513.1[ M + H ]]⁺。

(2- (4-chlorophenyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (12 fa): to a solution of (2- (4-chlorophenyl) -1- (phenylsulfonyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (11fa) (2.0mmol) in THF (20.0mL) was added 1.0M tetrabutylammonium fluoride (4.0mmol)l) and stirred overnight. The reaction mixture was taken up with 50mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (100 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 3: 1) or recrystallized from water and methanol to give a white solid. Yield: 80-95 percent. Yield: 36.9 percent; mp 193 and 195 ℃.¹H NMR(500MHz，CDCl₃)δ10.75(br，1H)，7.96(d，J＝8.5Hz，2H)，7.83(s，1H)，7.47(d，J＝9.0Hz，2H)，7.23(s，2H)，3.97(s，3H)，3.94(s，6H)，2.43(s，3H)。MS(ESI)：C₁₉H₁₇ClN₂O₄Calculated value of 372.1, found 395.1[ M + Na]⁺，370.9[M-H]^-. HPLC gradient: solvent a (water) and solvent B (methanol): 0-15min 40-100% B (linear gradient), 15-25min 100% B; t is t_R16.36min, the purity is more than 99 percent.

Synthesis of (2- (4-chlorophenyl) -1H-imidazol-4-yl) (4-fluorophenyl) methanone (12fb) (FIG. 9).

To a solution of (2- (4-chlorophenyl) -1- (phenylsulfonyl) -1H-imidazol-4-yl) (4-fluorophenyl) methanone (11fb, 440mg, 1.0mmol) in THF (12.0mL) was added 1.0M tetrabutylammonium fluoride (2.0mL, 2.0mmol) and stirred overnight. The reaction mixture was taken up with 20mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (60 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was recrystallized from water and methanol to give a white solid. Yield: 83.7 percent.

Physicochemical characterization of aryl-benzoyl-imidazole Compounds and intermediates

Example 3

(indolyl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17ya), (17yab) and (17yac) Synthesis of (FIG. 14)

Synthesis of (2- (1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17 ya):

synthesis of 1- (phenylsulfonyl) -1H-indole-3-carbaldehyde (8 ya): to a solution of indole 3-carbaldehyde (8y) (100mmol) in ethanol (500mL) was added potassium hydroxide (1.1 equiv.) at RT. The mixture was stirred until completely dissolved. The ethanol was completely removed in vacuo and the residue was dissolved in acetone (250mL) followed by the addition of benzenesulfonyl chloride (1.1 eq, 110 mmol). The reaction mixture was stirred for half an hour. The precipitate was filtered off, the filtrate was concentrated and recrystallized from methanol to give a white solid. Yield: 33 percent.¹H NMR(500MHz，CDCl₃)610.17(s，1H)，8.25-8.39(m，2H)，7.97-8.09(m，3H)，7.69(t，J＝7.33Hz，1H)，7.59(t，J＝7.5Hz，2H)，7.39-7.54(m，2H)。MS(ESI)C₁₅H₁₁NO₃Calculated value of S is 285.1, found 286.0[ M + H ]]⁺。

Synthesis of 3- (1H-imidazol-2-yl) -1- (phenylsulfonyl) -1H-indole (9 ya): to a solution of 1- (phenylsulfonyl) -1H-indole-3-carbaldehyde (8ya) (100mmol) in ethanol (400mL) at 0 deg.C were added 40% aqueous glyoxal (oxalaldehyde or glyoxal) solution (1.1 equiv., 110mmol) and 29% aqueous ammonium hydroxide solution (10 equiv., 1000 mmol). After stirring at RT for 2-3 days, the reaction mixture was quenched with water and extracted with dichloromethane. The organic layer was removed in vacuo and the residue was flash column chromatographed using hexane/ethyl acetate (4: 1-2: 1) as eluent to give the title compound as a yellow powder. Yield: 12 percent.¹H NMR(500MHz，DMSO-d₆)δ8.33(d，J＝2.9Hz，2H)，8.13(d，J＝7.8Hz，2H)，7.98-8.04(m，1H)，7.62-7.67(m，1H)，7.55(d，J＝7.82Hz，2H)，7.22-7.34(m，4H)。MS(ESI)C₁₇H₁₃N₃O₂Calculated value of S323.1, found 324.0[ M + H ]]⁺。

Synthesis of 1- (phenylsulfonyl) -3- (1- (phenylsulfonyl) -1H-imidazol-2-yl) -1H-indole (10 ya): to a solution of 3- (1H-imidazol-2-yl) -1- (phenylsulfonyl) -1H-indole (9ya) (20mmol) in anhydrous THF (300mL) at 0 ℃ was added sodium hydride (60% dispersion in mineral oil, 1.2 eq, 24mmol) and stirred for 20 min. Benzenesulfonyl chloride (1.2 eq, 24mmol) was added and the reaction mixture was stirred overnight. With 200mL saturated NaHCO₃After dilution of the solution (aqueous), the reaction mixture was extracted with ethyl acetate (600 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 5: 1) to give a white solid. Yield: 40 percent.¹H NMR(CDCl₃，300MHz)δ8.02-8.08(m，4H)，7.72(d，J＝1.5Hz，1H)，7.35-7.60(m，8H)，7.23(d，J＝1.5Hz，1H)，7.10-7.16(m，3H)。MS(ESI)C₂₃H₁₇N₃O₄S₂Has a calculated value of 463.1 and an observed value of 486.0[ M + Na ]]⁺。

Synthesis of (1- (phenylsulfonyl) -2- (1- (phenylsulfonyl) -1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17 yaa): to a solution of 1- (phenylsulfonyl) -3- (1- (phenylsulfonyl) -1H-imidazol-2-yl) -1H-indole (10ya) (5.0mmol) in dry THF (100mL) at-78 deg.C was added 1.7M t-butyllithium in pentane (1.2 equiv., 6.0mmol) and stirred for 10 min. A solution of 3, 4, 5-trimethoxybenzoyl chloride (1.2 eq., 6.0mmol) in THF was added at-78 deg.C and stirred overnight. The reaction mixture was taken up with 100mL of saturated NaHCO₃The solution (aq) was quenched and extracted with ethyl acetate (300 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 3: 1) to give a white solid. Yield: 30 percent.¹H NMR(500MHz，CDCl₃)δ8.09(d，J＝10Hz，1H)，8.04(d，J＝10Hz，2H)，7.91(s，1H)，7.76(d，J＝5Hz，2H)，7.65(t，J＝10Hz，1H)，7.55-7.58(m，5H)，7.40(s，2H)，7.33-7.36(m，3H)，7.25(t，J＝10Hz，1H)，4.05(s，3H)，4.03(s，6H)。MS(ESI)C₃₃H₂₇N₃O₈Calculated value of (d) 657.0, found 680.1[ M + Na ]]⁺。

Synthesis of (2- (1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17 ya): to a solution of (1- (phenylsulfonyl) -2- (1- (phenylsulfonyl) -1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17yaa) (1mmol) in ethanol (40mL) and water (4mL) was added sodium hydroxide (10 equiv., 10mmol) and stirred under reflux overnight in the dark. The reaction mixture was diluted with 50mL of water and extracted with ethyl acetate (200 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash column chromatography (hexane: ethyl acetate 1: 1) to give a yellow solid. Yield: 60 percent.¹H NMR(500MHz，CD₃OD)δ8.31(d，J＝6.5Hz，1H)，7.99(s，1H)，7.90(s，1H)，7.48-7.52(m，3H)，7.24-7.28(m，2H)，4.00(s，6H)，3.93(s，3H)。MS(ESI)C₂₁H₁₉N₃O₄Calculated value of 377.1, found 400.1[ M + Na]⁺。Mp 208-210℃。

Synthesis of (2- (1- (phenylsulfonyl) -1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17 yab):

to a solution of compound 17yaa (66mg) in THF (1.0mL) was added 1.0M tetrabutylammonium fluoride (0.4mL, 0.4mmol) and stirred overnight. The reaction mixture was taken up with 20mL of saturated NaHCO₃The solution (aqueous) was diluted and extracted with ethyl acetate (20 ml). The organic layer was dried over magnesium sulfate and concentrated. Flash column chromatography (hexane: ethyl acetate,2: 1) purifying the residue to obtain a pale solid. Yield: 45 percent. Mp 110-.¹H NMR(CDCl₃，500MHz)δ8.40-8.42(m，2H)，8.09(d，J＝8.0Hz，1H)，7.93-7.98(m，4H)，7.59(t，J＝7.5Hz，1H)，7.41-7.49(m，5H)，4.01(s，3H)，3.97(s，6H)。MS(ESI)C₂₇H₂₃N₃O₆Calculated S is 517.1, found 540.0[ M + Na ]]⁺。HPLC：t_R6.81min, the purity is 96.3%.

Synthesis of (1-methyl-2- (1- (methyl) -1H-indol-3-yl) -1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl) methanone (17 yac):

to a solution of 17ya (75mg, 0.2mmol) in anhydrous THF (20ml) was added sodium hydride (60% dispersion in mineral oil, 20mg, 0.5mmol) at 0 deg.C and stirred for 20 min. Methyl iodide (70mg, 0.5mmol) was added and the reaction mixture was stirred for 1 h. 20mL of saturated NaHCO was used₃After the solution (aqueous) was diluted, the reaction mixture was extracted with ethyl acetate (60 ml). The organic layer was dried over magnesium sulfate and concentrated. The residue was recrystallized from water and methanol to give a white solid. Yield 75%. Mp 164-.¹H NMR(CDCl₃，500MHz)δ8.30(d，J＝7.5Hz，1H)，8.01(s，1H)，7.87(s，1H)，7.41(t，J＝8.5Hz，1H)，7.39(s，1H)，7.35(t，J＝7.0Hz，1H)，7.23(t，J＝7.0Hz，1H)，3.98(s，6H)，3.95(s，3H)，3.91(s，3H)，3.89(s，3H)。MS(ESI)C₂₃H₂₃N₃O₄Calculated value of (d) 405.2, found 406.4[ M + H ]]⁺。HPLC：t_R4.80min, and the purity is more than 99 percent.

Example 4

Antiproliferative activity of selected ABI compounds of the invention

Cell culture cytotoxicity assays

Materials and methods

The antiproliferative activity of the ABI compounds in three melanoma cell lines (A375 and WM-164, human melanoma cell line; B16-F1, mouse melanoma cell line) and four human prostate cancer cell lines (LNCaP, DU145, PC-3 and PPC-1) was investigated. All of these cell lines, except the PPC-1 cell line, were purchased from ATCC (American type culture Collection, Manassas, Va.). Robert Clarke friend of Georgetown University School of Medicine, Washington, provides MDA-MB-435 and MDA-MB-435/LCCMDR1 cells. Melanoma cells were cultured in DMEM (Cellgro Mediatech, Herndon, VA) and prostate cancer cells were cultured in RPMI1640(Cellgro Mediatech, Hemdon, VA) supplemented with 10% fbs (Cellgro Mediatech). The culture was maintained at 37 ℃ and contained 5% CO₂In a humid atmosphere. Depending on the growth rate, 1000 to 5000 cells were plated into each well of a 96-well plate and exposed to different concentrations of test compound for 48 hours (fast-growing melanoma cells) and 96 hours (slow-growing prostate cancer cells), either in triplicate or in penta. The number of cells at the end of the drug treatment was measured by the sulforhodamine b (srb) assay. Briefly, cells were fixed with 10% trichloroacetic acid and stained with 0.4% SRB, and absorbance at 540nm was measured using a plate reader (DYNEX Technologies, Chantilly, VA). The% cell viability was plotted versus drug concentration and IC was obtained by nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, Calif.)₅₀Values (concentration that inhibited 50% of cell growth of untreated control).

Results

Tables 8-10 summarize the results of the in vitro antiproliferative activity of the compounds of the invention using three melanoma cell lines (one murine melanoma cell line, B16-F1; and two human metastatic melanoma cell lines, A375 and WM-164) and four human prostate cancer cell lines (LNCaP, PC-3, Du145 and PPC-1).

Table 8 in vitro growth inhibitory effect of ring a unsubstituted compounds.

According to Table 8, compounds 12aa-12ai showed moderate activity, IC₅₀Values were in the μ M range (average of all seven cell lines). The most potent compound in this series is 12aa, average IC₅₀The value was 160 nM. Removal of one of the methoxy groups (12ad, 12ae) from the 3, 4, 5-trimethoxy group of the C-ring resulted in a significant loss of activity (IC for 12ae)₅₀More than 10 mu M; average IC of 12ad₅₀3.1 μ M). The compound having 4-fluoro in the C ring (12af) also showed relatively good activity (IC)₅₀0.91 μ M), which is an important implication since the replacement of the trimethoxy moiety with 4-fluoro group provides good activity and improved metabolic stability. The position of the fluorine on the C-ring is critical for activity, since the change from 4-to 3-fluoro results in a complete loss of activity (IC of 12ag compared to 0.91. mu.M for 12af, IC of 12ag₅₀> 10. mu.M). This result indicates the presence of a potential hydrogen bond donor near the 4-position of the ring.

As is clear from table 8, the positions of the a and C rings are critical. A simple shift of the C-ring part from position 4 to position 1 in the imidazole ring (ring B) leads to a complete loss of activity (IC of 12aba, 12aaa, 10a, 10x, 10 j)₅₀＞10μM)。

TABLE 9 in vitro growth inhibitory Effect of Compounds having substitutions on the A ring.

ND-not determined

According to Table 9, compounds having 3, 4, 5-trimethoxy and 4-fluoro substitutions on the C ring show good activity with different substitutions on the A ring. These compounds show excellent antiproliferative activity against WM164 cell line IC₅₀Values as low as 8.0nM (12 da). In general, for example, from 12ca, 12cb, 12da, 12db, 12fa, 12fb, 12ga and 12gb (IC)₅₀7.9-110nM), the compound in which a single substituent is introduced para to the a ring is more active. With the corresponding free base 12db (IC)₅₀109nM) in comparison with 12db-HCl salt (IC)₅₀172nM) showed a slight decrease in activity. Compound 12fb (IC) with a single halogen substituent in the para position of the A and C rings and no methoxy group₅₀63.7nM) showed activity. Compounds with 3, 4, 5-trimethoxy substituents on the A ring lost activity completely (IC of 12ea, 12 eb)₅₀> 10 μ M), indicating a very different binding environment around the a and C loops. Removal of the 5-methoxy substituent from the A ring significantly improves activity (IC of 12ha, 12 ea)₅₀330nM and greater than 10 μ M, respectively). Demethylation of 3, 4, 5-trimethoxy dramatically reduced activity from 43nM (12fa) to 3.89. mu.M (13 fa). Similar results due to demethylation of substituents on the A or C ring were also observed for 13ea, 12ka, 12kb and 13 ha. The electron donating groups (4-methoxy, 4-dimethylamino, 4-methyl) and electron withdrawing groups (4-chloro, 2-trifluoromethyl) on the a ring show no substantial difference in activity. Introduction of trifluoromethyl in the ortho position to the A ring resulted in a complete loss of activity (IC of 12ia, 12 ib)₅₀> 10. mu.M). And p-hydroxy compound 12kb (IC)₅₀33 μ M) in contrast to benzyloxy (12jb IC) present in the a ring para₅₀75nM) resulted in a 440-fold increase in activity. It is noteworthy that 12jb, a compound with 4-fluoro in the C ring, has a better activity than the corresponding 12ja with 3, 4, 5-trimethoxy in the C ring (IC of 12 jb)₅₀75nM, 12ja IC₅₀＝7.3μM)。

Table 10 in vitro growth inhibitory effect of B ring protected compounds.

TABLE 10 inhibitory Effect of reverse (reversed) arylbenzoylimidazoles (RABI)

TABLE 10B inhibitory Effect of Reverse Arylbenzoylimidazoles (RABI)

According to Table 10, the compounds (11cb, 11db, 11fb, 11ga, 11gb, 11ha, 11jb) having the phenylsulfonyl protecting group bonded to the nitrogen of the imidazole ring also had strong activity and IC₅₀In the nM range (Table 10). In general, the activity of these compounds is comparable to their corresponding unprotected counterparts by comparing the activity of 11cb (43nM), 11db (111 nM), 11fb (72nM), 11ga (285nM), 11gb (87nM), 11ha (268nM) and 11jb (61nM) to their corresponding unprotected counterparts, 12cb (36nM), 12db (109nM), 12fb (64nM), 12ga (131nM), 12gb (72nM), 12ha (330nM) and 12jb (75 nM). The activity of the other compounds (11ab-11ag, 11ea, 11eb, 11hb, 11ia and 11ib, 1-50. mu.M) was generally much lower, and was also consistent with that of their counterparts (12ab-12ag, 12ea, 12eb, 12hb, 12ia and 12ib, 1-50. mu.M).

The PC3 cell cycle distribution of the compounds of the invention is presented in fig. 15.

And (4) analyzing the cell cycle.

Cell cycle distribution was determined by Propidium Iodide (PI) staining. Treated cells were washed with PBS and fixed with 70% ice cold ethanol overnight. Then, the fixed cells were stained with 20. mu.g/mL PI in the presence of RNase A (300. mu.g/mL) at 37 ℃ for 30 minutes. Cell cycle distribution was analyzed by Fluorescence Activated Cell Sorting (FACS) analysis of the core services at the University of Tennessee Health Science Center, TN.

Results

Reverse ABI (RABI) arrested cells at G as confirmed by cell cycle analysis₂And a/M period. PC3 cells were treated with compounds 12q, 70a, 70f and 70m for 24h (fig. 15) and the distribution of PI stained cells was investigated by FACS analysis. For each compound, four different concentrations-1 nM, 10nM, 50nM and 100nM were chosen to examine the dose effect. In the vehicle-treated group, about 18% of PC3 cells were distributed in G₂And a/M period. RABI will be at G approximately in a concentration dependent manner₂The proportion of cells in the/M phase increased up to 70%. Different concentrations arrested cells at G₂The efficacy of the/M phase is positively correlated with the in vitro cell growth inhibitory activity. Antiproliferation of RABI is reported in table 10A and table 10B. Some RABI show a rather potent antiproliferation (see e.g. 70 a).

Example 5

Activity of aryl-benzoyl-imidazole (ABI) compounds in drug resistant melanoma cells

P-glycoprotein (Pgp) -mediated drug efflux is the major mechanism by which cancer cells prevent the intracellular accumulation of effective concentrations of anticancer drugs. The ABI compounds were compared for activity against multidrug resistant (MDR) melanoma cells (MDA-MB-435/LCCMDR1) and their parental non-resistant cancer cells (MDA-MB-435). Although MDA-MB-435 was originally named as a breast cancer cell line, it has been shown specifically to be derived from the M14 melanoma cell line. Compounds 12da, 12fb, 12cb, 11cb and 11fb and other agents targeting tubulin (including colchicine, paclitaxel and vinblastine) were tested on both MDR melanoma cell lines and their parental melanoma cell lines (table 11A). Paclitaxel and vinblastine are clinically used anticancer drugs known to target cellular tubulin. Although colchicine is not an FDA-approved drug for cancer treatment, its prodrug, ZD6126, is already in clinical trials for solid tumors. Bortezomib was the first therapeutic proteasome inhibitor, approved by the FDA for multiple myeloma in 2003. ABT-751 is known to target the tubulin colchicine binding site. It is a promising drug candidate for childhood recurrent or refractory neuroblastoma in clinical trials. Compounds 12da, 12fb, 12cb, 11fb had much better resistance indices (3.0 for 12da, 0.9 for 12fb, 1.3 for 12cb, 0.8 for 11cb, 0.7 for 11 fb) than colchicine (65.8), paclitaxel (69.3) and vinblastine (27.5). Although colchicine, paclitaxel and vinblastine showed excellent activity in non-resistant melanoma cell lines (0.5-10nM), the efficacy of these compounds on MDR melanoma cell lines was significantly reduced (277-658 nM). In contrast, 12cb, 11fb were substantially equivalent in potency to MDR melanoma cell lines (15 nM, 38nM, 30nM, 35nM for 12da, 12fb, 11cb, and 11fb, respectively) and non-drug resistant melanoma cell lines (5 nM, 41nM, 24nM, 38nM, 50nM for 12da, 12fb, 11cb, and 11fb, respectively). Compound 12da was more active on A375 and WM-164 cells compared to paclitaxel and colchicine.

Table 11a. in vitro growth inhibitory effect of ABI compounds on multi-drug resistant melanoma cell lines (MDR cells) and corresponding sensitive parental cell lines (normal melanoma cells) compared to other anticancer drugs.

Resistance index by IC using cell line MDA-MB-435/LCC6MDR1 resistant to multidrug₅₀Value divided by IC for the corresponding sensitive parental cell line MDA-MB-435₅₀A value is calculated. Abbreviations: N/A, no numerical value is obtained; ND, not determined.

Table 11b. anticancer efficacy and colchicine site binding affinity of abi in different cancers and MDR cell lines with different mechanisms of resistance. ABI showed excellent efficacy against all melanoma cell lines tested, including highly metastatic cell lines and multidrug resistant cell lines. The high binding affinity of ABI to the colchicine binding site in tubulin confirms its targeting within the cell.

Note: *: (ii) parental cell lines to drug resistant cell sublines; MDR1 was overexpressed in MDA-MB-435/LCC6MDR1 and NCI/ADR-RES; MRP1, MRP2 and BCRP were overexpressed in HEK293-MRP1, HEK293-MRP2 and HEK293-482R 2. Resistance index (number in parentheses) by comparing IC against resistant cell sublines₅₀Value divided by IC for the corresponding parental cell line₅₀A value is calculated.⁺: IC for tubulin binding₅₀Composed of³H]Colchicine competition was calculated in combination with scintillation proximity assay.⁺⁺: binding affinities reported in the literature for ABT-751. Abbreviations: n/a, is not applicable because it binds to a different site of tubulin.

Table 11C: antiproliferative activity of methylene-linked compounds (aryl-benzyl-imidazoles) in melanoma cells.

Not applicable N/A

Not determined ND

Table 11D: antiproliferative activity of aryl-benzoyl-imidazoles in melanoma cells.

Not applicable N/A

Not determined ND

The results in Table 11A show that the cell line MDA-MB-435/LCCMDR1 is very resistant to colchicine, paclitaxel and vinblastine. The ABIs of the invention exhibit the same efficacy against drug resistant cell lines and sensitive parental cell lines. This result strongly suggests that ABI is not a substrate for P-gp. Thus, they overcome the multidrug resistance found in MDA-MB-435/LCCMDR1 cells. Fig. 16 shows the dose response curves for 12fb, 12da and 12 cb. Table 11B further discusses the mechanism of resistance to paclitaxel, SN-38, vinblastine and colchicine compared to ABI 12cb, 12da and 12 fb. MRP and BCRP confer moderate resistance to paclitaxel (resistance index 4 and 6, respectively), vinblastine (resistance index 6 and 5, respectively), and BCRP confers significant resistance to SN-38 (resistance index 41). However, none of the ABIs is susceptible to MRP or BCRP mediated resistance (resistance index 0.4-1.0). ABT-751 is as insensitive to MDR1, MRP or BCRP as ABI.

Example 6

In vitro microtubule polymerization assay

Materials and methods

Bovine brain tubulin (0.4mg) (Cytoskeleton, Denver, CO) was mixed with 10. mu.M of test compound and mixed at 110. mu.l of universal tubulin buffer (80mM PIPES, 2.0mM MgCl) pH6.9₂0.5mM EGTA and 1mM GTP). Absorbance at 340nm was monitored every 1min for 15min by a SYNERGY4 microplate reader (Bio-Tek Instruments, Winooski, VT). For tubulin polymerization, the spectrophotometer was set at 37 ℃.

Results

The inhibition of tubulin polymerization by aryl-benzoyl-imidazole (ABI) compounds was examined. Bovine brain tubulin (> 97% purity) was incubated with three potent ABI compounds 12cb, 12da and 12db at a concentration of 10 μ M to determine the effect of these ABI compounds on tubulin polymerization (fig. 17). Compound 12da completely inhibited tubulin polymerization, whereas about 80% inhibition was observed during incubation with compounds 12cb and 12 db.

This microtubule destabilization effect is similar to colchicine and vinblastine, but opposite to paclitaxel. This result not only confirms that ABIs interact directly with tubulin, but also suggests that they may share the same binding site with colchicine (or vinblastine).

Example 7

In vitro melanoma inhibition

Materials and methods

B16-F1 melanoma cells were plated at colony forming density (2000 cells per well, on six well plates) on top of 0.8% basal agar. Cells were cultured in 0.4% agar and DMEM medium at 37 ℃ in 95% air and 5% CO₂Is supplemented with fetal bovine serum and an antibiotic-antimycotic solution. Cells were treated with different concentrations (20nM, 100nM and 500nM) of compounds 12da, 12cb and 12 fb. Compounds were added to the medium from 1mM DMSO stock solutions and corresponding DMSO dilutions were used as controls. Cells were grown for 14 days. The plate was photographed and Colony counts were measured using an Aretek 880Automated Colony Counter (Aretek Systems, Inc., Farmingdale, NY).

Results

Fig. 18 shows four representative photographs. After 14 days of incubation, about 130 detectable colonies (greater than 100 μm in diameter) were formed in the control (untreated).

Compounds 12cb and 12da effectively inhibited the formation of B16-F1 melanoma colonies (p < 0.05 compared to control) even at the lowest tested concentration of 20 nM. 12fb showed potent inhibition at 100 nM. At 0.5 μ M, all three test compounds showed complete inhibition of colony formation, further demonstrating the anti-melanoma efficacy of ABI.

Example 8

In vivo antitumor Activity

Materials and methods

Animals: female C57/BL mice, 4-6 weeks old, were purchased from Harlan Laboratories (Harlan Laboratories, Inc., Indianapolis, IN). The Animal housing conditions meet the Association for Association and acceptance and Laboratory Animal Care specifications. All methods were performed according to the guidelines of the Institutional Animal Care and Use Committee.

And (4) evaluating the in vivo efficacy. In DMEM medium without FBS (Cellgro Mediatech) at 5X 10⁶Mouse melanoma B16-F1 cells were prepared at a concentration of individual viable cells/mL. The cell suspension (100 μ L) was injected subcutaneously into the right dorsal flank of each mouse. When the cells were seeded for about 7 days, the tumor size reached about 100-150mm³All tumor-bearing mice were divided into control and treatment groups (n-5 per group) according to tumor size. Each group had similar mean tumor size. Mice in the control group were injected intraperitoneally once daily with only 50 μ L of vehicle solution (negative control) or 60mg/kg of DTIC (positive control). Tumor volume was measured every two days with a traceable digital caliper (Fisher Scientific, Pittsburgh, Pa.) using the formula a x b²X 0.5, where a and b represent the larger and smaller diameters, respectively. Tumor volume is expressed in cubic millimeters. Each set of data is expressed as mean ± SE and plotted over time. Using the formula 100 × [ (T-T)₀)/(C-C₀)]The percent tumor reduction at the end of the experiment (14 days after the start of treatment) was calculated, where T represents the average tumor volume in the treated group on a certain day, T₀Mean tumor volume on the first day of treatment, C mean tumor volume of control on a certain day, and C₀Mean tumor volume on the first day of treatment for the same group. Throughout the experimental periodIn between, animal mobility and average body weight of each group were monitored to assess toxicity of the compounds. At the end of the treatment, CO is used₂Then cervical dislocation was performed to euthanize all mice and tumors were collected for further study.

Results

To evaluate the in vivo efficacy of ABI analogs, we tested compound 12cb for anti-tumor activity against mouse melanoma B16-F1 xenografts. Gold standard DTIC for malignant melanoma treatment was used as a positive control (fig. 19A). Twenty female C57/BL mice were divided into four groups: vehicle control group, DTIC (60mg/kg) treated group, 12cb (10mg/kg) treated group, and 12cb (30mg/kg) treated group. 50 ten thousand B16-F1 melanoma cells were injected subcutaneously into each mouse. Treatment was initiated 7 days after tumor inoculation by daily intraperitoneal injections of each compound (fig. 19). For 12cb (10mg/kg), DTIC (60mg/kg) and 12cb (30mg/kg), tumor volume was significantly (p < 0.05) reduced by 47%, 51% and 73%, respectively, after 14 days of treatment. No significant weight loss was observed in any of the treatment groups during the experiment.

Two dose levels of 12fb were selected: 15mg/kg and 45 mg/kg. DTIC at 60mg/kg was used as a positive control. The B16-F1 melanoma allograft model of C57BL/6 mice was first selected for study. After 13 days of treatment (FIG. 19B), 15mg/kg of compound 12fb inhibited melanoma tumor growth by 32% (TGI value), 82% at 45 mg/kg. The Student' st test p value of 12fb was less than 0.001 at 45mg/kg compared to the control, indicating a significant difference. At 15mg/kg, the t-test p value for 12fb was 0.08, indicating that this dose was not effective, compared to the control. Comparing 45mg/kg of 12fb with 60mg/kg of DTIC (TGI 51%), the p-value was about 0.001 in the t-test, indicating that 12fb has substantially better activity than DTIC. For the control and 12fb 15mg/kg treatment groups, there was a slight increase in mean body weight throughout the experiment.

To further confirm the in vivo activity of ABI, a375 human melanoma xenograft model of SHO mice was used and 25mg/kg of 12fb was tested. Again, 60mg/kg of DTIC was used as a positive control. After 31 days of treatment (fig. 19C), 12fb inhibited melanoma tumor growth by 69% (TGI value), while DTIC inhibited growth by 52%. Treatment with 12fb compared to control, p-value was less than 0.001 for the t-test, indicating that 25mg/kg of 12fb significantly inhibited melanoma tumor growth. The p-value for the t-test was less than 0.05 for 12fb treatment compared to DTIC, again indicating that 12fb has substantially better activity than DTIC. The average volume weight of all groups increased slightly throughout the experiment. Physical activity of the mice also appeared normal, indicating that 25mg/kg is a well tolerated dose in SHO mice.

Example 9

In vitro and in vivo pharmacology of compounds 17ya, 12fa, and 55

Materials and methods

Cell culture and cytotoxicity assays for prostate cancer. All prostate cancer cell lines (LNCaP, PC-3 and DU145, PPC-1) were obtained from ATCC (American type culture Collection, Manassas, Va., USA). Human PC-3_ TxR is resistant to paclitaxel and compared to PC-3 using the MDR model. Cell culture supplies were purchased from Cellgro Mediatech (Herndon, VA, USA). All cell lines were used to test the antiproliferative activity of compounds 17ya, 12fa and 55 by sulforhodamine b (srb) assay. All cancer cell lines were maintained in RPMI1640 medium with 2mM glutamine and 10% Fetal Bovine Serum (FBS).

In vitro microtubule polymerization assay. Porcine brain tubulin (0.4mg) (Cytoskeleton, Denver, CO) was mixed with 1. mu.M and 5. mu.M test compound or vehicle (DMSO) and buffered at 100. mu.L (80mM PIPES, 2.0mM MgCl)₂0.5mM EGTA, pH6.9 and 1mM GTP). Absorbance at a wavelength of 340nm was measured once per minute for 15 minutes (SYNERGY4 microplate reader, Bio-Tek Instruments, Winooski, VT). For tubulin polymerization, the spectrophotometer was maintained at 37 ℃.

And (4) metabolic incubation. Assay was performed by testing 0.5. mu.M in a shaking water bath at 37 ℃The compounds were incubated in 1mL total reaction volume containing 1.3mM NADP in reaction buffer [0.2M phosphate buffer (pH7.4) ]⁺3.3mM glucose-6-phosphate and 0.4U/mL glucose-6-phosphate dehydrogenase]1mg/mL microsomal protein. NADPH regeneration systems (solutions A and B) were obtained from BD Biosciences (Bedford, MA). For glucuronidation studies, 2mM UDP-glucuronic acid (Sigma, St. Louis, Mo.) cofactor in deionized water was combined with 8mM MgCl in deionized water₂25 μ g of prorocentsin (Sigma, St. Louis, MO) and NADPH regeneration solution as described above (BDbiosciences, Bedford, MA). The total DMSO concentration in the reaction solution was about 0.5% (v/v). Aliquots (100 μ L) of the reaction mixture used to determine metabolic stability were sampled at 5, 10, 20, 30, 60 and 90 minutes. Acetonitrile (150 μ L) containing 200nM internal standard was added to quench the reaction and precipitate the protein. The samples were then centrifuged at 4,000g for 30min at RT and the supernatants were directly analyzed by LC-MS/MS.

And (4) an analytical method. The sample solution (10. mu.L) was injected into an Agilent series HPLC system (Agilent 1100 series Agilent 1100 chemical workstation, Agilent Technology Co., Ltd.). All analytes were separated on a C18 pore column (Alltech Alltima HP, 2.1X 100mm, 3 μm, Fisher, Fair Lawn, NJ). Two gradient modes are used. For metabolic stability studies, mobile phase A [ ACN/H containing 0.1% formic acid ] was used₂O(5％/95％，v/v)]And mobile phase B [ ACN/H containing 0.1% formic acid₂O(95％/5％，v/v)]The separation of analytes is achieved using a gradient mode at a flow rate of 300. mu.L/min. Mobile phase a was used at 10% for 0 to 1min, then the gradient was programmed linearly to 100% mobile phase B over 4min, maintaining 100% mobile phase B for 0.5 min, then changing rapidly to 10% mobile phase a. Mobile phase a was continued for 10 minutes until the end of the analysis.

Triple quadrupole Mass spectrometer API Qtrap 4000 operating with a TurboIonSpray Source^TM(applied biosystems/MDS SCIEX, Concord, Ontario, Canada). For positive ion mode, the spray needle voltage is set to5 kV. The gas curtain gas is set to 10; gas 1 and gas 2 were set to 50. The Collision Assisted Dissociation (CAD) gas was medium and the ion source heater probe temperature was 500 ℃. Using the Multiple Reaction Monitoring (MRM) mode, m/z 378 → 210(17ya), m/z 373 → 205(12fa), m/z 410 → 242(55), and m/z 309 → 171 (internal standard) were scanned to obtain the most sensitive signals. With Analyst version 1.4.1^TMSoftware (Applied Biosystems) performs the data acquisition and quantification process.

Water-soluble. The Solubility of the drug was determined by MultiScreen Solubility Filter Plate (Millipore corporation, Billerica, Mass.) in combination with LC-MS/MS. Briefly, 198 μ L of Phosphate Buffered Saline (PBS) buffer (pH7.4) was loaded into a 96-well plate, 2 μ L of 10mM test compound (in DMSO) was dispensed, and mixed at RT for 1.5 hours with gentle shaking (200-. The plates were centrifuged at 800g for 10 minutes and the filtrate was used to determine its concentration and the solubility of the test compound by LC-MS/MS as described above.

Pharmacokinetic studies. Male ICR mice (n ═ 3 per group) 6 to 8 weeks old were purchased from Harlan corporation and examined for Pharmacokinetics (PK) of 17ya, 12fa and 55. All compounds (10mg/kg) were dissolved in DMSO/PEG300(1/9) and administered by a single intravenous (i.v.) injection (50 μ L) into the tail vein. Blood samples were collected at 5, 15, 30 minutes and 1, 1.5, 2, 3, 4, 8, 12 and 24 hours post intravenous administration. 20mg/kg of each test compound (Tween 80/DMSO/H at 2/2/6) was administered by oral gavage₂O) mice were given (p.o.) to evaluate their oral bioavailability. Blood samples were taken at 0.5, 1, 1.5, 2, 3, 4, 8, 12 and 24 hours post p.o. administration.

Female Sprague-Dawley rats (n ═ 3; 254 ± 4g) were purchased from Harlan corporation (Indianapolis, IN). Rat thoracic and jugular venous catheters were purchased from Braintree Scientific (Braintree, MA). Upon arrival at the animal facility, the animals were acclimatized in a controlled temperature chamber (20-22 ℃) for 3 days with a 12 hour light/dark cycle prior to any treatment. Compounds 17ya, 12fa and 55 were administered intravenously at a dose of 5mg/kg (in 1/9 DMSO/PEG 300) into the thoracic and jugular veins. Injection of the sameThe collected heparinized saline displaced the removed blood and blood samples (250 μ L) were collected via jugular vein catheter at 10, 20, 30 minutes and 1, 2, 4, 8, 12, 24 hours. 10mg/kg (Tween 80/DMSO/H at 2/2/6) was administered by oral gavage₂O) were administered (p.o.) to rats to evaluate their oral bioavailability. All blood samples (250 μ L) after oral administration were collected via jugular vein catheter at 30, 60, 90, 120, 150, 180, 210, 240 minutes and 8, 12, 24 hours. Heparinized syringes and vials were prepared prior to blood collection. Plasma samples were prepared by centrifuging blood samples at 8,000g for 5 minutes. All plasma samples were immediately stored at-80 ℃ until analysis.

Analytes were extracted from 100 μ L plasma with 200 μ L acetonitrile containing 200nM internal standard. The samples were mixed well, centrifuged, and the organic extracts transferred to an autosampler for LC-MS/MS analysis.

PC-3_ TxR xenograft study. Preparation of PC-3_ TxR cells (10X 10) in RPMI1640 growth Medium containing 10% FBS⁷one/mL) and mixed with Matrigel (BD Biosciences, San Jose, Calif.) at a ratio of 1: 1. 100 μ L of the mixture (5X 10.) was injected subcutaneously (s.c.) into the flank of 6-8 week old male athymic nude mice⁶Individual cells/animal) to establish tumors. Measuring the length and width of the tumor according to the formula pi/6 XLXW²Calculation of tumor volume (mm)³) Wherein the length (L) and width (W) are measured in mm. When the tumor volume reaches 300mm³When this is done, the vehicle [ Tween80/DMSO/H₂O(2/2/6)]Or 17ya (10mg/kg) orally treated PC-3_ TxR tumor-loaded animals. The dosing regimen was 3 times per week for 4 weeks.

Results

17ya and 55 showed extensive cytotoxicity in cells including multidrug resistant cells.

SRB assay was used to evaluate the ability of 17ya and 55 to inhibit the growth of cancer cell lines (table 12). The two compounds inhibit five prostate cancer cell lines and one glioma cancer cell lineHas a low nanomolar range of IC₅₀The value is obtained. 17ya showed 1.7-4.3 times higher potency than 55 in these cell lines. Paclitaxel-resistant PC-3(PC-3/TxR) cell lines that overexpress P-glycoprotein (P-gp) were used to study the resistance to 17ya and 55 and compared to their parental PC-3 cell lines. IC of docetaxel in PC-3 and PC-3/TxR cells₅₀The values were 1.2. + -. 0.1nM and 17.7. + -. 0.7nM, respectively. 17ya and 55 were equivalent to the parental PC-3 and PC-3/TxR, whereas paclitaxel and docetaxel showed 85-fold and 15-fold relative resistance, respectively. These data indicate that both 17ya and 55 prevent P-gp mediated drug resistance.

Table 12.17 ya and 55 cytotoxicity data.

Determination of IC after 96h treatment₅₀Value (mean ± SD) (N ═ 3). Paclitaxel was used as a positive control. The data in parentheses indicate the IC when compared between PC-3 and PC-3/TxR₅₀Resistance factor at value. NR, not reported.

17ya and 55 bind to the colchicine binding site on tubulin, inhibit tubulin polymerization and induce cells Apoptosis (fig. 20).

Competitive gravimetric binding assays were developed to study the interaction of small molecule inhibitors with tubulin. In this study, different concentrations of 17ya or 55 were used to compete with colchicine tubulin binding. Both compounds effectively competed with colchicine for tubulin binding (fig. 20A); however, their competitive binding curves deviate substantially from zero at higher concentrations when compared to the known potent colchicine site binding ligand podophyllotoxin (podophylltxin). This indicates that 17ya and 55 exhibit lower affinity than podophyllotoxin or that they partially bind to the colchicine binding site. The negative control vinblastine did not inhibit colchicine tubulin binding, which successfully confirmed the specificity of the competitive gravimetric binding assay.

Porcine brain tubulin (> 97% purity) was incubated with 17ya or 55(5 μ M) to test its effect on tubulin polymerization (fig. 20B). Tubulin polymerization was inhibited by 47% and 40% at 15min, 17ya and 55, respectively. Colchicine at 5 μ M was used as a positive control and inhibited tubulin polymerization by 32%. These data indicate that 17ya and 55 have slightly higher inhibition of tubulin polymerization than colchicine. Thus, the molecular mechanism of these compounds is to bind to the colchicine binding site, inhibit tubulin polymerization, and induce cytotoxicity.

PC-3 and PC-3/TxR cells were exposed to 0.8-600nmol/L of 17ya, 55 or docetaxel for 24 h. The level of DNA-histone complex is used to indicate apoptosis. 17ya and 55 were equivalent to inducing apoptosis in PC-3 (FIG. 20C) and PC-3/TxR (FIG. 20D) in 24 h. Although docetaxel strongly induces apoptosis of PC-3 cells, it has a weak effect on PC-3/TxR cells due to overexpression of P-gp.

17ya and 55 exhibited favorable drug-like (drug-like) properties.

17ya and 55 were examined for drug-like properties such as metabolic stability, permeability, water solubility and drug-drug interaction (Table 13A). 17ya showed higher metabolic stability and water solubility than 55. Both chemicals showed sufficiently large permeability values that they could be used orally. In addition, both 17ya and 55 showed high IC in micromolar range in CYP enzyme inhibition assay₅₀Values indicating that both compounds avoid drug-drug interactions through the major CYP liver enzymes. In summary, both compounds showed favorable drug-like properties.

Table 13a. drug-like properties of compounds 17a and 55. Metabolic stability, permeability, solubility and possible drug-drug interactions were evaluated. Each value represents the average of duplicate studies.

Table 13b.17ya, 12fa, 55 and 1h summary of drug-like properties and pharmacokinetic properties.

As shown in table 13B, 17ya had a half-life of 80min through the I phase reaction, indicating that 17ya was stable during the I phase metabolism. The half-life in the presence of UDP-glucuronic acid (90min) was similar to that observed in its absence. These data indicate that 17ya is stable in human liver microsomes and that it is expected to achieve low clearance and long half-life in humans. On the other hand, 55 showed half-lives of 30min and 43min, respectively, in the presence and absence of UDP-glucuronic acid. Compound 12fa exhibited a half-life of 44min in phase I. These data indicate that all three compounds exhibit acceptable stability in human liver microsomes, and that 17ya is more stable than 12fa and 55. When its metabolism was studied, 12fa and 55 were found to exhibit higher levels of ketone reduction (data not shown), indicating that 12fa and 55 are more unstable than 17 ya.

Compound 17ya showed high water solubility and 12fa and 55 showed acceptable solubility.

Compound 17ya contained an imidazole ring, and this ring improved water solubility, which resulted in a water solubility of > 75 μ g/mL (table 13A). Compounds 12fa and 55 exhibited poor water solubility and were present at 12. mu.g/mL and 19. mu.g/mL, respectively. In summary, 17ya showed high water solubility, 12fa and 55 showed acceptable water solubility, and the improvement was well over 1 h. The greater solubility of 12fa translates into greatly improved oral bioavailability compared to 1h (35% versus 3.3% in rats). For 17ya and 55, similarly, water solubility correlated with greatly improved oral bioavailability as discussed below (table 14).

Pharmacokinetic studies of 17ya and 55 in mice, rats and dogs.

Pharmacokinetic parameters for 17ya and 55 given in single (i.v. or p.o.) doses in ICR mice, Sprague-Dawley rats and beagle dogs are summarized in table 14. 17ya showed low clearance in mice and rats, indicating that 17ya showed metabolic stability and minimal first pass metabolism in these species. In addition, 17ya had a moderate volume of distribution in mice and rats, indicating that it can be distributed appropriately into tissues including tumors. Surprisingly, the total clearance of 17ya was higher in dogs than in mice and rats. The two major metabolites in canine plasma (hydroxylated metabolite and unknown metabolite with maternal +34m/z (data not shown)) are consistent with those found in canine liver microsomes. In conclusion, 17ya gave higher clearance and lower oral exposure in dogs than 55, but not in mice and rats. In addition, 17ya produced a large amount of metabolites only in canine liver microsomes, but not in mouse, rat or human liver microsomes (data not shown). 17ya showed acceptable oral bioavailability of 21%, 36% and 50% in rats, mice and dogs, respectively. Meanwhile, 55 has low clearance in rats and moderate clearance in mice and dogs. Similar to 17ya, 55 has a moderate volume of distribution in these species. 55 has constant oral bioavailability (24% -36%) in three species. These properties indicate that 17ya and 55 are both potential orally available tubulin inhibitors.

Table 14 pharmacokinetic studies of compounds 17ya and 55 in mice, rats and dogs.

17ya and 55 inhibited growth of paclitaxel resistant prostate (PC-3/TxR) xenografts.

PC-3 cells (FIG. 21A) and paclitaxel-resistant prostate cancer cells (PC-3/TxR) (FIG. 21B) were inoculated into nude mice and tumor volumes were brought to about 150-300mm³. Will be provided withDocetaxel (10mg/kg or 20mg/kg) used clinically for prostate cancer was used to assess its efficacy in a P-gp mediated drug resistance model in vivo. PC-3/TxR tumors were found to grow rapidly and reached a volume of 1500-³. Although docetaxel administered intravenously at 10mg/kg and 20mg/kg showed dose response in both models (fig. 21A and 21B), when administered intravenously at 10mg/kg, the Tumor Growth Inhibition (TGI) effect decreased from 84% TGI in PC-3 tumors to 14% TGI in PC-3/TxR tumors (table 15). In addition, at higher doses (20mg/kg), docetaxel caused partial regression (> 100% TGI) of PC-3 tumors, but only 56% TGI in PC-3/TxR tumors. The efficacy of docetaxel in PC-3/TxR tumors was significantly reduced compared to that in PC-3 tumors, indicating a reduced efficacy of drug resistance mediated by P-gp, and these results are in close agreement with our in vitro cytotoxicity or apoptosis data. In contrast to the lack of efficacy of docetaxel in PC-3/TxR tumors, orally administered 17ya (6.7mg/kg) showed over 100% TGI with no effect on its body weight (fig. 21B and table 15). In addition, 2 of 4 nude mice with PC-3/TxR tumors had no tumor at day 19 (data not shown).

The efficacy of 17ya (on other dosing schedules) and 55 was further evaluated using the PC-3/TxR xenograft model. When orally administered once daily for four days, the maximum tolerated dose (> 20% weight loss) of 17ya was found to be 10 mg/kg; or 3.3mg/kg when administered twice daily (b.i.d.) for five days (data not shown). As shown in FIG. 21C, 3.3mg/kg 17ya was administered twice daily for the first four consecutive days of the first week, and then the schedule was changed to once daily for weeks 2 to 4. The results showed that partial regression was obtained in days 4-19 with a TGI of 97%, and one of seven mice was tumor-free on day 26. Higher doses (10mg/kg) of 17ya (fig. 21D) at lower dosing frequency (q2D) caused partial regression on days 13 to 29. These data indicate that a regimen with optimal dose and schedule of administration would favor successful inhibition of PC-3/TxR tumors by 17 ya. 55 was orally administered to nude mice at 10mg/kg or 30mg/kg b.i.d. five times a week on weeks 1 to 4. As shown in fig. 21C, the inhibition curve shows dose response in PC-3/TxR tumors. For the treatment group with the lower dose (10mg/kg), the TGI value was 59%. Moreover, higher doses (30mg/kg) showed partial regression (> 100% TGI) starting from day 19 to study termination (day 26). Some mice in the vehicle group lost weight at the end due in part to cancer cachexia. In contrast, mice treated with 17ya (3.3mg/kg) or 55(30mg/kg) gained weight (table 15), indicating that these optimal doses of 17ya or 55 are well tolerated and prevent cancer cachexia.

Table 15. in vivo anti-tumor activity of compounds 17ya and 55 relative to concomitantly evaluated docetaxel.

Dosing schedule: qd × 5/w — once weekly for five consecutive days; b.i.d. × 5/w given twice weekly for five consecutive days; or q2d × 3/w is administered every other day or three times a week.

^aThe dosing schedule was twice four consecutive days for the first week and changed (due to toxicity) to once five consecutive days per week for the second to fourth weeks.

Brain penetration of 17ya and 55 in nude mice.

The whole brain concentration in nude mice was determined 1h and 4h after oral administration of 20mg/kg 17ya or 55 (Table 16). The ratio of brain to plasma concentrations was determined and compared to docetaxel in nude mice. 55 showed higher brain penetration than 17ya and docetaxel. 17ya showed slightly higher brain/plasma concentration ratios than docetaxel at only 1h and 4 h. The 55 brain concentration reached 14-19% plasma concentration at 1h and 4h, respectively, showing a 3.2-fold higher brain/plasma ratio at 1h and 4h compared to docetaxel. These data indicate that 55 exhibits potentially advantageous properties for treating gliomas due to its higher brain penetration and high potency in glioma cells (22nM, table 12).

TABLE 16 Blood Brain Barrier (BBB) studies of compounds 17ya and 55. Brain and plasma concentrations were determined in nude mice at 1h and 4h after docetaxel (IP, 10mpk), 17ya (PO, 20mpk) and 55(PO, 20mpk) administration. Each value represents the mean ± SD of 3 nude mice.

Example 10

In vivo efficacy in leukemia (HL60) xenografts (fig. 22).

HL60 cells (10X 10) were prepared in RPMI1640 growth medium containing 10% FBS⁷one/mL) and mixed with Matrigel (BD Biosciences, San Jose, Calif.) at a ratio of 1: 1. 100 μ L of the mixture (5X 10) was injected subcutaneously into the flank of 6-8 week old male athymic nude mice⁶Individual cells/animal) to establish tumors. Measuring the length and width of the tumor according to the formula pi/6 XLXW²Calculation of tumor volume (mm)³) Wherein the length (L) and width (W) are measured in mm. When the tumor volume reaches about 200mm³When this is done, the vehicle [ Tween80/DMSO/H₂O(2/2/6)]Or 17ya (20mg/kg) orally treated animals loaded with HL60 tumor. The dosing schedule was once a week for two weeks. Vincristine (1mg/mL) was administered once a week via intraperitoneal injection.

Results

Human promyelocytic leukemia cells, HL60 cells, were seeded in nude mice to reach a tumor volume of about 200mm³. Vincristine (1mg/kg), clinically used for hematological cancers including leukemia, was used to assess the response of this in vivo model relative to positive control drugs. Tumor volume (mm)³) Curves were plotted against time and the tumor volume was the mean ± SD of 4-5 animals. HL60 tumor growth was found to be rapid and the volume reached 2000-300 in two weeks0mm³. However, intraperitoneal injection of 1mg/kg vincristine showed a very strong tumor growth inhibition effect (fig. 22), and the Tumor Growth Inhibition (TGI) was 84%. Orally administered 17ya (20mg/kg) showed 40% inhibition of tumor growth. The size of the HL60 tumor remained unchanged for up to 5 days after 17ya treatment without significant increase, but the tumor size increased significantly (60-100%) over the next two days. This indicates that the tumor growth inhibitory effect of 17ya was enhanced by the more frequent dosing schedule.

Example 11

Combinations of BRAFi and tubulin inhibitors targeting the alternative pathway delay or prevent vemurafenib resistance And (5) progressing.

The treatment specific goals for this study are summarized in figure 27.

Docetaxel (approved tubulin inhibitor) and BRAFi (Verofenib or Dalafinil) or MEKi (tramet) Tinib) can suppress melanoma patient-derived xenograft (PDX or "xenopatient (xenopatient)") tumor Acquired vemurafenib resistance in the model.

The usefulness of any new therapeutic strategy ultimately depends on its ability to demonstrate sustained clinical efficacy. Melanoma is a heterogeneous tumor that is well known to exhibit high phenotypic and functional plasticity in response to microenvironment or epigenetic factors. In fact, recent reports indicate that vemurafenib resistant tumors can develop a multi-drug resistance mechanism within one patient or even within the same tumor biopsy taken from a single metastatic site. Unlike melanoma tumors in patients, tumors grown from established cell lines (e.g., a375) have two major limitations: (a) they lose their original tumor heterogeneity which can significantly affect the efficacy of the drug; and (b) they often develop irreversible genetic changes as a result of their adaptation to cell culture conditions different from the native tumor microenvironment. Numerous studies have shown that early passage PDX tumors (< 5 passages) maintain the genetic fidelity of the patient's tumor, retain the original tumor morphology and heterogeneity, and have a similar response pattern to treatment as observed clinically. Therefore, it must be confirmed that the strong synergy and efficacy observed in a375RF21 tumors (see fig. 6 and 7) is still effective in PDX tumors. Also important, PDX tumors are used to assess the possible progression of drug resistance for more direct laboratory-to-hospital bed transformation (stem-to-stem transformation) than tumors grown from established cell lines.

Since there are approved tubulin inhibitors, to facilitate the development of this innovative combination to rapidly benefit patients, an assessment of the efficacy of the combination of docetaxel with the three BRAFi/MEKi currently approved was first established to determine the optimal combination. Furthermore, if a new combination strategy is used to treat both groups with BRAF^V600EPatients with mutated melanoma, then they have a higher impact. The first group is patients initially treated for BRAFi who do not take BRAFi and/or MEKi. Such patients benefit most if the combination can significantly delay or even prevent the development of possible BRAFi resistance. The second group is BRAFi-resistant patients who have been treated with BRAFi and/or MEKi in clinical reality. These patients benefit if the combination is effective to overcome acquired resistance.

The progression of combined efficacy and possible resistance in vivo in vemurafenib-sensitive PDX tumors was determined to mimic clinical use (e.g., prior use of combination therapy prior to vemurafenib resistance development) to treat patients with BRAFi naive melanoma. As the best strategy to overcome acquired resistance is to significantly delay or even prevent its progression.

Early PDX tumors were established based on PDX tumor-loaded mice.

In this field, efforts have recently been made to standardize some of the schemes used to build successful PDX models. A variety of PDX models are now available from commercial sources. Purchased NSG mice (typically 1-2 mice) implanted with small second generation (P2) PDX tumors were used. Growing the tumor to about 1,500mm³And bred toUp to five new NSG mice to provide sufficient P3 tumor.Genetic profiles and histology of five randomly selected P3 tumors were characterized and data from the original P0 tumor was used Validation is performed to ensure overall genetic and histological fidelityTumor masses were then implanted into a large number of mice for subsequent studies (fig. 27). Histological analysis was performed to ensure the clarity of these validation analyses.

a. Determining vemurafenib sensitivity in combination therapy with docetaxel and vemurafenib, dalafinib or trametinib In vivo efficacy in sensitive PDX tumors.

Three currently approved BRAFi or MEKi were tested to rank their combined efficacy for future clinical optimization. Although NSG mice are the preferred mice for tumor propagation, they have fur and are more expensive (2-3 fold) than nude mice. Numerous studies have shown that the end-point results using nude mice are equally good in PDX studies compared to using more expensive and difficult NSG mice. Therefore, nude mice were used in the terminal efficacy study. Standard combination therapy of dabrafenib + trametinib was included as a reference combination therapy. In addition, all drugs used in this step are approved drugs and their pharmacokinetic properties are known. The study followed the dosages and routes of administration established thereby. Specifically, vemurafenib (45mg/kg), dabrafenib (30mg/kg), and trametinib (0.3mg/kg) were administered to mice by oral gavage. Docetaxel (10mg/kg) was injected intravenously via the tail vein because it was not orally available.

Each group of 7 mice was used to assess whether sustained tumor regression was achieved in three independent vemurafenib sensitive PDX tumors. Statistical analysis and sample size calculations were performed to ensure statistical significance in these important animal studies.

Briefly, 6 week old male athymic nude mice were purchased from Charles River. P3 Verofini sensitive PDX melanoma tumors were minced into small pieces (about 3 mm)³) Then, according to established recipes reported in the literatureSurgically implanted subcutaneously into the flank of 63 anesthetized nude mice. When implanted for 2-3 weeks, the tumor reaches about 100-200mm³When mice were randomized into 9 groups (n-7) to minimize the difference in weight and tumor size: negative control group with vehicle only (group 1); four single agent treatment groups (groups 2-5) using continuous daily oral treatment with vemurafenib, dabrafenib, trametinib, or docetaxel (i.v.); and four combination treatment groups (groups 6-9) using dabrafenib + trametinib (reference combination), docetaxel + vemurafenib, docetaxel + dabrafenib and docetaxel + trametinib, continuously daily using the same dose and route of administration as the single agents.

Due to the nature of PDX, it is impractical to attach any in vivo luminescent probe for tumor monitoring. Therefore, every three days the tumor was measured with a caliper and its volume was calculated using the formula: (Width)²X length)/2. Based on previous reports, vemurafenib-sensitive tumors clearly develop acquired resistance to single agent vemurafenib treatment in mice within 60 days. Thus, tumor monitoring is up to 90 days, or until the tumor has completely regressed. The progression of potential drug resistance is closely monitored by tumor growth kinetics. In addition, a series of tumor biopsies were taken every two weeks from each tumor with a fine needle according to the reported method. The levels of BRAF and pERK/tERK in these biopsies were determined by western blotting to monitor the progression of possible resistance to BRAFi and MEKi. Mice were closely monitored for weight, activity and appearance for potential toxicity.

At the end of the experiment, terminal blood samples (0.8-1 mL/mouse) were collected by cardiac puncture for comprehensive clinical pathology analysis provided by charles river laboratory. Immediately after blood collection all animals were passed through CO₂Inhalation followed by cervical dislocation was sacrificed and the major organs (brain, heart, lung, liver, spleen, kidney) of each mouse were collected and stored in 10% buffered formalin phosphate solution, respectively. These organs were carefully examined and analyzed for potential drug toxicity (e.g., hepatotoxicity) and signs of metastasis. Tumors were carefully collected, weighed, and processed to determineThe effect of drugs on key indicators of cell proliferation, anti-angiogenesis and apoptosis, and fixation and handling for histopathological examination. The above experiment was repeated using two additional vemurafenib sensitive PDX tumor models to ensure that efficacy was not correlated with a particular PDX tumor model, therefore, all mice used in this step were estimated to be as many as 63 x 3 ═ 189.

b. Determining vemurafenib resistance in combination therapy with docetaxel and vemurafenib, dabrafenib or trametinib In vivo efficacy in drug-induced PDX tumors.

An ideal strategy to overcome acquired vemurafenib resistance is to forego the use of very effective therapies to prevent their development. Unfortunately, this is not practical with current therapies and most patients develop vemurafenib resistance rapidly. Stuart et al have recently suggested that an intermittent high dose schedule of vemurafenib, a single agent, may attenuate the progression of drug resistance compared to a continuous schedule. However, the psychological impact and long-term clinical efficacy of this "drug holiday" strategy on patients remains to be observed. Since drug resistant melanoma tumors often prefer the activated MEK-ERK pathway, a suitable combination of a tubulin inhibitor with BRAFi/MEKi still suppresses tumor growth very effectively, however the use of a single agent may not be sufficient (fig. 6 and 7). Thus, it was confirmed that docetaxel in combination with BRAFi/MEKi is important for vemurafenib resistant tumors to induce substantial tumor regression. Such a result may translate into prolonged patient survival even when the tumor becomes vemurafenib resistant.

Similar to the experimental approach described above, 63 mice divided into 9 groups (one vehicle only group, four single agent treatment groups, and four combination treatment groups) were used to determine whether docetaxel in combination with approved BRAFi or MEKi drugs was effective in the vemurafenib resistant PDX model after early PDX tumors were established in vemurafenib resistant PDX tumor-loaded mice. Tumor size, series of tumor biopsies, terminal blood samples and major organs were measured or collected to assess efficacy and potential toxicity similar to those described in the previous section. Repeating the combination test in two additional vemurafenib resistant PDX tumor models; therefore, 63 × 3 ═ 189 mice were used in this step.

Although these vemurafenib resistant PDX tumors are expected to be resistant to the combination of the single agents BRAFi/MEKi or dabrafenib + trametinib as shown in clinical trials and results presented above with the a375RF21 xenograft model, these are considered valuable references for objectively assessing the potential efficacy of the combination containing docetaxel. Similar to experiments using the vemurafenib sensitive PDX model, tumor size, potential acute toxicity, clinical pathology, and levels of pERK/ERK in tumor biopsies were determined to rank the efficacy of these combinations.

Determination of potential toxicity and focused western blot analysis to assess treatment efficacy and identify use for clinical disease Potential biomarkers for disease monitoring.

Determination of potential toxicity of combination therapy in PDX model:

in addition to closely monitoring the weight and activity of PDX tumor-bearing mice during treatment, blood samples collected at the endpoint were sent for comprehensive clinical pathology (blood chemistry) within 24 hours after collection. The complete pathological, chemical and hematological (complete blood count by differential) properties were examined and provided detailed results. Results were analyzed for potential toxicity indications similar to those described above.For pathological analysisFormalin-fixed tumor tissue is processed into paraffin blocks, and sections are stained with hematoxylin and eosin. Use ofXT (Aperio Technologies, CA) scans the slides at 0.25 pixels/μm to create digital copies of the entire tissue on glass microscope slides. The scanning process enables the display and analysis of tissue images at different magnifications to closely simulate the conventional observation of tissue with a conventional microscope.

Pathological assessment of cell proliferation and apoptosis in PDX tumor sections.

To assess whether the drug combination retained enhanced efficacy, harvested PDX tumors were processed into tumor sections and immunohistochemical analysis was performed.

For anti-proliferation assessment, tumor sections were examined for their decreased pERK levels and Ki67 staining, which is a marker of tumor cell proliferation, following standard immunohistochemical methods. The proportion of melanoma cells within tumor sections was determined using melanoma specific markers, i.e., S100 and HMB-45 binding H & E staining.

For apoptosis assessment, nuclear morphology was assessed for evidence of nuclear fragmentation by fixing tumor sections, staining the sections with Hoechst 33342 and counting nuclei that showed fragmented or normal morphology. Alternatively, changes in the nucleus are assessed by TUNEL followed by analysis of apoptotic pathways. Changes in mitochondrial transmembrane potential were measured using the flow cytometric kit. Cytochrome c concentration and subcellular distribution (translocation from mitochondria to cytosol) were assessed by western blotting. Expression of the anti-apoptotic proteins Bcl-2 and Bcl-x 1; expression of pro-apoptotic proteins Bax and Noxa; and the phosphorylation state of pro-apoptotic protein Bad were assessed by western blotting. Activation of the initiator caspase 9 and the effector caspase 3 was assessed by measuring the specific proteolytic cleavage of the fluorogenic substrates Ac-LEHD-AFC (caspase 9) and Ac-DEVD-AMC (caspase 3).

Focused western blot analysis to assess combined treatment efficacy and identify potential for clinical disease monitoring In the context of biomarkers.

Specifically, based on the data given above, the focus was on examining elements in the MAPK, PDGFB, PI3K/AKT and apoptotic pathways, as they are known to be associated with vervafenib resistance, the expression levels of RAS, RAF, MEK1/2, phospho-MEK 1/2(Ser217/221), ERK1/2, phospho-ERK 2/2 (Thr202/Tyr204), AKT, phospho-AKT (Ser473), β, lytic PARP or caspase-3 proteins were analyzed for vervafenib resistance PDX tumors, the gene profile of their resistance was obtained and the resistance of tumors was monitored by a baseline protein blot 0, the baseline resistance mechanism of the treatment, and the focus was determined by western blot.

In addition, the use of taxanes including docetaxel gives only a moderate survival advantage, with most patients eventually progressing due to inherent acquired resistance. An important drug resistance mechanism is mediated by ABC transporters, which can reduce the intracellular concentration of docetaxel. Thus, in addition to monitoring the levels of proteins responsible for BRAFi/MEKi resistance, the production of potential docetaxel was also monitored by examining the expression of important ABC transporters including P-glycoprotein (Pgp), Multidrug Resistance Protein (MRP) and Breast Cancer Resistance Protein (BCRP) using methods similar to those described in the above studies. Protein extraction, western blotting and antigen detection were performed according to standard protocols, with the target antigen changed. Protein levels (average from triplicate experiments) were accurately quantified by densitometric analysis.

Expected results, defects, and other alternatives.

Docetaxel in combination with BRAFi or MEKi is expected to be effective against both vemurafenib sensitive PDX models and vemurafenib resistant PDX models. Since all the drugs used in this study have been approved, the results of this goal would be very easy to translate if proven to be effective, and clinical testing could be performed quickly to immediately benefit patients as a first-line combination therapy.

The combination of ABI (a novel tubulin inhibitor) with BRAFi or MEKi suppresses acquired BRAFi resistance and secondary taxane resistance in PDX tumors.

Table 17.ABI showed excellent efficacy against all melanoma cell lines tested, including highly metastatic cell lines and multidrug resistant cell lines.

Note: *: (ii) parental cell lines to drug resistant cell sublines; MDR1 was overexpressed in MDA-MB-435/LCC6MDR1 and NCI/ADR-RES; MRP1, MRP2 and BCRP were overexpressed in HEK293-MRP1, HEK293-MRP2 and HEK293-482R 2. Drug resistance index (number in parentheses) by using IC against drug resistant cell sublines₅₀Value divided by IC for the corresponding parental cell line₅₀A value is calculated. Abbreviations: n/a, is not applicable because it binds to a different site of tubulin. ND, not determined.

Clinical use of docetaxel may lead to secondary taxane resistance in combination therapy of docetaxel with BRAFi/MEKi. ABI binds to different sites of tubulin than approved tubulin inhibitors and has unique advantages including high potency, acceptable oral bioavailability, excellent pharmacokinetic properties, and efficacy in overcoming ABC transporter mediated multidrug resistance (table 17). Preliminary toxicity studies indicate that ABI has significantly lower toxicity compared to existing tubulin inhibitors such as docetaxel or vinblastine. Therefore, to develop a new generation combination therapy to overcome potential secondary resistance to the first-line combination containing docetaxel, the efficacy of BRAFi/MEKi in combination with two advanced ABIs (compound 12da and compound 17ya) was determined in the PDX model. ABI and approved BRAFi/MEKi are both orally active agents. No adverse drug-drug interactions between tubulin inhibitors and BRAFi/MEKi were reported. Thus, based on the a375RF21 xenograft model, oral co-administration of ABI with vemurafenib, dabrafenib, or trametinib maintained strong synergy in suppressing melanoma tumor growth in PDX tumors.

Following the same approach described above, a brief description follows:

c. the assay was performed using a combination of ABI (Compounds 12da and 17ya) and Verofenib, dabrafenib or trametinib In vivo efficacy in treating vemurafenib sensitive PDX tumors.

NSG mice (typically 1-2 mice) implanted with small second generation (P2) PDX tumors were used for this study. Growing the tumor to about 1,500mm³And propagated into up to five new NSG mice to provide sufficient P3 tumor. The genetic profile and histology of five randomly picked P3 tumors were characterized and validated with data from the original P0 tumor to ensure overall genetic and histological fidelity, and the tumor masses were then implanted into a large number of mice for subsequent studies (fig. 27).

Since the pharmacokinetic properties of ABI and BRAFi/MEKi have been established, combined efficacy, potential toxicity and possible disease monitoring biomarkers were determined in three independent vemurafenib sensitive PDX models, similar to those described above. Since the results of standard single agent vemurafenib, dabrafenib or trametinib treatments have been obtained as described above, the combination treatment continues to be studied by including the best efficacy determined in the docetaxel combination as well as the reference combination (dalrafenib + trametinib) when the evaluation of the combination containing docetaxel is complete. Thus, for compound 12da, a small piece (about 3 mm) was added³) Vemurafenib-sensitive P3 tumor was surgically implanted subcutaneously in the flank of 49 anesthetized nude mice (6 week old male athymic nude mice). When implanted for 2-3 weeks, the tumor reaches about 100-200mm³When mice were randomly divided into 7 groups (n-7) (to minimize weight and tumor size differences): negative control group with vehicle only (group 1); a single medicineAgent Compound 12da (15 mg/kg)⁵³Group 2); and five combination treatment groups including the following consecutive daily treatments: dabrafenib + trametinib (reference combination, group 3), the most potent docetaxel-containing combination identified above (group 4), and the combination of compound 12da with vemurafenib, dabrafenib or trametinib (groups 5-7). Every three days the tumors were measured with calipers and their volume was calculated using the formula: (Width)²X length)/2. tumor monitoring for up to 90 days, or until complete tumor regression, progress of potential resistance is closely monitored by tumor growth kinetics.the body weight, activity and appearance of mice are closely monitored for potential toxicity.terminal blood samples are sent for blood chemistry analysis and tumor sections are processed for clinical pathology analysis.additionally, a series of tumor biopsies are taken weekly from each tumor with fine needles according to the reported methods focused western blot analysis examining elements in the MAPK, PDGF β -PI3K/AKT and apoptotic pathways is performed to monitor acquired resistance and potential biomarkers for assessing therapeutic efficacy the experiment is repeated using two additional vemurafenib sensitive PDX tumors, thus, for testing with compound 12da, up to 49 x 3-147 mice are used, to complete the test with compound 17ya, up to 147 x 2-294 mice are used.

d. Determination of Victoria in combination treatment with ABI (12da or 17ya) and Verofenib, Dalafinib or trametinib In vivo efficacy in rofenib resistant PDX tumors.

Similar to the experimental method described briefly in the section above, the experiment was repeated using three vemurafenib resistant PDX tumor models and up to 294 mice were used to determine the efficacy of ABI (compound 12da or 17ya) in combination with vemurafenib, dabrafenib or trametinib. Tumor size, series of tumor biopsies, terminal blood samples and major organs were measured or collected to assess efficacy and potential toxicity similar to those described in the previous section. Potential toxicity was determined by monitoring weight loss during the experiment and after pathological analysis. Focused western blot analysis was performed as detailed above to assess treatment efficacy and identify potential biomarkers.

Expected results, defects, and other alternatives

ABI in combination with BRAFi or MEKi is expected to be effective against both vemurafenib sensitive PDX models and vemurafenib resistant PDX models. It is expected that the efficacy of such combinations will be comparable to or higher than those containing docetaxel, but with the benefit of overcoming the potential secondary resistance associated with the use of docetaxel.

Combinations of tubulin inhibitors with BRAFi or MEKi are effective in melanoma metastasis models.

A major challenge in providing extended survival for melanoma patients is finding an effective treatment for melanoma metastases. Due to the subcutaneous nature, the PDX model rarely metastasizes. Thus, in addition to evaluating the efficacy of the systemically administered drug combination against melanoma tumors in these subcutaneous PDX models as described above, the combined efficacy in experimental lung metastasis models was also determined using nude mice. The lung metastasis model was chosen because it is one of the major metastatic sites of malignant melanoma and has the worst 5-year survival rate among all melanoma metastases. The PI laboratory has a well established protocol to assess melanoma lung metastases (fig. 28), and ABI as a single agent has been shown to be effective in suppressing melanoma lung metastases (compound 17ya is shown as an example). Similar models have been widely used in the literature. In addition, unlike cells cultured from long-established cell lines (e.g., a375), single cell suspensions from early passages were never grown in plastic directly from PDX tumor isolation. Thus, they can retain tumor heterogeneity, genetic fidelity, and the expected response to drug treatment to cells in the original patient's tumor.

Single cell suspensions were isolated from early passage PDX tumors.

Single cell suspensions were prepared from PDX tumors according to standard protocols. Briefly, NSG mice (usually 1-2 mice) were purchased and implanted with small, second generation (P2) PDX tumorsThereafter, the tumor was grown to about 2,000mm³. Within 1 hour of surgical removal from NSG mice, fresh tumors were washed 3 times with DMEM medium to avoid contamination (5 min each on ice). PDX tumors without necrosis and connective tissue were minced under sterile conditions into small pieces (1 x 1mm or less) using sterile crossed blades. The tumor mass was mixed with a 1mg/mL solution of ultrapure collagenase type IV (Gibco, 17104-. After digestion, the resulting mixture was filtered through a nylon mesh cell strainer (BDBiosciences, 70 μm pore, 352340) to obtain a single cell suspension. The number of cells was counted using an Auto T4 cell counter, pelleted, and resuspended in DMEM for tail vein injection. The viability of cells obtained by this method is typically over 95% in the trypan blue test.

Lung metastases were arrested using either vemurafenib-sensitive or vemurafenib-resistant single cell suspensions.

Since the best combination has been arranged in the previous study described above, the best combination is tested. The approved combination of dabrafenib + trametinib is always included as a reference combination. Briefly, 7.5 million single cells generated from either vemurafenib-sensitive or vemurafenib-resistant PDX tumor models suspended in100 μ L DMEM were injected via their tail vein into each of 50 nude mice (6 weeks old, either sex), and the mice were divided into 5 groups (n ═ 10): negative control group with vehicle only (group 1); treatment group with a reference combination of dabrafenib + trametinib (group 2); the best combination of docetaxel and BRAFi/MEKi identified above (group 3); optimal combination of compounds 12da or 17ya with BRAFi/MEKi as determined above (groups 4 and 5). The number of mice in each group was increased from 7 to 10 to ensure statistical significance, since large variations are expected with this experimental lung metastasis model. After 7-10 days of tumor cell injection, mice were treated orally with vehicle or drug combination daily (except docetaxel, which was not available orally and was administered by i.v. route) for four weeks. At the end of the treatment, all mice were passed through CO₂Inhaling and then dislocating the neckAnd (6) killing. Mice were dissected to remove lungs. The number of tumor nodules in the lung was accurately counted and treatment efficacy was assessed based on the absence of tumor nodules or the reduction in the number of tumor nodules in the lung. Paraffin-embedded sections were also fixed and processed, followed by H&After E staining the nodules were examined for melanoma nature and tumor morphology. For this experiment using cells from three vemurafenib-sensitive PDX models and three vemurafenib-resistant PDX models, up to 50 × 3 × 2 ═ 300 nude mice are expected to be used.

Expected results, defects, and other alternatives

These combinations are expected to be effective in reducing the number of lung metastases in vemurafenib-sensitive tumors, and to a lesser extent in vemurafenib-resistant tumors. The combination containing the tubulin inhibitor (docetaxel, compound 12da or 17ya) may be more effective than the reference combination of dabrafenib + trametinib, especially for vemurafenib resistant tumor metastases. In the unlikely event that single cell suspensions isolated from PDX tumors are unable to form lung metastases, as an alternative approach, established early passage BRAF is used^V600EHuman melanoma cell lines (YUGEN8, yusc 2, YUKOLI and YUSIK).

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims.

Claims

1. A pharmaceutical composition comprising (a) a tubulin inhibitor, which is a compound represented by the structure of formula II:

wherein

A is aryl or indolyl;

R₁is H, C₁-C₆Linear or branched alkyl, aryl, phenyl, benzyl, haloalkyl, aminoalkylRadical, -OCH₂Ph、SO₂Aryl, SO₂-phenyl, - (C ═ O) -aryl, - (C ═ O) -phenyl or OH;

n is an integer from 1 to 4;

or a pharmaceutically acceptable salt, N-oxide, hydrate, tautomer, or isomer thereof;

(b) at least one of a BRAF inhibitor or a MEK inhibitor; and (c) a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the BRAF inhibitor is vemurafenib, dabrafenib, or a combination thereof, and the MEK inhibitor is trametinib or RO5068760, or a combination thereof.

3. Use of a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor and at least one compound of formula II in the manufacture of a medicament for treating a BRAF mutant cancer in a subject, wherein the compound of formula II is represented by the following structure:

wherein

A is aryl or indolyl;

R₁is H, C₁-C₆Linear or branched alkyl, aryl, phenyl, benzyl, haloalkyl, aminoalkyl, -OCH₂Ph、SO₂-aryl radical、SO₂-phenyl, - (C ═ O) -aryl, - (C ═ O) -phenyl or OH;

n is an integer from 1 to 4;

4. The use of claim 3, wherein the BRAF mutant cancer is a BRAF inhibitor-resistant cancer.

5. Use of a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and at least one compound represented by the structure of formula II:

wherein

A is aryl or indolyl;

n is an integer from 1 to 4;

6. The use of claim 5, wherein the melanoma is drug resistant melanoma.

7. The use of claim 4, wherein the medicament delays or prevents BRAF inhibitor resistant cancer.

8. Use of a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and at least one compound represented by the structure of formula II:

wherein

A is aryl or indolyl;

R₄and R₅Each independently is hydrogen, C₁-C₆Linear or branched alkyl, C₁-C₆Linear or branched haloalkyl, C₁-C₆Linear or branched alkoxy, C₁-C₆Linear or branched haloalkoxy、F、Cl、Br、I、CF₃、CN、-CH₂CN、NH₂、OH、-OC(O)CF₃Alkylamino, aminoalkyl, -OCH₂Ph, -NHCO-alkyl, COOH, -C (O) Ph, C (O) O-alkyl, C (O) H, -C (O) NH₂Or NO₂(ii) a And is

n is an integer from 1 to 4;

9. Use of a composition comprising at least one of a BRAF inhibitor or a MEK inhibitor, and at least one compound represented by the structure of formula II, in the manufacture of a medicament for treating secondary cancer resistance to a taxane drug in a subject having cancer previously treated with the taxane drug:

wherein

A is aryl or indolyl;

n is an integer from 1 to 4;

10. The use of claim 9, wherein the taxane is docetaxel.

11. The use of any of claims 3-10, wherein the BRAF inhibitor is vemurafenib, dabrafenib, GDC-0879, PLX-4720, sorafenib tosylate, LGX818, or any combination thereof; and the MEK inhibitor is trametinib, semetinib, RO5068760, MEK162, PD-325901, cotinetinib, CI-1040, or any combination thereof.

12. The use of any one of claims 3, 4 or 7-10, wherein the cancer is melanoma, thyroid cancer, colorectal cancer, breast cancer, colon cancer, biliary tract cancer, non-small cell lung cancer (NSCLC) or ovarian cancer.

13. The use of any one of claims 3, 4 or 7-10, wherein the cancer is melanoma, thyroid cancer, colorectal cancer or ovarian cancer.

14. The use of any one of claims 3, 4 or 7-10, wherein the cancer is melanoma.

15. The use of claim 5, wherein the melanoma is drug resistant melanoma.

16. The use of claim 11, wherein the melanoma is V600E positive melanoma.

17. The use of any one of claims 3, 4, or 7-10, wherein the cancer is a drug-resistant cancer.

18. The use of any of claims 3-10, wherein the BRAF inhibitor is vemurafenib, dabrafenib, or a combination thereof; and the MEK inhibitor is trametinib, RO5068760, or a combination thereof.