Microtubule-targeting agents

Abstract

A method is provided for treating cancers that respond to modulation of microtubule assembly and P-gp-mediated resistant cancers using 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 525,377, filed Jul. 7, 2023, and U.S. Provisional Patent Application No. 63 / 459,173, filed Apr. 13, 2023, each of which is incorporated herein by reference in its entirety.

Background Art

[0002] Microtubule targeting agents (MTAs) are one of the most effective chemotherapeutic agents used in cancer treatment. However, the clinical utility of current MTAs such as microtubule targeting vinca alkaloids (e.g., vinblastine and vincristine) and taxanes (paclitaxel and docetaxel) is often limited due to adverse side effects or multidrug resistance (MDR). Previous studies have established that a broad range of resistance to these drugs largely results from overexpression of P-glycoprotein (P-gp), of which paclitaxel, vinblastine, vincristine, docetaxel, and others are substrates. See, e.g., Gottesman et al. (2002), Nat Rev Cancer 2 48-58, and Mol Pharmacol. 2009 Jan;75(1):92-100. This presents a major challenge in cancer chemotherapy, particularly when managing patients with metastatic cancer who are resistant to conventional MTA therapy.

[0003] To overcome P-gp-mediated MDR, many small molecule drugs that modulate the activity of P-gp have been tested. See, e.g., Darby et al., Curr. Drug. Metab 2011, 12, 722-731. However, most of these programs have failed clinical trials due to lack of efficacy and / or toxicity issues. Thus, there is a need for new microtubule interacting substances, particularly those that are not substrates for P-gp.

Summary of the Invention

[0004] Herein, the evidence suggests that 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide, referred to herein as compound 1, inhibits microtubule assembly formation. See, for example, Figure 1.

[0005] Accordingly, this specification provides a method for treating cancers that respond to the modulation of microtubule assembly using 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof. Such cancers include, for example, prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer.

[0006] The evidence also indicates that compound 1 is not a substrate for P-gp, and that its overexpression leads to P-gp-mediated multidrug resistance to certain vinca alkaloids and taxane chemotherapeutic agents. Therefore, methods for treating P-gp-mediated resistant cancers using compound 1 or a pharmaceutically acceptable salt thereof are also provided.

[0007] In certain embodiments, the present disclosure relates to a method for treating cancer in response to modulation of microtubule assembly, comprising administering a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof to a subject in need thereof. In some embodiments, the cancer is selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is sarcoma.

[0008] In certain embodiments, the Disclosure relates to a method for treating P-gp-mediated resistant cancer, comprising administering a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof to a subject in need thereof. In some embodiments, the cancer is taxane-resistant cancer. In some embodiments, the cancer is resistant to paclitaxel, vinblastine, vincristine, or docetaxel. In some embodiments, the cancer is resistant to paclitaxel. In some embodiments, the cancer is selected from ovarian cancer, prostate cancer, breast cancer, bladder cancer, head and neck cancer, and lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is vinca alkaloid-resistant cancer. In some embodiments, the cancer is resistant to vinblastine or vincristine. In some embodiments, the cancer is selected from lymphoma, acute lymphoblastic leukemia (ALL), and solid tumors. [Brief explanation of the drawing]

[0009] [Figure 1] The Vmax values ​​for compound 1, paclitaxel, and the positive control nocodazole in the microtubule polymerization assay are shown. [Figure 2] This Western blot supports the binding of compound 1 to the colchicine binding site of tubulin. [Figure 3] Figure 2 shows the significance of the upper band intensity (relative to total B-tubulin) from the Western blot. [Figure 4] This illustrates the effect of compound 1 on kinetochore assembly in HCT116 cells treated with the compound. [Figure 5]This illustrates the effect of compound 1 on kinetochore assembly in HCT116 cells treated with the compound. [Figure 6] This shows spindle assembly checkpoint activation in HCT116 cells treated with compound 1. [Figure 7A] This study demonstrates the efficacy of compound 1 in patient-derived organoids (PDOs) representing multiple tumor types. PDOs were treated with compound 1 or a control (vehicle control - 0.1% DMSO, positive control - 10% DMSO) for 5 days. Survival rates were assessed using the CellTiter-Glo® assay. IC50 and max inhibition values ​​were estimated from survival curves. Examples of models considered highly responsive (>60% max inhibition; A), responsive (40-60% max inhibition; B), and unresponsive (<40% max inhibition; C) to compound 1 are shown. A summary of all tested models (D): NR (no regression, ambiguous nonlinear regression curve or R2 < 0.65) and NA (not applicable, ambiguous max inhibition). N = 4 copies of compiled data. [Figure 7B] This study demonstrates the efficacy of compound 1 in patient-derived organoids (PDOs) representing multiple tumor types. PDOs were treated with compound 1 or a control (vehicle control - 0.1% DMSO, positive control - 10% DMSO) for 5 days. Survival rates were assessed using the CellTiter-Glo® assay. IC50 and max inhibition values ​​were estimated from survival curves. Examples of models considered highly responsive (>60% max inhibition; A), responsive (40-60% max inhibition; B), and unresponsive (<40% max inhibition; C) to compound 1 are shown. A summary of all tested models (D): NR (no regression, ambiguous nonlinear regression curve or R2 < 0.65) and NA (not applicable, ambiguous max inhibition). N = 4 copies of compiled data. [Figure 7C]This study demonstrates the efficacy of compound 1 in patient-derived organoids (PDOs) representing multiple tumor types. PDOs were treated with compound 1 or a control (vehicle control - 0.1% DMSO, positive control - 10% DMSO) for 5 days. Survival rates were assessed using the CellTiter-Glo® assay. IC50 and max inhibition values ​​were estimated from survival curves. Examples of models considered highly responsive (>60% max inhibition; A), responsive (40-60% max inhibition; B), and unresponsive (<40% max inhibition; C) to compound 1 are shown. A summary of all tested models (D): NR (no regression, ambiguous nonlinear regression curve or R2 < 0.65) and NA (not applicable, ambiguous max inhibition). N = 4 copies of compiled data. [Figure 7D] This study demonstrates the efficacy of compound 1 in patient-derived organoids (PDOs) representing multiple tumor types. PDOs were treated with compound 1 or a control (vehicle control - 0.1% DMSO, positive control - 10% DMSO) for 5 days. Survival rates were assessed using the CellTiter-Glo® assay. IC50 and max inhibition values ​​were estimated from survival curves. Examples of models considered highly responsive (>60% max inhibition; A), responsive (40-60% max inhibition; B), and unresponsive (<40% max inhibition; C) to compound 1 are shown. A summary of all tested models (D): NR (no regression, ambiguous nonlinear regression curve or R2 < 0.65) and NA (not applicable, ambiguous max inhibition). N = 4 copies of compiled data. [Figure 8] This shows the tissue distribution results at various time points resulting from oral administration of compound 1 to SD rats. [Figure 9A] This study demonstrates the efficacy of compound 1 in a taxane-resistant patient-derived organoid (PDO) model. Patient-derived organoids were treated for 5 days with compound 1, paclitaxel, or a control (0.1% DMSO, positive control - 10% DMSO). Survival rates were assessed using the CellTiter-Glo® assay. IC50 and maximum inhibitory values ​​were estimated from the survival curves. Model summaries and dose-response curves for compound 1 and paclitaxel are also provided. [Figure 9B] This study demonstrates the efficacy of compound 1 in a taxane-resistant patient-derived organoid (PDO) model. Patient-derived organoids were treated for 5 days with compound 1, paclitaxel, or a control (0.1% DMSO, positive control - 10% DMSO). Survival rates were assessed using the CellTiter-Glo® assay. IC50 and max inhibitory values ​​were estimated from survival curves. A summary of CellTiter-Glo results for all three models tested is provided. Compiled data from N=4 replicates. [Figure 10A] This study demonstrates the efficacy of compound 1 in a patient-derived organoid model with taxane resistance. Summary of the study design. [Figure 10B] This study demonstrates the efficacy of compound 1 in a taxane-resistant patient-derived organoid model. CTG-1520 was subcutaneously implanted into nude mice, and tumors were allowed to grow to a volume of 210 mm³. These mice were then randomized into three groups (vehicle control, 75 mg / kg, and 150 mg / kg of compound 1), each containing 12 animals. A solid dispersion of compound 1 was prepared before each dose and administered twice daily (BID) by forced oral administration. The mean tumor volume and tumor growth in each animal group over time were graphed. For each animal in the vehicle group, tumor growth inhibition (TGI)% was calculated based on the tumor growth inhibition of individual tumors on each given study day, compared to the group mean on that study day. P-values ​​were calculated for each group treated with compound 1 on each given study day in relation to the vehicle control, using two-sided distributions and two-sample equal variances (equal variances). ns p>0.05, *p≦0.05, **p≦0.01. [Figure 10C] This study demonstrates the efficacy of compound 1 in a patient-derived organoid model with taxane resistance. Body weight of each animal was measured on days 0, 3, 6, 10, 13, and 17 and graphed using Graphpad Prism. [Figure 11A]This demonstrates the efficacy of compound 1 in a colorectal cancer xenograft model (COLO205). The in vitro efficacy of compound 1 in COLO205 cells was evaluated by ATPlite 1Step® Perkin Elmer (performed by NTRC Oncolines). [Figure 11B-1] This study demonstrates the efficacy of compound 1 in a colorectal cancer xenograft model (COLO205). COLO205 cells were subcutaneously transplanted into nude mice, and tumors were allowed to grow to a volume of 210 mm³. Subsequently, the mice were randomized into three groups (vehicle control, 75 mg / kg, and 150 mg / kg of compound 1), each containing 12 animals. A solid dispersion of compound 1 was prepared before each dose and administered by forced oral administration twice daily (BID). The mean tumor volume and tumor growth in each animal over time were graphed. For each animal in the vehicle group, TGI (%) was calculated based on the inhibition of tumor growth in the individual tumor on each given study day, compared to the group mean on that study day. P-values ​​were calculated for each group treated with compound 1 on each given study day in relation to the vehicle control, using two-sided distributions and two-sample equal variances (equal variances). NS p>0.05, *p≦0.05, **p≦0.01, ***pp≦0.001, ****p≦0.0001. [Figure 11B-2] Same as above. [Figure 11C] This study demonstrates the efficacy of compound 1 in a colorectal cancer xenograft model (COLO205). Plasma and tumor tissue were collected at the end of the study from the vehicle (n=11), 75 mg / kg (n=12), and 150 mg / kg (n=11) groups. The concentrations of compound 1 in plasma or tumor tissue were plotted as column plots (mean ± SEM) or scatter plots with medians. [Figure 11D]Demonstrates the efficacy of Compound 1 in a colorectal cancer xenograft model (COLO205). Tumor tissues from the vehicle (n = 11), 75 mg / kg (n = 12), and 150 mg / kg (n = 11) groups were collected at the end of the study when each animal was sacrificed. The levels of two biomarkers, pHH3 and CCNB1, in each tissue were measured and graphed as minimum - maximum plots and scatter plots of the levels of each biomarker. The lines within the scatter plots indicate the group means. [Figure 11E] Demonstrates the efficacy of Compound 1 in a colorectal cancer xenograft model (COLO205). The body weight of each individual animal was measured on each study day and graphed using Graphpad Prism. [Figure 12A] Demonstrates the efficacy of Compound 1 in a prostate cancer xenograft model (DU145). In vitro potency of Compound 1 in DU145 cells as evaluated by ATPlite 1Step™ Perkin Elmer (performed by NTRC Oncolines). [Figure 12B-1] Demonstrates the efficacy of Compound 1 in a prostate cancer xenograft model (DU145). DU145 cells were subcutaneously implanted into nude mice, and the tumors were allowed to grow to a volume of 210 mm3. Subsequently, the animals were randomized into three groups (vehicle control, 75 mg / kg, 150 mg / kg of Compound 1), each containing 12 animals per group. A solid dispersion of Compound 1 was prepared prior to each dosing and administered by forced oral administration twice a day (BID). The mean tumor volume of each group over time and the tumor growth in each individual animal were graphed. For each individual animal in the vehicle group, the TGI (%) was calculated based on the tumor growth inhibition of the individual tumor on each given study day and compared to the mean of the group on that given study day. P - values were calculated for each group treated with Compound 1 on each given study day relative to the vehicle control using two - tailed distribution and two - sample equal variance (equal variance). NS p>0.05, *p≦0.05, **p≦0.01, ***p p≦0.001, ****p≦0.0001. [Figure 12B-2] Same as above. [Figure 12C]Demonstrates the efficacy of compound 1 in a prostate cancer xenograft model (DU145). Plasma and tumor tissues from the vehicle (n = 11), 75 mg / kg (n = 12), and 150 mg / kg (n = 12) groups were collected at the end of the study. The concentrations of compound 1 in plasma or tumor tissues were plotted as column plots (mean ± SEM) or scatter plots with median. [Figure 12D] Demonstrates the efficacy of compound 1 in a prostate cancer xenograft model (DU145). Tumor tissues from the vehicle (n = 12), 75 mg / kg (n = 12), and 150 mg / kg (n = 12) groups were collected at the end of the study when each animal was sacrificed. The levels of two biomarkers, pHH3 and CCNB1, in each tissue were measured and graphed as minimum - maximum plots and scatter plots of the levels of each biomarker. The lines within the scatter plots indicate the group means. [Figure 12E] Demonstrates the efficacy of compound 1 in a prostate cancer xenograft model (DU145). The body weight of each individual animal was measured on days 0, 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, and 26 and graphed using Graphpad Prism. [Figure 13A] Demonstrates the efficacy of compound 1 in a lung adenocarcinoma xenograft model (A549). The in vitro potency of compound 1 in A549 cells when evaluated by ATPlite 1Step™ Perkin Elmer (performed by NTRC Oncolines). [Figure 13B-1]This study demonstrates the efficacy of compound 1 in a lung adenocarcinoma xenograft model (A549). A549 cells (5 × 10⁶) were subcutaneously transplanted into athymic female nude mice, and tumors were allowed to grow to approximately 112 mm³. These mice were then randomized into three groups (vehicle control, 75 mg / kg, and 150 mg / kg of compound 1), each containing 12 animals. A solid dispersion of compound 1 was prepared before each dose and administered twice daily (BID) by forced oral administration. The mean tumor volume and tumor growth in each animal group over time were graphed. For each animal in the vehicle group, TGI (%) was calculated based on tumor growth inhibition of the individual tumor on each given study day, compared to the group mean on that study day. P-values ​​were calculated for each group treated with compound 1 on each given study day in relation to the vehicle control, using two-sided distributions and two-sample equal variances (equal variances). NS p>0.05, *p≦0.05, **p≦0.01, ***pp≦0.001, ****p≦0.0001. [Figure 13B-2] Same as above. [Figure 14A] This demonstrates the efficacy of compound 1 in vitro in glioblastoma cell lines and glioblastoma rat model (C6). The in vitro efficacy of compound 1 in four different glioblastoma cell lines was evaluated by the Realtime-Glo® assay (C6 and U87 cells) or ATPlite 1Step® Perkin Elmer (T98G and A-172 cells, performed by NTRC Oncolines). [Figure 14B] This study demonstrates the efficacy of compound 1 in vitro in glioblastoma cell lines and in a glioblastoma rat model (C6). Study Design Summary: C6 rat glioma cells (3 × 10⁵) were transplanted into the right entorhinal cortex / hippocampal region of male Sprague Dawley rats. Tumor load was assessed 14 days post-inoculation using MRI, and the animals were randomized into four groups (vehicle control, compound 1 at 5 mg / kg, 10 mg / kg, and 20 mg / kg), each containing four animals. A solid dispersion of compound 1 was prepared before each dose and administered by forced oral administration twice daily (BID). [Figure 14C]The efficacy of compound 1 in vitro in glioblastoma cell lines and in a glioblastoma rat model (C6) was demonstrated. Body weight was measured every two days. [Figure 14D] This shows the efficacy of compound 1 in vitro in glioblastoma cell lines and in a glioblastoma rat model (C6). Survival curves of vehicle versus treated animals. [Figure 14E] This study demonstrates the efficacy of compound 1 in vitro in glioblastoma cell lines and in a glioblastoma rat model (C6). MRI images of long-term survivors at baseline (D0), 16 days after treatment (D17), and 31 days after treatment are shown. [Modes for carrying out the invention]

[0010] In certain embodiments, the present disclosure relates to a method for treating cancers responsive to modulation of microtubule assembly, comprising administering a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof to a subject in need thereof. As used herein, the term “cancers responsive to modulation of microtubule assembly” refers to cancers responsive to microtubule-targeting agents such as vinca alkaloids (e.g., vinblastine or vincristine) or taxanes (e.g., paclitaxel and docetaxel). In some embodiments, cancers responsive to modulation of microtubule assembly are selected from the group consisting of prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer.

[0011] In one embodiment, a method is provided for treating a cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer in a subject, comprising administering to the subject a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof. Also provided are a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof for treating a cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. Furthermore, the use of a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof is provided in the manufacture of a drug for treating cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. In one embodiment, the cancer treated by the aforementioned method is prostate cancer. In another embodiment, the cancer treated by the aforementioned method is head and neck cancer. In yet another embodiment, the cancer treated by the aforementioned method is lung cancer, such as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In yet another embodiment, the cancer treated by the aforementioned method is endometrial cancer. In yet another embodiment, the cancer treated by the aforementioned method is glioblastoma multiforme. In yet another embodiment, the cancer treated by the aforementioned method is sarcoma.

[0012] One embodiment provides a method for treating P-glycoprotein (P-gp)-mediated resistant cancer in a subject, comprising administering to the subject a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof. Also provided are a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof for treating P-gp-mediated resistant cancer. Furthermore, the use of a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating P-glycoprotein (P-gp)-mediated resistant cancer is also provided.

[0013] In one embodiment, the P-gp-mediated resistance cancer described herein is a taxane-resistant cancer. In another embodiment, the P-gp-mediated resistance cancer described herein is a vinca alkaloid-resistant cancer. In some embodiments, the P-gp-mediated resistance cancer described herein is a paclitaxel, vinblastine, vincristine, and / or docetaxel-resistant cancer.

[0014] As used herein, “P-glycoprotein-mediated resistant cancer” or “Pg-p-mediated resistant cancer” as used herein synonymously refers to cancer that is resistant to treatment with one or more anticancer agents that are substrates for P-glycoprotein (Pg-p). Such anticancer agents include, but are not limited to, paclitaxel, vinblastine, vincristine, and docetaxel.

[0015] In the context of resistant cancer, the term "resistant" means that the cancer no longer responds to treatment. This includes cancers that are unresponsive or show disease progression while receiving a given treatment. In one aspect, resistant cancer refers to cancer that has become resistant during the course of treatment, i.e., the cancer initially responded but no longer responds to treatment after a certain period of time.

[0016] As used herein, “taxane-resistant cancer” refers to cancer that is resistant to treatment with taxane anticancer drugs. Taxanes are known in the art and include small molecules containing tetradecahydro-6,10-methanobenzo

[10] anulene and its derivatives as a central nucleus. Examples of taxane-resistant cancers include ovarian cancer, prostate cancer, breast cancer, bladder cancer, head and neck cancer, and lung cancer.

[0017] As used herein, “vinca alkaloid-resistant cancer” refers to cancer that is resistant to treatment with vinca alkaloid anticancer agents. Vinca alkaloids are known in the art and include small molecules consisting of two polycyclic units, an indole nucleus and a dihydroindole nucleus, joined together with other complex systems. Examples of vinca alkaloid-resistant cancers include lymphoma, acute lymphoblastic leukemia (ALL), and solid tumors.

[0018] For use in pharmaceuticals, the pharmaceutically acceptable salts described herein refer to non-toxic "pharmaceutically acceptable salts." Forms of pharmaceutically acceptable salts include pharmaceutically acceptable basic / cationic salts.

[0019] The terms "subject" and "patient" are used synonymously and can refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats), farm animals (e.g., cows, pigs, horses, sheep, goats), and laboratory animals (e.g., rats, mice, guinea pigs). Typically, the subject is a human being in need of treatment.

[0020] As used herein, the terms “treatment,” “to treat,” and “to treat” mean to reverse, alleviate, delay the onset of, or inhibit the progression of the cancer described herein. In some embodiments, treatment may be administered after the onset of one or more symptoms, i.e., a therapeutic measure. In other embodiments, treatment may be administered when there are no symptoms. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., considering a history of symptoms and / or exposure to a particular organism or other susceptibility factors), i.e., a prophylactic measure. Treatment may also be continued after the disappearance of symptoms, for example, to delay recurrence.

[0021] The terms “effective dose” or “therapeutic effective dose” refer to the amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide that elicits a biological or medical response in the subject, e.g., a dose of 0.01 to 100 mg / kg body weight / day. In one embodiment, the effective dose of compound 1 is in the range of about 50 mg / kg to about 250 mg / kg. In one embodiment, the effective dose of compound 1 is about 75 mg / kg, about 100 mg / kg, about 150 mg / kg, or about 200 mg / kg.

[0022] In some embodiments, compound 1 may be administered as part of a pharmaceutical composition. The pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, or via an implanted reservoir. As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the composition is administered orally, intraperitoneally, or intravenously. In some embodiments, the composition is administered orally. The sterile injectable forms of the compositions described herein may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents.

[0023] The specific dosage and treatment plan for any particular patient will depend on a variety of factors, including the activity of the specific compound being used, age, weight, general health status, sex, diet, administration time, excretion rate, drug combinations, and the judgment of the treating physician, as well as the severity of the specific disease being treated. [Examples]

[0024] 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide (compound 1) has the chemical structure shown below and can be synthesized by following the procedure described for compound 126 in U.S. Patent No. 11,091,447, the entirety of which is incorporated herein by reference. [ka] 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide may exist in various tautomers, each of which is explicitly included as part of the present invention.

[0025] Example 1. Effect of Compound 1 on tubular phosphate polymerization An in vitro tubulin polymerization assay (using >99% pure tubulin, OD-based, porcine (BK006P) from Cytoskeleton Inc.) demonstrated that certain anticancer effects from compound 1 are a result of cytoskeletal targeting to inhibit microtubule assembly.

[0026] method The protocol followed for tubulin polymerization was the manufacturer's protocol. The following components were diluted as follows: The lyophilized GTP vial was diluted with 100 μl of sterile water, prepared into 10 μl aliquots, and stored at -80°C. Tubulin was diluted to 10 mg / ml in buffer + GTP as recommended, flash-frozen in liquid nitrogen, and stored at -80°C. Paclitaxel was diluted to 2 mM in DMSO.

[0027] The half-area 96-well plate provided by the kit manufacturer was heated to 37°C for 30 minutes in a Molecular Devices M5 plate reader. Compound 1 and nocodazole were diluted to a concentration of 2 mM in DMSO, and then these working stocks were diluted 10-fold to the final well concentration and 10 μl final well volume in general tubulin buffer (GTB - provided in the kit). The same was done with the 2 mM paclitaxel stock. Once the plate was heated, the compounds were added to the wells in duplicate. 10 μl of GTB was added to the control well (no ligand control). The plate was placed in the plate reader at 37°C for 3 minutes. Flash-frozen tubulin was diluted as follows to have a final concentration of 10% glycerol and 1 mM GTP: Regarding 3 mg / ml tubulin: 200 μl reconstituted tubulin + 420 μl tubulin polymerization buffer (750 μl GTB + 250 μl tubulin lyserol buffer + 10 μl 100 mM GTP) • For 2 mg / ml tubulin: 200 μl reconstituted tubulin + 800 μl tubulin polymerization buffer (950 μl GTB + 250 μl tubulin lyserol buffer + 12 μl 100 mM GTP) • 100 μl of diluted tubulin was added to each well containing the warmed compound, and the experiment was started immediately. The experimental setup was as follows: Absorbance reading: 340nm Temperature: 37℃ Shake: 5 seconds at a moderate speed before the first read. Readings: Absorbance was read every minute for 90 minutes (total of 91 readings).

[0028] The values ​​were normalized to the first read by specifying the first read as "0" and subtracting the value of the first read from all subsequent reads.

[0029] result Microtubule polymerization, as readouts of absorbance at 340 nm under standard conditions (without ligand control) and in the presence of 10 μM paclitaxel, a microtubule polymerization agent, as well as high (10 μM) and low (20 nM) concentrations of compound 1 and the positive control nocodazole, are shown in Figure 1 as representative Vmax values ​​for N=3 replicates. As shown, compound 1 inhibits microtubule polymerization to a similar degree to nocodazole in the assay.

[0030] Example 2. Binding of compound 1 to the colchicine binding site of tubulin. material ·MIA PaCa2 cells • DMEM growth medium (Gibco, catalog number 1195-065, lot number 2366044) • Fetal bovine serum (FBS) Penicillin / Streptomycin DMSO ·RIPA lysis buffer • BRG519, Compound 1, Colchicine, Vinblastine, Paclitaxel, and Nocodazole stock (1000x or 2000x) • EBI (N,N'-ethylene-bis(iodoacetamide)) ·PBS • Halt protease inhibitor cocktail (Thermo Fisher, catalog number 87786)

[0031] method Cell culture: MIA PaCa2 cells were cultured in DMEM + 10% FBS and 1% P / S. The cells were placed in 60 mm dishes in a 1.5–2 × 10⁶ container. 6 The cells were plated. For drug treatment, the cells were treated for 2 hours the day after plating with 0.1% DMSO or the compounds tested at the concentrations shown in Figure 2.

[0032] EBI-labeled assay The cells were treated with the compound (in a cell culture incubator) at 37°C and 5% CO2 for 2 hours, followed by a further 2 hours with 100 μM EBI (in the presence of the compound) under the same conditions. Next, the cells were isolated using trypsin and pelletized in 500g containers for 3 minutes. The cells were rinsed with 1 mL of PBS and transferred to a clean epitube. The cells were pelleted again at 500 g for 3 minutes, and then lysed in 300 μL of RIPA + Halt protease inhibitor. Each cell lysate was sonicated at 20% power for 10 seconds, and then kept on ice. • A BCA assay was performed to normalize protein concentrations and reduce the intensity of the paclitaxel bands (these bands were consistently much brighter than those in other samples and were consistent across replicated and troubleshooting samples). The BCA assay plates were incubated for 15 minutes in a 37°C incubator in the external molecular laboratory. After protein normalization (including the addition of reduced Laemmli buffer from a 6x stock), the sample was boiled at 95°C for 3 minutes and then frozen for subsequent Western blotting.

[0033] The significance of the difference between treated MIA PaCa-2 cells and 0.1% DMSO-treated cells was tested by Student's t-test (*p<0.05, **p<0.01, ***p<0.001).

[0034] result As shown in Figures 2 and 3, compound 1 inhibited the formation of the EBI(N,N'-ethylene-bis(iodoacetamide)):β-tubulin adduct, revealing the colchicine binding site occupied by tubulin. Nocodazole and colchicine also inhibited the formation of the EBI:β-tubulin adduct in the assay.

[0035] Example 3. Kinetochore assembly of cells treated with compound 1 material ·HCT116 cells • DMEM cell growth medium (Thermofisher catalog number 11995073, lot number 1930072) • Fetal bovine serum: Sigma Aldrich, • Trypsin-EDTA solution (1x dilution): Gibco, #25300096 (100ml) Penicillin / Streptomycin: Gibco, #15140-122 (100ml) ·Poly-D-Lysine German Glass Coverslip(#1.5, 18mm)(Electron Microscopy sciences #72294-04) • Dulbecco's phosphate-buffered saline (1x dilution) without calcium and magnesium (Fisher #BW17-512Q) ·Mad1 antibody (GeneTex #GTX105079) • α-tubulin antibody (Cell Signaling #3873S) • 8% aqueous solution of paraformaldehyde (Electron Microscopy Sciences (#157-8)) • Goat serum (Novus Biologicals #NBP2-23475) • Alexa Fluor (registered trademark) 594 AffiniPure donkey anti-human IgG (Jackson Immunoresearch #709-585-149) • Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (Jackson Immunoresearch #709-585-150) ·Triton(TM)X-100 (Molecular Biology Grade, Sigma #T8787) • ProLong Gold fade-resistant mounting agent containing DAPI (Fisher Scientific #S36938)

[0036] method Cell culture: HCT116 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were maintained at a maximum confluence of 80% before being divided and plated onto coverslips in 12-well plates. [Table 1]

[0037] Immunocytochemistry: 1. HCT116 cells were plated onto Poly-D-Lysine German Glass Coverslip (#1.5, 18 mm). 2. After 24 hours, the cells were treated with 300 nM of compound 1 and nocodozol. 3. After 24 hours, the cells were briefly washed with PBS and fixed in warm 4% paraformaldehyde at room temperature for 10 minutes. 4. The cells were permeabilized and blocked for 1 hour in PBS with 10% goat serum and 0.5% Triton-X. 5. The cells were incubated with the primary antibody overnight. 6. Wash with PBS for 10 minutes, three times. 7. The secondary antibody was incubated at room temperature for 1 hour. 8. Washed with PBS for 10 minutes, three times. 9. The coverslips were mounted with ProLong Gold anti-fading mounting medium containing DAPI and imaged using an Olympus FV1200 MPE microscope. 10. Images were acquired for all samples using the same confocal setting, and the Z-stacked images were projected using Fluor View software in maximum projection mode.

[0038] Calculation of corrected whole-cell fluorescence (CTCF) for Mad1 using ImageJ: 1. Use a drawing / selection tool (i.e., rectangle, circle, polygon, or freeform) to select the desired cells. 2. Select "Measurement" from the analysis menu. 3. I selected a region without fluorescence to serve as my background. 4. These steps were repeated for other cells in the field of view. 5. Mad1 expression was calculated using the CTCF formula: CTCF = Integrated density - (Area of ​​selected cells × Average fluorescence of background readings)

[0039] Microscopic images were taken using an Olympus FV1200 MPE confocal microscope at 63x oil immersion. White arrows indicate dividing cells. N=3 representative images of biological replication. The significance of the difference between treated HCT116 cells and untreated cells was tested by Student's t-test (**p<0.01).

[0040] result As shown in Figure 4, confocal microscopy evaluation of kinetochore assembly in cells treated with compound 1 or nocodazole revealed a lack of proper mitotic spindle formation (A), highlighted by increased Mad1 signal intensity (B) emphasizing metaphase arrest. See also Figure 5.

[0041] Example 4. Activation of spindle assembly checkpoints material ·HCT116 cell line DMEM Lonza #12-604F, Lot number 0001008586 • PBS: Lonza #17-512Q Lot number 02204 FBS: Gibco #26140-087 Lot number 2206642RP Trypsin EDTA, Gibco catalog number 25200-056, lot number 2323073 Penicillin / Streptomycin Gibco #14140-122 Lot number 02204 ·Compound 1 • Nocodazole: MedChemExpress: #HY-13520 Lot number 10555 • Ro-3306: MedChemExpress: #HY-12529 Lot number 14923 • Hesperazine, MedChemExpress: #HY-12054 Lot number 08617 DMSO:Invirogen #D12345 • Bovine serum albumin, Boston Bioproducts (#P-753), Lot: 21CN127 Triton X-100: Sigma Catalog: T8787-100mL Lot: SLC06163 • CS&T Beads: BD Biosciences #661415 Lot 1182462 • EDTA Invitrogen #15575-038 Lot 2393343 • 200-proof purity ethyl alcohol for molecular biology (Sigma-Aldrich, catalog number E7023) ·PBT buffer (PBS, 1%BSA, 0.25%TritonX100) ·PBE buffer (PBS, 1%BSA, 2mM EDTA) • Alexa Fluor® 488 anti-histone H3 phospho(Ser10) antibody (Biolegend catalog number 650804) • Alexa Fluor® 488 mouse IgG2b, κ isotype control antibody (Biolegend catalog number 400329) • 7AAD (BD Pharmingen catalog number 559925) • Accuri C6 Plus Flow Cytometer

[0042] method 1. Hct116 was grown in DMEM containing 10% FBS and 1% penicillin / streptomycin. 2. Cells were plated into 6-well dishes with the cell counts indicated below (i.e., plate 1 = 300k cells per well), and treatment was initiated later on the same day. Asynch control cells (plate 1) were collected 1 day after plating. Compound 1 and nocodazole were both 300 nM, CDK1i Ro-3306 was 10 μM, and Aurora Bi(ABi) hesperazine was 50 μM. 3. Cells were treated with compound 1 or nocodazole for 20 hours, then subsequently treated with CDK1i or ABi for 4 hours without changing the medium. Alternatively, cells were treated with compound 1 or nocodazole for 20 hours, then washed twice with warm medium and grown in fresh medium for 4 hours to allow mitotic passage to G1 as diploid cells. 4. It should be noted that the treatment of plate 4 was generally either highly toxic or inconclusive to the cells.

[0043] Flow cytometry collection and staining: 1. All culture media and cells were collected in the same conical tube. The cells were centrifuged at 1000Xg for 3 minutes. 2. The supernatant was aspirated and washed with PBS. 3. The cells were centrifuged as described above. 4. The cells were resuspended in 400 μl of cold water, and 1 mL of cold 100% ethanol was slowly added dropwise while gently vortexing (for fixation and permeabilization). Stored at 5.4°C for a minimum of 1 hour or a maximum of 1 week. 6. Antibody staining: First, the PBT (PBT buffer (PBS, 1% BSA, 0.25% Triton X100)) was washed once, and the supernatant was aspirated. Finally, the samples were labeled with 7AAD and pH 3-488. 1 ml of PBT was added, mixed, pelletized, and the supernatant was removed. A master mix of 100 μL per sample in PBT was prepared: 90 μl PBT 5 μl of α-phosphohistone H3-Alexa488 (1:25) 7.5 μl of 7AAD For the isotyped sample, 5 μl of isotyped control was used. The solution was added to the cells and incubated in the dark at room temperature for 1 hour. After incubation, the cells were washed once with PBT and once with PBE, and then resuspended in 600 μl of PFE. 7. Next, the cells were subjected to flow cytometry analysis on Accuri C6 Plus, which was performed at a relatively slow rate of approximately 200 single-cell events per second. 8. Approximately 10,000 single-cell events were collected. 9. Data was analyzed using FlowJo 10 software, and graphs were created using GraphPad Prism.

[0044] result As shown in Figure 6, the mechanism of microtubule-kinetochore binding during or prior to the M phase of the cell cycle induced by compound 1 was demonstrated.

[0045] Example 5. Efficacy of Compound 1 in multiple tumor types of patient-derived organoids (PDOs) method The efficacy of compound 1 was evaluated using a panel of patient-derived organoids (PDOs) representing multiple tumor types. [Table 2]

[0046] Test drug information Test substance: A fresh test substance was prepared each week. [Table 3]

[0047] Experimental Design 1. Frozen samples (200-300 mg or more, depending on the research design) were prepared as described below. 2. The collected tumors were dissociated manually (using a previously developed tumor pulverization process) or using the Gentle MACS dissociation device and the Human Tumor Dissociation Kit (Miltenyi). After preparation, the tumor fragments were cultured for up to 7 days in DMEM + 10% FBS + 1x antibiotic / antifungal agent ("anti-anti") + 1:500 Primosin antimicrobial agent before filtering out larger fragments (using a 500 μm filter followed by a 200 μm filter). 3. Flow-through samples were cultured in low-volume, flat-bottomed 384-well assay plates coated with Cell-Tak. A 4.10-fold concentration of the test drug (or DMSO as a vehicle) was added to the culture medium in a volume that varied depending on the culture conditions. 5. Each group had four wells containing 0.1% DMSO (as the final vehicle) and 10% DMSO as a positive control. 6. PBS was plated into the surrounding wells to avoid peripheral effect artifacts. 7. Cells were treated for 5 days, and viability was read via Cell Titer Glo. Viability was quantitatively assessed via Cell Titer Glo on day 0 (baseline), day 6 (baseline), and day 6 (compound treatment - total dose response). Cell Titer Glo data were supplemented with biodye imaging palettes for positive and negative controls (Hoechst, Cell Tracker Green, MitoTracker Red CMXRos, Draq7), as well as two doses of the study drug (high and medium doses).

[0048] result Figures 7A–7C show examples of survival curves from PDO models that were highly responsive (Figure 7A, SCLC), responsive (Figure 7B, prostate cancer), and unresponsive (Figure 7C, colorectal cancer) to compound 1. Cancers considered highly responsive (>60% maximum inhibition), responsive (40–60% maximum inhibition), and unresponsive (<40% maximum inhibition) to compound 1 are shown in Figure 7D. For example, tumor samples from patients with endometrial cancer, small cell lung cancer (SCLC), sarcoma, breast cancer, non-small cell lung cancer (NSCLC), and ovarian cancer were shown to be highly responsive to compound 1. Tumor samples from patients with glioblastoma multiforme, SCLC, ovarian cancer, endometrial cancer, breast cancer, prostate cancer, or renal cell carcinoma were found to respond to compound 1. A summary of all models tested using highly responsive models (highly highlighted in green), responsive models (highly highlighted in blue), and unresponsive models (highly highlighted in white) is shown in Figure 7D. N = 4 copies of compiled data. NR = No regression, NA = No relevant data.

[0049] Example 6. In vivo distribution of compound 1 in rats method Six male Sprague-Dawley rats (210–227 g) aged 6–8 weeks were used in this in vivo distribution study. Compound 1 was administered to all animals at a dose of 75 mg / kg via oral administration at a dose volume of 10 ml / kg. Compound 1 formulations were freshly prepared in Tween-80 + 0.5% methylcellulose and maintained at room temperature until administration. The dose was administered to all animals under fasting conditions. Two rats were euthanized at 0.25 hours, 1 hour, and 8 hours, respectively. The heart, lungs, liver, kidneys, stomach, small intestine, large intestine, skeletal muscle, brain, spleen, pancreas, and fat were collected, while blood was processed into plasma for the biological quantification of Compound 1.

[0050] A bioanalytical assay for compound 1 was developed and validated using a SCIEX 6500 LC-MS / MS. The mobile phase consisted of acetonitrile and 0.2% formic acid in MilliQ water within an Atlantis dC18-HPLC column (50 × 4.6 mm, 3 μM) using an isocratic flow with a flow rate of 0.9 mL / min and a run time of 2.20 min. Mass transitions (m / z) at 375.040 and 188.100 were used to detect compound 1 at 2.14 ng / mL LLOQ.

[0051] result The results are shown in Tables 1-3 below and are represented graphically in Figure 8. Drug distribution was revealed across all tissues with pharmacologically significant blood-brain barrier permeability observed over 8 hours. [Table 4] [Table 5] [Table 6]

[0052] Example 7. Permeability of Compound 1 across the blood-brain barrier method 1) Donor solution preparation: a) A 0.2 mM working solution was prepared by diluting a 10 mM stock solution with DMSO. b) A 10 μM donor solution (5% DMSO) was prepared by diluting 20 μL of the working solution with 380 μL of PBS. 2) Replications were prepared by adding 150 μL of 10 μM donor solution to each well of a donor plate, where the PVDF membrane was pre-coated with 5 μL of 1% brain polar lipid extract (porcine) / dodecane mixture. 3) 300 μL of PBS was added to each well of a PTFE acceptor plate. 4) The donor plate and acceptor plate were combined and incubated at room temperature for 4 hours with shaking at 300 rpm. 5) Preparation of T0 sample: 20 μL of donor solution was transferred to a new well, followed by the addition of 250 μL of PBS (DF: 13.5) and 130 μL of ACN (containing internal standard) as the T0 sample. 6) Preparation of acceptor samples: The plate was removed from the incubator. 270 μL of solution was transferred from each acceptor well and mixed with 130 μL of ACN (containing an internal standard) as the acceptor sample. 7) Preparation of donor samples: 20 μL of solution was transferred from each donor well and mixed with 250 μL of PBS (DF: 13.5) and 130 μL of ACN (containing an internal standard) as the donor sample. 8) All acceptor and donor samples were analyzed by LC / MS / MS. 9) The formula used to determine the permeation rate (Pe) is shown below.

number

[0053] result As shown in the results in Table 4, compound 1 is highly permeable across the blood-brain barrier. [Table 7]

[0054] Example 8. Antitumor activity of Compound 1 in ex vivo PDO and in vivo xenograft models of taxane-resistant breast cancer. method While taxane resistance can be derived in vitro, this approach has limitations because it does not faithfully reproduce all aspects of taxane resistance observed in patients. Therefore, we conducted a study designed to test the efficacy of compound 1 in patient-derived organoid (PDO) ex vivo models of taxane-resistant cancer. CTG-1520 and CTG-0896 were derived from patients with post-treatment triple-negative breast cancer (TNBC), and model progression with taxane therapy and resistance to paclitaxel were confirmed by testing in vivo PDX (patient-derived xenograft) tumor growth. CTG-1017 was derived from the same patient as CTG-1520 but at an earlier stage in the treatment course and has been shown to be sensitive to paclitaxel in vivo (CRO history data). Compound 1 and paclitaxel were directly compared in each model using dose-curve settings. 10% DMSO was used as a positive control for induction of 100% cytotoxicity.

[0055] In the next step, we evaluated the ability of compound 1 to inhibit the proliferation of CTG-1520 PDX in nude mice. CTG-1520 was subcutaneously implanted into nude mice, and the tumor was reduced to 210 mm. 3 The animals were allowed to grow to a certain volume and then randomized into three groups containing 12 animals each (vehicle control, compound 1 at 75 mg / kg, and compound 1 at 150 mg / kg) (see Figure 10A). A solid dispersion of compound 1 was administered by forced oral administration twice daily (BID). All animals except one from the vehicle control group survived throughout the study (17 days). The one animal from the vehicle control group survived at 2000 mm before day 17. 3The endpoint was reached and it was excluded from all calculations.

[0056] result As shown in Figure 9, compound 1 was potent in all three PDO models tested. Furthermore, compound 1 maintained efficacy in matched PDO models derived from the same patients before (CTG-1017) and after (CTG-1520) taxane-based chemotherapy when paclitaxel failed to maintain efficacy.

[0057] Figure 10B shows the mean tumor volume and tumor growth in each animal group over time. Based on the patient's history of treatment refractory, CTG-1520 is a paclitaxel-resistant model confirmed ex vivo (maximum effect 29.1%) and in vivo (TGI 10%) (data from Champion Oncology Lumen Platform database.championsoncology.com / models / filter). As shown in Figure 10B, treatment of CTG-1520 PDX tumors with compound 1 led to significant inhibition of tumor growth at the highest dose (150 mg / kg) throughout the various study days. Compound 1 at a dose of 75 mg / kg also reduced tumor volume compared to the vehicle control, but to a lesser extent than the 150 mg / kg dose. No weight loss associated with compound 1 treatment was observed in this study (Figure 10C).

[0058] Example 9. Investigation of the antitumor efficacy of compound 1 in xenograft models of solid tumor cancer cell lines of colorectal cancer, prostate cancer, and lung adenocarcinoma. method The antitumor efficacy of compound 1 was evaluated in three different preclinical oncology mouse models of colorectal cancer, prostate cancer, and lung adenocarcinoma (see Table 5 below). For this purpose, an oral formulation of compound 1 was used. [Table 8]

[0059] Human colorectal cancer model - COLO205 Previous screening of 102 cancer cell lines revealed that colorectal cancer cell lines were one of the cancer indications with the highest response percentage and lowest IC50 for compound 1. One of the most responsive cell lines was COLO205, which showed a maximum response of 95% and a low IC50 of 67 nM (Figure 11A). COLO205 is an epithelial-derived colorectal adenocarcinoma (Dukes type D) cell line isolated from the human colon. COLO205 is a well-established model in the oncology field for evaluating the in vivo efficacy of oncological treatments. COLO205 cells were subcutaneously transplanted into nude mice, and tumors were reduced to 113 mm. 3 The tumors were allowed to grow to an average tumor volume and then randomized into three groups (12 animals per group): vehicle control, and compound 1 at 75 mg / kg and 150 mg / kg. A solid dispersion of compound 1 was administered by forced oral administration twice daily (BID). Initially, the study was planned for 28 days of treatment, but the tumor size reached 2000 mm. 3 Due to reaching their limits, several animals withdrew from the study after 17 days of medication. Therefore, only data from the first 17 days of medication were used for analysis. In addition, one animal from the vehicle group and one animal from the 150 mg / kg group withdrew from the study before day 17 and were therefore excluded from the analysis. The percentage of tumor growth inhibition (TGI%) was calculated for each individual animal.

[0060] Human prostate cancer model - DU145 DU145 is a human prostate cancer cell line that responded well to compound 1 in vitro with an IC50 response of 77 nM and a maximum effect of 91% (Figure 12A). DU145 is an epithelial cell carcinoma originating from metastatic sites (brain). DU145 cells were subcutaneously transplanted into nude mice, and the tumor was reduced to 106 mm. 3The tumors were allowed to grow to an average volume and then randomized into three groups (12 animals per group): a vehicle control, and compound 1 at 75 mg / kg and 150 mg / kg. A solid dispersion of compound 1 was administered by forced oral administration twice daily (BID). All animals except one from the vehicle group survived the entire 26-day study. Any animal that withdrew from the study before day 26 was excluded from all calculations. The percentage of tumor growth inhibition (TGI%) was calculated for each individual tumor. The average tumor volume for each group over time and tumor growth in each individual animal are plotted below. The percentage of tumor growth inhibition (TGI%) was calculated for each individual animal.

[0061] Human lung adenocarcinoma model - A549 A549 is a human lung adenocarcinoma cell line isolated from lung tissue of their origin. A549 cells showed a biphasic response to compound 1 in vitro, with an IC50 response of 53 nM and a maximum effect of 52% in phase 1, followed by 100% at the highest concentration tested (Figure 13A). A549 cells were subcutaneously transplanted into nude mice, and tumors were reduced to 112 mm². 3 The animals were allowed to grow to an average volume and then randomized into three groups (12 animals per group) consisting of a vehicle control, and Compound 1 at 75 mg / kg and 150 mg / kg. A solid dispersion of Compound 1 was administered by forced oral administration twice daily (BID). All animals remained in the study until day 28, with the exception of one animal (animal number 29) from group 3, which discontinued the study on day 11 due to a weight loss exceeding 20%. This animal was excluded from all analyses. Data from days 14–18 were excluded from the analysis due to inconsistencies in data collection methods.

[0062] result As shown in Figure 11B, compound 1 was able to inhibit the growth of COLO205 tumors in a dose-dependent manner (e.g., 20.13% at 75 mg / kg versus 35.38% at 150 mg / kg on day 17). This was further reflected in dose-dependent increases in the levels of compound 1 in plasma and tumor tissue (Figure 11C), as well as increases in cyclin B1 and phosphohistone H3 (pHH3), both markers of increased M-phase arrest in tumor tissue (Figure 11D). The lowest body weight was observed in the 150 mg / kg treatment group during the study, but this effect was transient and within 20% body weight fluctuations, as reflected by the absence of significant weight changes by the end of the study (Figure 11E). In conclusion, compound 1 demonstrated effective efficacy in inhibiting the growth of COLO205 tumors.

[0063] As shown in Figure 12B, in nude mice, compound 1 was able to inhibit the growth of DU145 tumors in a dose-dependent manner (e.g., 22% using 75 mg / kg on day 26 vs. 38% using 150 mg / g). Significant inhibition of tumor growth was observed throughout the various study days, particularly at the highest dose. The dose-dependent efficacy reflected a dose-dependent increase in drug levels in plasma and tumor tissue as the dose of compound 1 increased from 75 mg / kg to 150 mg / kg (Figure 12C), and correlated with increases in the levels of two biomarkers, CCNB1 and pHH3 (Figure 12D). Only one animal from the vehicle group discontinued the study early, and data from this animal were subsequently excluded from all calculations. There was no drug-related weight loss in this study, indicating that the drug was well tolerated throughout the duration of the study (Figure 12E). In conclusion, compound 1 demonstrated effective efficacy in inhibiting the growth of DU145 tumors.

[0064] Data from a human lung adenocarcinoma (A549) study demonstrated that treatment with compound 1 reduced mean tumor growth at all evaluated doses (Figure 13B). Peak TGI (%) were observed on day 7 of treatment at 36.61% at 75 mg / kg and 55.43% at 150 mg / kg, respectively. Data from body weight measurements did not demonstrate a drug effect in any of the animals.

[0065] conclusion Compound 1 demonstrated antitumor efficacy in various preclinical solid tumor models, including those of the colon, prostate, and lung.

[0066] Example 10. Investigation of the antitumor efficacy of compound 1 in an animal model of glioblastoma. method Glioblastoma (GBM) is the most common and highly malignant form of primary brain tumor, with a poor prognosis despite advances in treatment. Treatment resistance and recurrence remain significant clinical challenges, necessitating the development of novel therapeutic approaches not limited by the blood-oncological barrier. Therefore, the in vitro efficacy of compound 1 was investigated in four different glioblastoma cell lines when evaluated by the Realtime-Glo® assay (C6 and U87 cells) or ATPlite 1Step® Perkin Elmer (T98G and A-172 cells, performed by NTRC Oncolines). Inactive compound BRG396 was used as a negative control, and nocodazole was used as a positive control. Nocodazole is known to be a mitotic agent that induces cell death. In particular, the rat C6 glioma cell line is an intensively studied model used to evaluate the therapeutic efficacy of various modalities. Therefore, the in vivo tumor activity of compound 1 was next evaluated in Sprague Dawley rats in which C6 rat glioma cells were transplanted into the right entorhinal cortex / hippocampal region (AP: -7 mm, ML: 4.5 mm, DV: 5 mm). Magnetic resonance imaging (MRI) was performed on post-transplant day 14 to ensure successful transplantation, and animals were randomized to receive either vehicle or compound 1 treatment (5, 10, and 20 mg / kg groups) twice daily via forced oral administration for 16 days (post-transplant day 30). An overview of the study design is shown in Figure 14B.

[0067] result The anticancer activity of compound 1 in an in vitro model of GBM was evaluated to be within a two-order-of-magnitude nanomolar range (ranging from 42 nM to 89 nM) in four different cell lines tested: C6 cells: 42 nM, U87 cells: 89 nM, T98G cells: 39 nM, and A-172 cells: 48 nM (Figure 14A).

[0068] Figure 14C shows graphed body weight recorded every two days throughout the in vivo study. Sequential MRI was performed on survivors on days 17 and 31 post-transplant. As can be seen in Figure 14D, administration of compound 1 resulted in a significant improvement in survival (7.7±2.6 days to 25.5±15.6 days, log-rank test, p:0.007). Furthermore, six of the animals treated with compound 1 (two in each treatment group) were still alive at day 60 after the start of treatment. MRI imaging performed on the surviving animals showed tumor size reduction and tumor fragmentation (Figure 14E, upper and lower panels show images from two different animals). These results demonstrate the desirable therapeutic effect of compound 1 on rats with intracranial gliomas and demonstrate the therapeutic potential of compound 1 in the treatment of GBM.

[0069] conclusion Compound 1 demonstrated efficacy in treating gliomas in an orthotopic glioma model.

[0070] While numerous embodiments have been described, it is clear that the basic embodiments of the present invention may be modified to provide other embodiments utilizing the compounds and methods of the present invention. Therefore, it will be understood that the scope of the present invention is defined by the appended claims, rather than by the specific embodiments presented as examples.

[0071] The contents of all references cited throughout this application (including references to documents, published patents, published patent applications, and concurrently pending patent applications) are expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein are given the meanings commonly known to those skilled in the art.

Claims

1. A method for treating cancer that responds to the regulation of microtubule assembly, comprising administering a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof to a subject in need thereof.

2. The method according to claim 1, wherein the cancer is selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer.

3. The method according to claim 1 or 2, wherein the cancer is prostate cancer.

4. The method according to claim 1 or 2, wherein the cancer is head and neck cancer.

5. The method according to claim 1 or 2, wherein the cancer is lung cancer.

6. The method according to claim 5, wherein the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

7. The method according to claim 1 or 2, wherein the cancer is endometrial cancer.

8. The method according to claim 1 or 2, wherein the cancer is glioblastoma multiforme.

9. The method according to claim 1 or 2, wherein the cancer is a sarcoma.

10. A method for treating P-gp-mediated resistant cancer, comprising administering a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazole-3-yl]methyl]benzamide or a pharmaceutically acceptable salt thereof to a subject in need thereof.

11. The method according to claim 10, wherein the cancer is a taxane-resistant cancer.

12. The method according to claim 10 or 11, wherein the cancer is resistant to paclitaxel, vinblastine, vincristine, or docetaxel.

13. The method according to any one of claims 10 to 12, wherein the cancer exhibits resistance to paclitaxel.

14. The method according to any one of claims 10 to 13, wherein the cancer is selected from ovarian cancer, prostate cancer, breast cancer, bladder cancer, head and neck cancer, and lung cancer.

15. The method according to any one of claims 10 to 14, wherein the cancer is ovarian cancer.

16. The method according to any one of claims 10 to 14, wherein the cancer is breast cancer.

17. The method according to any one of claims 10 to 14, wherein the cancer is bladder cancer.

18. The method according to any one of claims 10 to 14, wherein the cancer is head and neck cancer.

19. The method according to any one of claims 10 to 14, wherein the cancer is lung cancer.

20. The method according to claim 10, wherein the cancer is a vinca alkaloid-resistant cancer.

21. The method according to claim 10 or 16, wherein the cancer exhibits resistance to vinblastine or vincristine.

22. The method according to any one of claims 10, 20, and 21, wherein the cancer is selected from lymphoma, acute lymphoblastic leukemia (ALL), and solid tumors.

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