Isoxazole derivatives targeting TACC3 as anticancer agents

Abstract

To provide methods of treating TACC3 mediated diseases and disorders.SOLUTION: There is provided compounds of Formula (I) or a pharmacetuically acceptable salt thereof, wherein X1 is N or CR6; X2 is N or CR3; R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amide, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide; and R5 is heterocyclyl, alkyl, or amino.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of EP Patent Application No. 19209120.5, filed November 14, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to substituted compounds of formula (I) useful as transforming acidic coiled-coil protein 3 (TACC3) inhibitors, pharmaceutical compositions of such compounds, methods for their preparation, and uses. More specifically, TACC3 inhibitors are useful for treating or ameliorating TACC3-mediated cancers, including breast cancer, leukemia cancer, lung cancer, colon cancer, melanoma cancer, prostate cancer, ovarian cancer, renal cancer, and CNS cancer. [ka] [Background technology]

[0003] Cancer is a complex disease characterized by uncontrolled cell division. Among cancer types, breast cancer is the most common cancer among women and one of the leading causes of cancer death. Understanding tumor biology has continuously led to the development of targeted medical therapies to improve patient survival.

[0004] Although the Food and Drug Administration (FDA) has approved approximately two dozen drugs for the treatment of breast cancer, breast cancer still accounts for 500,000 deaths worldwide each year. The development of less toxic targeted therapies has become a major focus in recent years, especially considering the side effects of currently available chemotherapy drugs. Because cancer is characterized as abnormal and uncontrollable cell growth that can invade or spread to other parts of the body or to malignant tumors, drugs or substances that target and inhibit the function of specific macromolecules involved in tumor cell proliferation and survival are used in targeted breast cancer treatments.

[0005] Because microtubule reorganization is a critical step during cell division, drugs that disrupt this process have become a major focus of cancer research. Cytotoxic drugs disrupt microtubule polymerization dynamics by activating the spindle assembly checkpoint (SAC), which prevents the metaphase-to-anaphase transition. As a result, cells cease division, and these mitotically arrested cells ultimately die. Continued investigation of the mechanisms underlying mitotic events may lead to new target proteins and / or pathways and is crucial for providing more effective treatment options for cancer patients. Microtubule inhibitors, such as vinca alkaloids, maytansinoids, and taxanes, are examples of such drugs and are widely used as chemotherapeutic agents for various tumors (Marzo & Naval, 2013). However, a significant concern regarding these drugs is their toxicity to non-tumorigenic cells, which can cause serious side effects.

[0006] Drug resistance is another major issue, leaving patient responses to these drugs highly unpredictable (Gascoigne & Taylor, 2009). To overcome these issues and improve chemotherapy response, antimitotic, cancer-specific therapies targeting mitosis-specific kinases and microtubule motor proteins have been identified (Dominguez-Brauer et al., 2015). Importantly, because phosphorylation is a critical step in cell cycle regulation and spindle assembly, kinases that play a role in these processes have long been investigated as potential targets. Among these, specific inhibitors against cyclin-dependent kinases (CDKs), Aurora kinases, and Polo-like kinases (PLKs) have been developed and clinically tested (Sanchez-Martinez, Gelbert, Lallena, & de Dios, 2015; Strebhardt & Ullrich, 2006; Tang et al., 2017). Compared with microtubule inhibitors, all of these antimitotic drugs, despite their low toxicity profiles, have not shown impressive clinical outcomes and have limited clinical efficacy (Chan, Koh, & Li, 2012). Therefore, alternative targeting molecules that selectively and effectively target dividing cancer cells remain to be elucidated and developed.

[0007] TACC3, a TACC member, is a nonkinase microtubule-binding protein that plays a key role in centrosome regulation, ensuring microtubule stability (Singh, Thomas, Gireesh, & Manna, 2014). The TACC3 gene also plays a key role in nucleating centrosomal microtubules. Elevated levels have been observed in many cancer types, including prostate cancer, hepatocellular carcinoma, non-small cell lung cancer, and breast cancer. Accordingly, knockdown of TACC3 suppresses tumorigenesis and cell growth in renal cell carcinoma (RCC) (Guo & Liu, 2018). Disruption of TACC3 function also results in a range of distinct cellular outcomes, including multipolar spindle formation leading to mitotic arrest (Yao et al., 2012), chromosome misalignment resulting in caspase-dependent apoptosis (Schneider et al., 2007), and, in some cases, senescence (Schmidt et al., 2010). These studies indicate that TACC3 is a key molecule participating in spindle formation in cancer cells, making it an important and potential target for cancer targeted therapy.

[0008] KHS101, a small molecule TACC3 inhibitor, was first identified to promote neural differentiation in rats (Wurdak et al., 2010). Tumor growth in glioblastoma (GBM) xenografts was suppressed by KHS101 treatment (Polson et al., 2018), but its poor systemic stability and high workload require pharmacological optimization before it can be translated into the clinic (Wurdak et al., 2010). Another TACC3 inhibitor, SPL-B, has been shown to inhibit centrosomal microtubule nucleation in ovarian cancer cells and suppress tumor growth in ovarian cancer xenografts (Yao et al., 2014). Two currently available TACC3 inhibitors, KHS101 and SPL-B, have been shown to reduce tumor growth in glioblastoma and ovarian cancer xenografts, respectively. However, neither of these inhibitors has yet reached the clinical stage due to their low potency or poor systemic stability.

[0009] All of the above evidence supports the important role of TACC3 in cancer, and inhibition of TACC3 function is effective in treating or ameliorating various human cancers. Furthermore, there remains a need for TACC3 inhibitor compounds with pharmacokinetic and pharmacodynamic properties suitable for use as human pharmaceuticals. Summary of the Invention

[0010] In one aspect, the present disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: X1 is N or CR6, X2 is N or CR3, R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R5 is heterocyclyl, alkyl, or amino.

[0011] In another aspect, the present disclosure relates to methods of treating TACC3-mediated diseases and disorders using the compounds disclosed herein. In certain embodiments, the TACC3-mediated disease or disorder is cancer.

[0012] In one aspect, it is an object of the present disclosure to reduce undesirable side effects by using lower doses of TACC3 inhibitors that have higher potency as mitotic blockers than certain inhibitors available in cancer treatment.

[0013] As an example of all compounds, compound 5 showed superior antiproliferative effects to known TACC3 inhibitors in various breast cancer cell lines of various subtypes, while having minimal effects on normal breast cell lines. In addition to breast cancer cells, compound 5 exhibited highly effective cytotoxicity (approximately 90% with a GI of less than 1 μM) against multiple cancer types, including colon, melanoma, lung, central nervous system, ovarian, leukemia, renal, and prostate cancer cells in the NCI-60 panel. 50 Furthermore, compound 5 exhibited significant anticancer effects against cells harboring the FGFR3-TACC3 fusion protein, and its activity correlated with the TACC3 levels in these cells. Compound 5 also reduced ERK1 / 2 phosphorylation, a marker of activated FGFR signaling, along with potent induction of mitotic arrest and apoptosis.

[0014] Furthermore, compound 5 was found to induce mitotic arrest, apoptosis, and DNA damage at lower doses than the other two TACC3 inhibitors. Compound 5 also induced aberrant spindle formation in a dose-dependent manner. Importantly, oral administration of compound 5 suppressed tumor growth in both immunodeficient and immunocompetent mouse models of breast cancer. Compound 5 also impaired metastatic growth and significantly improved overall survival in mice bearing highly aggressive breast cancer metastases. Similar to breast cancer tumor models, compound 5 significantly suppressed tumor growth in colon cancer xenografts and immunocompetent syngeneic models. Thus, the present disclosure provides novel TACC3 inhibitors with high potency as mitotic blockers for the treatment of both primary and metastatic breast cancer, and potentially other cancers.

[0015] In certain aspects, the present disclosure (i) provides a compound selected from the group represented by general formula I as a TACC3 inhibitor, (ii) provides a compound selected from the group represented by general formula I as an anti-cancer agent responsive to TACC3 inhibition, (iii) provides a comprehensive analysis of compound 5 as an example of all compounds against breast cancer cell lines, (iv) reveals that this compound exhibits superior effects on various cellular processes such as mitotic arrest, DNA damage, and apoptosis compared to other available TACC3 inhibitors, (v) demonstrates the in vivo anti-tumor efficacy of compound 5 without observable toxicity when orally administered in animal models of breast cancer and colon cancer, and (vi) demonstrates its ability to impair metastatic growth and improve overall survival in mice bearing metastases, suggesting that it can be used as a mitotic blocker for the treatment of breast cancer and other cancers responsive to TACC3 inhibition. [Brief explanation of the drawings]

[0016] [Figure 1A] Overall, Figure 1 shows that TACC3 is upregulated in several different cancer types, and its high levels are associated with poor overall survival. Figure 1A shows the differential mRNA expression plot of TACC3 between tumor and normal tissues from TCGA patients, expressed as Reads Per Kilobase Million (RPKM) (log2) values. ***: p<0.001. (BLCA: bladder and urinary tract cancer; BRCA: invasive breast cancer; ESCA: esophageal cancer; HNSC: head and neck squamous cell carcinoma; KIPAN: pan-renal cohort (KICH+KIRC+KIRP); KIRC: kidney renal clear cell carcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; STAD: gastric adenocarcinoma; STES: gastric and esophageal cancer; UCEC: endometrial carcinoma). Figure 1B shows the effect of TACC3 levels on overall survival (OS) in breast (B-1) and gastric (B-2) cancer patients, recurrence-free survival (RFS) in lung (B-3) cancer patients, and disease-free survival (DFS) in prostate (B-4) cancer patients, retrieved from the METABRIC, KM Plotter database, GSE31210, and TCGA datasets, respectively. Log-rank tests were used for statistical analysis. [Figure 1B] Overall, Figure 1 shows that TACC3 is upregulated in several different cancer types, and its high levels are associated with poor overall survival. Figure 1A shows the differential mRNA expression plot of TACC3 between tumor and normal tissues from TCGA patients, expressed as Reads Per Kilobase Million (RPKM) (log2) values. ***: p<0.001. (BLCA: bladder and urinary tract cancer; BRCA: invasive breast cancer; ESCA: esophageal cancer; HNSC: head and neck squamous cell carcinoma; KIPAN: pan-renal cohort (KICH+KIRC+KIRP); KIRC: kidney renal clear cell carcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; STAD: gastric adenocarcinoma; STES: gastric and esophageal cancer; UCEC: endometrial carcinoma). Figure 1B shows the effect of TACC3 levels on overall survival (OS) in breast (B-1) and gastric (B-2) cancer patients, recurrence-free survival (RFS) in lung (B-3) cancer patients, and disease-free survival (DFS) in prostate (B-4) cancer patients, retrieved from the METABRIC, KM Plotter database, GSE31210, and TCGA datasets, respectively. Log-rank tests were used for statistical analysis. [Figure 2] Figure 1 shows a multivariate analysis performed on METABRIC patients by selecting TACC3 levels, tumor grade, tumor stage, ER, PR, and HER2 status as covariates. TACC3 expression is separated based on the 25th percentile. [Figure 3A]Overall, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A shows enrichment plots from GSEA performed using a set of mitosis- and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. Data significance is expressed as normalized enrichment scores (NES) and FDR (q) values. N indicates the total number of genes used in the analysis. Figure 3B shows qRT-PCR experiments demonstrating the knockdown efficiency of TACC3-specific siRNA in breast cancer cell lines. Cells were transfected with 20 nM of two different siRNAs against TACC3, and TACC3 mRNA levels were examined 48 h after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C shows a graph demonstrating the growth inhibition of breast cancer cells upon TACC3 knockdown using two different siRNAs. Cells were transfected with siRNA targeting TACC3, and cell viability was measured 72 hours after transfection. Figure 3D shows Western blot analysis of mitotic arrest, apoptosis, and DNA damage markers in breast cancer cells upon TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3B]Overall, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A shows enrichment plots from GSEA performed using a set of mitosis- and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. Data significance is expressed as normalized enrichment scores (NES) and FDR (q) values. N indicates the total number of genes used in the analysis. Figure 3B shows qRT-PCR experiments demonstrating the knockdown efficiency of TACC3-specific siRNA in breast cancer cell lines. Cells were transfected with 20 nM of two different siRNAs against TACC3, and TACC3 mRNA levels were examined 48 h after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C shows a graph demonstrating the growth inhibition of breast cancer cells upon TACC3 knockdown using two different siRNAs. Cells were transfected with siRNA targeting TACC3, and cell viability was measured 72 hours after transfection. Figure 3D shows Western blot analysis of mitotic arrest, apoptosis, and DNA damage markers in breast cancer cells upon TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3C]Overall, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A shows enrichment plots from GSEA performed using a set of mitosis- and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. Data significance is expressed as normalized enrichment scores (NES) and FDR (q) values. N indicates the total number of genes used in the analysis. Figure 3B shows qRT-PCR experiments demonstrating the knockdown efficiency of TACC3-specific siRNA in breast cancer cell lines. Cells were transfected with 20 nM of two different siRNAs against TACC3, and TACC3 mRNA levels were examined 48 h after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C shows a graph demonstrating the growth inhibition of breast cancer cells upon TACC3 knockdown using two different siRNAs. Cells were transfected with siRNA targeting TACC3, and cell viability was measured 72 hours after transfection. Figure 3D shows Western blot analysis of mitotic arrest, apoptosis, and DNA damage markers in breast cancer cells upon TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3D]Overall, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A shows enrichment plots from GSEA performed using a set of mitosis- and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. Data significance is expressed as normalized enrichment scores (NES) and FDR (q) values. N indicates the total number of genes used in the analysis. Figure 3B shows qRT-PCR experiments demonstrating the knockdown efficiency of TACC3-specific siRNA in breast cancer cell lines. Cells were transfected with 20 nM of two different siRNAs against TACC3, and TACC3 mRNA levels were examined 48 h after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C shows a graph demonstrating the growth inhibition of breast cancer cells upon TACC3 knockdown using two different siRNAs. Cells were transfected with siRNA targeting TACC3, and cell viability was measured 72 hours after transfection. Figure 3D shows Western blot analysis of mitotic arrest, apoptosis, and DNA damage markers in breast cancer cells upon TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 4A]This figure shows the binding of Compound 5, a novel TACC3 inhibitor, to TACC3. Figure 4A shows the target engagement of Compound 5 with TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with vehicle, Compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, then harvested, heated at the indicated temperatures, and lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. CETSA curves show the relative band intensity of TACC3 and indicate shifts between treatment groups. Figure 4B shows the determination of the binding constant for the interaction of TACC3 and Compound 5 using isothermal titration calorimetry (ITC). The top trace shows raw data, while the bottom trace shows integrated data from titration of TACC3 with Compound 5. Model fitting using a single interaction model was applied using Origin7 software provided with the ITC200 instrument. Figure 4C shows the Western blot results of a DARTS assay using JIMT-1 cell protein extracts to confirm the interaction between compound 5 and TACC3. CDK4 was used as a non-target protein whose levels did not change between treatment groups. 10 μM of drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing quantification of TACC3 relative band intensity between treatment groups normalized to β-actin, representative of two independent experiments. Data are presented as mean ± SD. **p<0.01 [Figure 4B]This figure shows the binding of Compound 5, a novel TACC3 inhibitor, to TACC3. Figure 4A shows the target engagement of Compound 5 with TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with vehicle, Compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, then harvested, heated at the indicated temperatures, and lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. CETSA curves show the relative band intensity of TACC3 and indicate shifts between treatment groups. Figure 4B shows the determination of the binding constant for the interaction of TACC3 and Compound 5 using isothermal titration calorimetry (ITC). The top trace shows raw data, while the bottom trace shows integrated data from titration of TACC3 with Compound 5. Model fitting using a single interaction model was applied using Origin7 software provided with the ITC200 instrument. Figure 4C shows the Western blot results of a DARTS assay using JIMT-1 cell protein extracts to confirm the interaction between compound 5 and TACC3. CDK4 was used as a non-target protein whose levels did not change between treatment groups. 10 μM of drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing quantification of TACC3 relative band intensity between treatment groups normalized to β-actin, representative of two independent experiments. Data are presented as mean ± SD. **p<0.01 [Figure 4C]This figure shows the binding of Compound 5, a novel TACC3 inhibitor, to TACC3. Figure 4A shows the target engagement of Compound 5 with TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with vehicle, Compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, then harvested, heated at the indicated temperatures, and lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. CETSA curves show the relative band intensity of TACC3 and indicate shifts between treatment groups. Figure 4B shows the determination of the binding constant for the interaction of TACC3 and Compound 5 using isothermal titration calorimetry (ITC). The top trace shows raw data, while the bottom trace shows integrated data from titration of TACC3 with Compound 5. Model fitting using a single interaction model was applied using Origin7 software provided with the ITC200 instrument. Figure 4C shows the Western blot results of a DARTS assay using JIMT-1 cell protein extracts to confirm the interaction between compound 5 and TACC3. CDK4 was used as a non-target protein whose levels did not change between treatment groups. 10 μM of drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing quantification of TACC3 relative band intensity between treatment groups normalized to β-actin, representative of two independent experiments. Data are presented as mean ± SD. **p<0.01 [Figure 4D]This figure shows the binding of Compound 5, a novel TACC3 inhibitor, to TACC3. Figure 4A shows the target engagement of Compound 5 with TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with vehicle, Compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, then harvested, heated at the indicated temperatures, and lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. CETSA curves show the relative band intensity of TACC3 and indicate shifts between treatment groups. Figure 4B shows the determination of the binding constant for the interaction of TACC3 and Compound 5 using isothermal titration calorimetry (ITC). The top trace shows raw data, while the bottom trace shows integrated data from titration of TACC3 with Compound 5. Model fitting using a single interaction model was applied using Origin7 software provided with the ITC200 instrument. Figure 4C shows the Western blot results of a DARTS assay using JIMT-1 cell protein extracts to confirm the interaction between compound 5 and TACC3. CDK4 was used as a non-target protein whose levels did not change between treatment groups. 10 μM of drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing quantification of TACC3 relative band intensity between treatment groups normalized to β-actin, representative of two independent experiments. Data are presented as mean ± SD. **p<0.01 [Figure 5A]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5B]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5C]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5D]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5E]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5F]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5G]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 5H]Overall, Figure 5 shows that compound 5 is more potent than the currently available TACC3 inhibitors, SPL-B and KHS101. Figure 5A is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of breast cancer cell lines (IC50: inhibitory concentration 50%). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all following cell viability experiments. Figure 5B shows the colony formation assay of JIMT-1 cells treated with three different TACC3 inhibitors for 12 days. Colonies were stained with crystal violet. The number of colonies was counted and analyzed using ImageJ software (lower panel). Data are presented as mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001). Figure 5C shows Western blot analysis of JIMT-1 cells treated with compound 5, SPL-B, or KHS101 to examine the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. The Western blot experiment compared the effects of the three drugs on these markers using the same amount of protein and exposure time. Figure 5D is a plot showing Annexin V / PI staining of JIMT-1 cells treated with compound 5 (500 nM). The percentage of Annexin V / PI double-positive cells is shown in the graph. Figure 5E shows the induction of abnormal spindle formation in JIMT-1 cells treated with compound 5. Cells were treated with two different doses of compound 5 for 12 hours, fixed with methanol, and then stained with anti-α-tubulin (green) antibody. DNA was stained with DAPI (blue). Scale bar: 10 μm. Figure 5F is a graph showing quantification of spindle abnormalities as seen in Figure 5D. Data represent the mean and standard deviation (SD) of three independent experiments (t-test). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of SAC markers, BubR1 (red) and α-tubulin (green), in JIMT-1 cells treated with vehicle versus compound 5 (500 nM).Figure 5H shows Western blot analysis of mitotic arrest, DNA damage, and apoptosis markers in JIMT-1 cells treated with compound 5 upon treatment with the spindle checkpoint kinase (Mps1) inhibitor, TC Mps1. JIMT-1 cells were treated with 200 nM compound 5 for 24 hours, followed by treatment with 1 μM TC Mps1 for 12 hours. GAPDH was used as a loading control. [Figure 6A] Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 6B]Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 6C]Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 6D]Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 6E]Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 6F]Overall, Figure 6 shows the significant anticancer activity of compound 5 in cell lines harboring the FGFR3-TACC3 fusion protein and the NCI-60 cancer cell line. Figure 6A shows Western blot analysis of TACC3 protein levels in RT112 and RT4 cells. β-Actin was used as a loading control. Figure 6B is a graph showing the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of the FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested with drug incubation for 3 days and measured by SRB assay. Figure 6C shows images of representative wells stained with SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of the mitotic arrest marker, p-Histone H3, and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24 hours of treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 values (M) determined from the NCI-60 5-dose screen of compound 5. The black dotted line indicates the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 values of compound 5 and the TACC3 dependency scores obtained from DepMap.org. A lower score indicates a higher likelihood of TACC3 dependency in a given cell line. [Figure 7A]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7B]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7C]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7D]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7E]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7F]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 7G]Overall, this figure demonstrates that TACC3 levels are important for the response of Compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on cell viability of a normal breast cell line, MCF-12A. Figure 7B shows the IC50 levels of Compound 5 in breast normal epithelial cell line, MCF-12A, and cancer cell lines separated according to their subtypes. Figure 7C shows the TACC3 protein levels in these cell lines. TACC3 protein levels were analyzed by Western blot. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected with either control or TACC3 vector (250 ng) for 48 hours. Transfected cells were later seeded into 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows the number of colonies counted and analyzed using ImageJ software. Data are presented as the mean ± SD of three independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001. ns: not significant. Figure 7F is a graph showing cell viability assays of MCF-12A cells overexpressing TACC3 upon treatment with Compound 5. Figure 7G shows doubling time evaluations of MCF-12A and breast cancer cell line models. Cells were plated at low density and grown for one week. Cells were then counted daily. One-way ANOVA was used. [Figure 8A] Overall, compound 5 significantly impaired tumor growth compared with SPL-B. Figure 8A shows the tumor volume changes of JIMT-1 xenografts orally treated with vehicle, 5 mg / kg of compound 5, or SPL-B for 30 days. Treatment was initiated when tumors reached 90-100 mm. Figure 8B shows the percent body weight changes of JIMT-1 xenografts followed for 30 days. *: p<0.05; **: p<0.01; ***: p<0.001; ns: not significant. [Figure 8B]Overall, compound 5 significantly impaired tumor growth compared with SPL-B. Figure 8A shows the tumor volume changes of JIMT-1 xenografts orally treated with vehicle, 5 mg / kg of compound 5, or SPL-B for 30 days. Treatment was initiated when tumors reached 90-100 mm. Figure 8B shows the percent body weight changes of JIMT-1 xenografts followed for 30 days. *: p<0.05; **: p<0.01; ***: p<0.001; ns: not significant. [Figure 9A] This figure shows that different doses and administration routes of Compound 5 generally inhibit tumor growth in JIMT-1 xenografts in vivo without affecting mouse body weight. Figure 9A shows the change in tumor volume after administration of all three groups of Compound 5 at different doses and administration routes. Figure 9B shows the change in mouse body weight over the course of treatment at different doses and administration methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts in female nude mice after treatment with vehicle or 25 mg / kg of Compound 5. Treatment was initiated when tumors reached 90-100 mm3. Figure 9D shows the tumor weight of mice treated with either vehicle or Compound 5. Figure 9E shows the change in mouse body weight (%) after treatment. [Figure 9B] This figure shows that different doses and administration routes of Compound 5 generally inhibit tumor growth in JIMT-1 xenografts in vivo without affecting mouse body weight. Figure 9A shows the change in tumor volume after administration of all three groups of Compound 5 at different doses and administration routes. Figure 9B shows the change in mouse body weight over the course of treatment at different doses and administration methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts in female nude mice after treatment with vehicle or 25 mg / kg of Compound 5. Treatment was initiated when tumors reached 90-100 mm3. Figure 9D shows the tumor weight of mice treated with either vehicle or Compound 5. Figure 9E shows the change in mouse body weight (%) after treatment. [Figure 9C]This figure shows that different doses and administration routes of Compound 5 generally inhibit tumor growth in JIMT-1 xenografts in vivo without affecting mouse body weight. Figure 9A shows the change in tumor volume after administration of all three groups of Compound 5 at different doses and administration routes. Figure 9B shows the change in mouse body weight over the course of treatment at different doses and administration methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts in female nude mice after treatment with vehicle or 25 mg / kg of Compound 5. Treatment was initiated when tumors reached 90-100 mm3. Figure 9D shows the tumor weight of mice treated with either vehicle or Compound 5. Figure 9E shows the change in mouse body weight (%) after treatment. [Figure 9D] This figure shows that different doses and administration routes of Compound 5 generally inhibit tumor growth in JIMT-1 xenografts in vivo without affecting mouse body weight. Figure 9A shows the change in tumor volume after administration of all three groups of Compound 5 at different doses and administration routes. Figure 9B shows the change in mouse body weight over the course of treatment at different doses and administration methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts in female nude mice after treatment with vehicle or 25 mg / kg of Compound 5. Treatment was initiated when tumors reached 90-100 mm3. Figure 9D shows the tumor weight of mice treated with either vehicle or Compound 5. Figure 9E shows the change in mouse body weight (%) after treatment. [Figure 9E] This figure shows that different doses and administration routes of Compound 5 generally inhibit tumor growth in JIMT-1 xenografts in vivo without affecting mouse body weight. Figure 9A shows the change in tumor volume after administration of all three groups of Compound 5 at different doses and administration routes. Figure 9B shows the change in mouse body weight over the course of treatment at different doses and administration methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts in female nude mice after treatment with vehicle or 25 mg / kg of Compound 5. Treatment was initiated when tumors reached 90-100 mm3. Figure 9D shows the tumor weight of mice treated with either vehicle or Compound 5. Figure 9E shows the change in mouse body weight (%) after treatment. [Figure 10A] Overall, this figure shows that compound 5 significantly impaired tumor growth and improved survival in immunocompetent mice. Figure 10A shows tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly aggressive syngeneic mouse breast cancer model. Significance was calculated using multiple t-tests for each data point on each day compared to vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows Kaplan-Meier survival curves for EMT6 xenografts treated with vehicle (median survival 14 days) or compound 5 (median survival 22 days). P values were calculated using the log-rank test. Figure 10C shows the percent body weight change of EMT6 xenografts after treatment. [Figure 10B] Overall, this figure shows that compound 5 significantly impaired tumor growth and improved survival in immunocompetent mice. Figure 10A shows tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly aggressive syngeneic mouse breast cancer model. Significance was calculated using multiple t-tests for each data point on each day compared to vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows Kaplan-Meier survival curves for EMT6 xenografts treated with vehicle (median survival 14 days) or compound 5 (median survival 22 days). P values were calculated using the log-rank test. Figure 10C shows the percent body weight change of EMT6 xenografts after treatment. [Figure 10C]Overall, this figure shows that compound 5 significantly impaired tumor growth and improved survival in immunocompetent mice. Figure 10A shows tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly aggressive syngeneic mouse breast cancer model. Significance was calculated using multiple t-tests for each data point on each day compared to vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows Kaplan-Meier survival curves for EMT6 xenografts treated with vehicle (median survival 14 days) or compound 5 (median survival 22 days). P values were calculated using the log-rank test. Figure 10C shows the percent body weight change of EMT6 xenografts after treatment. [Figure 11A] Overall, targeting TACC3 with Compound 5 significantly impaired tumor growth in mouse models of colorectal cancer. Figure 11A shows tumor volume assessment of the effects of Compound 5 on colorectal cancer xenografts in immunocompromised (HCT-116) and syngeneic immunocompetent (CT-26) mouse models. Mice were orally administered vehicle or Compound 5 (25 and / or 50 mg / kg) daily. Treatment began when tumor volumes reached 90-100 mm3. Significance was calculated using Student's t-test. *: p<0.05; **: p<0.01. Figure 11B shows the percent body weight change of HCT-116 and CT-26 xenografts after treatment. [Figure 11B] Overall, targeting TACC3 with Compound 5 significantly impaired tumor growth in mouse models of colorectal cancer. Figure 11A shows tumor volume assessment of the effects of Compound 5 on colorectal cancer xenografts in immunocompromised (HCT-116) and syngeneic immunocompetent (CT-26) mouse models. Mice were orally administered vehicle or Compound 5 (25 and / or 50 mg / kg) daily. Treatment began when tumor volumes reached 90-100 mm3. Significance was calculated using Student's t-test. *: p<0.05; **: p<0.01. Figure 11B shows the percent body weight change of HCT-116 and CT-26 xenografts after treatment. [Figure 12A]Figure 12A shows the effect of targeting TACC3 with Compound 5 on metastatic growth in the 4T1.luc2 model. Figure 12A shows bioluminescence (BLI) images of mice injected with 4T1.luc2 cells via the tail vein. After metastases were established in the lungs, the mice were orally treated daily with either vehicle or Compound 5 (50 mg / kg). Metastatic growth was monitored weekly by BLI using an IVIS (in vivo imaging system). Figure 12B shows Kaplan-Meier survival curves for 4T1.luc2 syngeneic models treated with vehicle (median survival 19 days) or Compound 5 (median survival 28 days). P values were calculated using the log-rank test. [Figure 12B] Figure 12A shows the effect of targeting TACC3 with Compound 5 on metastatic growth in the 4T1.luc2 model. Figure 12A shows bioluminescence (BLI) images of mice injected with 4T1.luc2 cells via the tail vein. After metastases were established in the lungs, the mice were orally treated daily with either vehicle or Compound 5 (50 mg / kg). Metastatic growth was monitored weekly by BLI using an IVIS (in vivo imaging system). Figure 12B shows Kaplan-Meier survival curves for 4T1.luc2 syngeneic models treated with vehicle (median survival 19 days) or Compound 5 (median survival 28 days). P values were calculated using the log-rank test. [Figure 13A] 13A shows the effects of high-dose Compound 5 on mouse body weight and internal organs. Figure 13A shows the body weight (g) of mice after 7 days of daily oral administration of 100 mg / kg of Compound 5. Figure 13B shows the body weight (g) of mice after a single dose of Compound 5 (500 mg / kg). Mice were sacrificed on different days. Figure 13C shows photographs of mice and their organs (lower panel) treated with either vehicle or 500 mg / kg of Compound 5. Mice were sacrificed 48 hours after drug treatment. [Figure 13B]13A shows the effects of high-dose Compound 5 on mouse body weight and internal organs. Figure 13A shows the body weight (g) of mice after 7 days of daily oral administration of 100 mg / kg of Compound 5. Figure 13B shows the body weight (g) of mice after a single dose of Compound 5 (500 mg / kg). Mice were sacrificed on different days. Figure 13C shows photographs of mice and their organs (lower panel) treated with either vehicle or 500 mg / kg of Compound 5. Mice were sacrificed 48 hours after drug treatment. [Figure 13C] 13A shows the effects of high-dose Compound 5 on mouse body weight and internal organs. Figure 13A shows the body weight (g) of mice after 7 days of daily oral administration of 100 mg / kg of Compound 5. Figure 13B shows the body weight (g) of mice after a single dose of Compound 5 (500 mg / kg). Mice were sacrificed on different days. Figure 13C shows photographs of mice and their organs (lower panel) treated with either vehicle or 500 mg / kg of Compound 5. Mice were sacrificed 48 hours after drug treatment. [Figure 14] A summary of the drug-specific physicochemical properties of compound 5 is shown. a Kinetic solubility in sodium phosphate buffer at pH 7.4 at 25 °C after 2 h (Buttar et al., 2010); b LogD: partition coefficient in octanol / sodium phosphate buffer (50 mM, pH 7.4) (Unger et al., 1978); c PPB: plasma protein binding assessed by equilibrium dialysis in humans at 37 °C (Buttar et al., 2010); dT1 / 2: half-life in mouse and human liver microsomes (MLM and HLM, respectively) (Kalvass et al., 2001); e Clint: intrinsic clearance in MLM and HLM (Kalvass et al., 2001); f CYP: cytochrome P450 isozyme inhibition by compound 5 assessed in HLM (Bourrie et al., 1996); g Papp: apparent permeability coefficient in Caco-2 cell monolayers (Camenisch et al., 1998). DETAILED DESCRIPTION OF THE INVENTION

[0017] Elevated levels of TACC3 have been observed in many different cancer types, making it a highly attractive target for cancer therapy. TACC3 plays a key role in regulating microtubules and centrosomes, maintaining mitotic spindle stability (Schneider et al., 2007; Thakur et al., 2013).

[0018] To further investigate TACC3 levels in different tumor types, we analyzed TACC3 levels in many different cancer types and their normal tissue counterparts (Figure 1A). We found that TACC3 was significantly overexpressed in many different cancer types, including breast cancer. We then analyzed different patient survival datasets (GSE31210 and TCGA) using different databases (METABRIC and KM Plotter). High TACC3 levels were found to be significantly correlated with poor overall survival in breast cancer (Figure 1B-1) and gastric cancer (Figure 1B-2). Furthermore, high TACC3 levels were associated with poor recurrence-free survival in lung cancer patients (Figure 1B-3) and disease-free survival in prostate cancer patients (Figure 1B-4). Importantly, multivariate Cox regression analysis demonstrated that TACC3 expression was an independent prognostic factor in breast cancer (Figure 2). Collectively, these results indicate that TACC3 is a clinically relevant target with strong prognostic value in several different cancers, and its expression level is a key factor defining disease severity and patient survival. Therefore, inhibiting TACC3 function is a promising therapeutic strategy for improving patient survival in breast and other cancers.

[0019] Decreased TACC3 levels in Hela cells have been shown to cause mitotic arrest (Schneider et al., 2007) and caspase-dependent apoptosis (Kimura et al., 2013). In this study, we found that breast cancer patients expressing high TACC3 levels exhibited enhanced mitotic progression and DNA repair genes, supporting the oncogenic role of TACC3 in breast cancer progression (Figure 3A). Next, we investigated the effects of TACC3 deficiency on cell viability in different breast cancer cell lines. We utilized four different breast cancer cell line models with different molecular subtypes. JIMT-1, a HER2-positive breast cancer cell line, and BT-474 T-DM1R (T-DM1-resistant), a luminal B subtype breast cancer cell line, were shown to have elevated TACC3 levels (Saatci et al., 2018). On the other hand, MDA-MB-436 and MDA-MB-157 are both triple-negative breast cancer (TNBC) cell lines. In this study, we first used small interfering RNA (siRNA) to reduce TACC3 levels in these cell lines. Figure 3B shows that overall TACC3 levels were reduced by approximately 60–70% in all cell lines after 48 hours of siRNA treatment. Consistent with this, we observed that TACC3 knockdown resulted in significant growth inhibition of breast cancer cells (Figure 3C). Next, we investigated the possible mechanisms underlying TACC3 knockdown. Suppression of TACC3 levels by siRNA in all breast cancer cell lines tested activated mitotic arrest, apoptosis, and DNA damage (Figure 3D).

[0020] We conducted an in-house screening of a series of small molecules by testing their antiproliferative effects in breast cancer cells in which TACC3 is aberrantly expressed (Ma et al., 2003; Song et al., 2018). Specifically, the JIMT-1 cell line was selected to screen the effects of compounds on cell viability due to its high TACC3 protein levels compared to other tested breast cancer cell lines, as well as its in vivo tumorigenicity (Saatci et al., 2018; Tanner et al., 2004) (depicted in Figures 7 and 8-9, respectively). Through experiments using cultured cells, we found that compound 5 inhibited cell growth (IC of 190 nM). 50 ), which has been shown to exhibit antitumor effects. Therefore, by using this compound as an active pharmaceutical ingredient (API) for anticancer drugs, effective anticancer drugs can be developed. Therefore, to obtain novel compounds with anticancer activity against cancer cells expressing high levels of TACC3, we synthesized a compound having the basic structure of formula (I).

[0021] In one aspect, the present disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: X1 is N or CR6, X2 is N or CR3, R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R5 is heterocyclyl, alkyl, or amino.

[0022] In certain embodiments, the compound is [ka] isn't it.

[0023] In certain embodiments, R1 is aryl (e.g., phenyl). In other embodiments, R1 is heteroaryl (e.g., benzodioxole, dihydrobenzofuran, benzofuran, or pyrimidinyl).

[0024] In certain embodiments, R1 is substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl (e.g., methylsulfonyl), or sulfonamido. In certain embodiments, R1 is substituted with alkyl (e.g., methyl, ethyl, isopropyl, fluoroethyl, or trifluoromethyl), alkyloxy (e.g., methoxy, trifluoromethyloxy, difluoromethyloxy, ethoxy, or propyloxy), alkylthio (e.g., methylthio), aralkyloxy (e.g., benzyloxy), hydroxyl, halo (e.g., fluoro or chloro), or amino (e.g., dimethylaminoalkyl). In certain preferred embodiments, R1 is substituted with halo (e.g., fluoro). In certain embodiments, halo (e.g., F) is para to the isoxazole. In other embodiments, the halo (e.g., F) is ortho to the isoxazole. In other embodiments, the halo (e.g., F) is meta to the isoxazole. In certain preferred embodiments, R1 is substituted with two halo (e.g., F). In certain embodiments, one halo (e.g., F) is meta to the isoxazole and one halo (e.g., F) is ortho to the isoxazole. In other preferred embodiments, R1 is substituted with alkyloxy (e.g., methoxy). In certain embodiments, the alkoxy (e.g., methoxy) is para to the isoxazole. In other embodiments, the alkoxy (e.g., methoxy) is ortho to the isoxazole. In certain embodiments, the alkoxy (e.g., methoxy) is meta to the isoxazole. In still other preferred embodiments, R1 is substituted with alkyl (e.g., methyl, ethyl, or trifluoromethyl). In still other preferred embodiments, R1 is substituted with halo (e.g., fluoro) and alkyloxy (e.g., methoxy).In further preferred embodiments, R1 is substituted with a methoxy moiety and one or two fluoro moieties. In the most preferred embodiments, R1 is substituted with a methoxy moiety and two fluoro moieties. In certain embodiments, the alkoxy (e.g., methoxy) is para to the isoxazole and F is meta to the isoxazole. In yet other modifications, the alkoxy (e.g., methoxy) is para to the isoxazole and F is ortho to the isoxazole. In other embodiments, the halo (e.g., F) is para to the isoxazole and the alkoxy (e.g., methoxy) is meta to the isoxazole. In yet other embodiments, the alkoxy (e.g., methoxy) is para to the isoxazole, one halo (e.g., F) is meta to the isoxazole, and one halo (e.g., F) is ortho to the isoxazole.

[0025] In certain embodiments, R2 is alkyl (e.g., methyl or ethyl). In certain embodiments, R2 is substituted with amino (e.g., dimethylamino or diethylamino), or nitrile. In other embodiments, R2 is H.

[0026] In certain embodiments, X1 is N. In other embodiments, X1 is CR6. In certain embodiments, R6 is H.

[0027] In certain embodiments, X2 is N. In other embodiments, X2 is CR3. In certain embodiments, R3 is H or halo (e.g., fluoro or chloro).

[0028] In certain embodiments, R3 is H or halo (eg, fluoro or chloro).

[0029] In certain embodiments, R4 is alkyl (eg, methyl).

[0030] In certain embodiments, R5 is heterocyclyl (e.g., azetidinyl, morpholino, pyrrolidinyl, piperazinyl, piperidinyl, oxaazabicyclooctanyl, oxaazabicycloheptnyl, thiomorpholino, thiomorpholino dioxide, hexahydrofuropyrrolyl, or azabicyclohexanyl). In certain embodiments, R5 is a 6-membered heterocyclyl, and the backbone of the cycle contains one nitrogen. In certain embodiments, R5 is a 6-membered heterocyclyl, and the backbone of the cycle contains one nitrogen and one oxygen. In other embodiments, R5 is a 7-membered heterocyclyl, and the backbone of the cycle contains one nitrogen. In certain embodiments, R5 is a 7-membered heterocyclyl, and the backbone of the cycle contains one nitrogen and one oxygen. In other embodiments, R5 is an 8-membered heterocyclyl and the backbone of the cycle contains one nitrogen. In certain embodiments, R5 is an 8-membered heterocyclyl and the backbone of the cycle contains one nitrogen and one oxygen. In certain preferred embodiments, R5 is a nitrogen-containing heterocyclyl and the nitrogen is directly bonded to the aryl or heteroaryl ring bearing the R4 substituent. In certain preferred embodiments, R5 is 2,6-dimethylmopholine, 4-methylpiperidine, or 4-(trifluoromethyl)piperidine.

[0031] In certain embodiments, R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or sulfonamide. In certain embodiments, R5 is substituted with ester (e.g., ethyl ester), carboxyl, alkyl (e.g., methyl or trifluoromethyl), hydroxyalkyl (e.g., hydroxyethyl), halo (e.g., fluoro), cycloalkyl (e.g., cyclopropyl or cyclobutyl), or heterocyclyl (e.g., oxetnyl or tetrahydrofuranyl). In certain preferred embodiments, R5 is substituted with halo (e.g., fluoro). In certain preferred embodiments, R5 is substituted with two alkyl moieties. In even more preferred embodiments, R5 is substituted with two methyl moieties.

[0032] In other embodiments, R5 is amino. In certain embodiments, R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, sulfonamido, cycloalkyl, or heterocyclyl. In certain embodiments, R5 is substituted with alkyl (e.g., difluoroethyl, or isobutyl), alkyloxyalkyl (e.g., methyloxyethyl), hydroxyalkyl (e.g., hydroxyethyl), cyclopropyl (e.g., cyclopropyl), or heterocyclyl (e.g., pyranyl).

[0033] In certain embodiments, the compound of formula I has a structure represented by formula II: [ka]

[0034] Anti-cancer agents of the present disclosure include compounds represented by general formula (I), wherein: R1: unsubstituted phenyl, or o-, m-, or p-CH3, C2H5, CH(CH3)2, OCH3, OC2H5, OC3H7, SCH3, CF2CH3, CF3, OCF3, OCHF2, N(CH3)2, F, Cl, OH mono- or di-substituted phenyl, pyridyl, benzyloxy, or piperonyl group; R2: H, CH3; R3: H, F, Cl; R4: H, CH3; X1: CH, N; R5: morpholine, 2,6-dimethylmorpholine, thiomorpholine, thiomorpholine 1,1-dioxide, morpholin-4-amine, piperidine, tetrahydro-2H-pyran-4-amine, piperidin-1-amine 4-piperidine, 4-fluoropiperidine, 4,4-difluoropiperidine, 4-methylpiperidine, 4-(trifluoromethyl)piperidine, piperazine, N-methylpiperazine, pyrrolidine, 2-(4-piperidine)ethanol, 2-(1-piperazinyl)ethanol, 4-piperidinecarboxylic acid, ethyl 4-piperidinecarboxylate, (1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane, (1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane, 3-oxa-8-azabicyclo[3.2.1]octane or 8-oxa-3-azabicyclo[3.2.1]octane group.

[0035] In certain embodiments, R1 is phenyl. In other embodiments, R1 is pyridyl. In yet other embodiments, R1 is benzyloxy. In still other embodiments, R1 is piperonyl. In certain embodiments, R1 is substituted with CH3, C2H5, CH(CH3)2, OCH3, OC2H5, OC3H7, SCH3, CF2CH3, CF3, OCF3, OCHF2, N(CH3)2, F, Cl, or OH. In certain preferred embodiments, R1 is substituted with CH3. In other preferred embodiments, R1 is substituted with OCH3. In certain embodiments, OCH3 is para to the isoxazole. In certain embodiments, OCH3 is ortho to the isoxazole. In certain embodiments, OCH3 is meta to the isoxazole. In still other preferred embodiments, R1 is substituted with F. In certain embodiments, R1 is substituted with one F. In certain embodiments, F is para to the isoxazole. In certain embodiments, F is ortho to the isoxazole. In certain embodiments, F is meta to the isoxazole. In certain embodiments, R1 is substituted with two F. In certain embodiments, the first F is meta to the isoxazole and the second F is ortho to the isoxazole. In further preferred embodiments, R1 is substituted with OCH3 and F. In certain embodiments, OCH3 is para to the isoxazole and F is meta to the isoxazole. In certain embodiments, OCH3 is para to the isoxazole and F is ortho to the isoxazole. In certain embodiments, F is para to the isoxazole and OCH3 is meta to the isoxazole. In further preferred embodiments, R1 is substituted with OCH3 and two F. In certain embodiments, OCH3 is para to the isoxazole and both F are meta to the isoxazole. In certain embodiments, OCH3 is para to the isoxazole, one F is meta to the isoxazole, and one F is ortho to the isoxazole.

[0036] In certain embodiments, R2 is H. In other embodiments, R2 is CH3.

[0037] In certain embodiments, R3 is H. In other embodiments, R3 is F. In yet other embodiments, R3 is Cl.

[0038] In certain preferred embodiments, R5 is morpholine. In other preferred embodiments, R5 is piperidine. In yet other preferred embodiments, R5 is 4-fluoropiperidine. In yet other preferred embodiments, R5 is 4,4-difluoropiperidine. In yet other preferred embodiments, R5 is 3-oxa-8-azabicyclo[3.2.1]octane. In yet other preferred embodiments, R5 is 8-oxa-3-azabicyclo[3.2.1]octane. In yet other preferred embodiments, R5 is 2,6-dimethylmorpholine. In yet other preferred embodiments, R5 is 4-methylpiperidine. In yet other preferred embodiments, R5 is 4-methylpiperidine.

[0039] In certain embodiments, X 1 is C. In other embodiments, X 1 is N.

[0040] The specific final components of the present disclosure are listed below. 3-(3-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 6) 3-(2-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 7) N-(2-morpholinopyrimidin-4-yl)-3-phenylisoxazol-5-amine (Compound 8) 3-(4-ethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 9) N-(2-morpholinopyrimidin-4-yl)-3-(4-propoxyphenyl)isoxazol-5-amine (Compound 10) 3-(4-Fluorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 11) 3-(4-chlorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 12) N-(2-morpholinopyrimidin-4-yl)-3-(p-tolyl)isoxazol-5-amine (Compound 13) 3-(4-Ethylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 14) 3-(4-Isopropylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 15) N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazol-5-amine (Compound 16) 3-(4-(1,1-difluoroethyl)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 17) 3-(4-(difluoromethoxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 18) N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazol-5-amine (Compound 19) 3-(4-(methylthio)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 20) 3-(4-(dimethylamino)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 21) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 22) 3-(3-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 23) 3-(3,4-Dimethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 24) 3-(2,3-Difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 25) 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (compound 26) 3-(6-Methoxypyridin-3-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 27) 4-(5-((2-morpholinopyrimidin-4-yl)amino)isoxazol-3-yl)phenol (Compound 30) 3-(4-(benzyloxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 32) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 33) N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (compound 34) N-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 35) N-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 36) N-(2-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 37) N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 38) N-(2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 39) 3-(4-Methoxyphenyl)-N-(2-thiomorpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 40) 4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)thiomorpholine 1,1-dioxide (Compound 41) 3-(4-Methoxyphenyl)-N-(2-(piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 42) 3-(4-Methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 43) 3-(4-Methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 44) 3-(4-Methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 45) 2-(1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidin-4-yl)ethan-1-ol (Compound 46), 2-(4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethan-1-ol (Compound 47), 3-(4-methoxyphenyl)-N-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 48) 3-(4-Methoxyphenyl)-N-(2-(piperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 49) Ethyl 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylate (Compound 50) 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid (Compound 51) N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -Morpholinopyrimidine-2,4-diamine (Compound 52) N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(Piperidin-1-yl)pyrimidine-2,4-diamine (Compound 53) N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(Tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine (Compound 54) N-(5-chloro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 59) N-(5-fluoro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 60) N-(2-(4-fluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 61) 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine (Compound 62) 3-(4-Methoxyphenyl)-N-methyl-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 63) 3-(4-(dimethylamino)phenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 64) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 65) N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 66) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 67) 3-(2,3-Dihydrobenzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 69) 3-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 70) N 2 -cyclopropyl-N 4 -(3-(4-Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine (Compound 71) N 2 -Isobutyl-N 4 -(3-(4-Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine (Compound 72) N 2 -(2-Methoxyethyl)-N 4 -(3-(4-Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine (Compound 73) N 1 ,N 1 -Diethyl-N 2 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine (Compound 74) N 1 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 ,N 2-Dimethyl-N1-(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine (Compound 75) 2-((3-(4-Methoxyphenyl)isoxazol-5-yl)(2-morpholinopyrimidin-4-yl)amino)acetonitrile (Compound 76) 3-(5-Methoxypyridin-2-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 77) 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine (Compound 78) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine (Compound 79) 3-(4-Methoxyphenyl)-N-(4-morpholino-1,3,5-triazin-2-yl)isoxazol-5-amine (Compound 80) N 2 -(2,2-difluoroethyl)-N 4 -(3-(4-Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine (Compound 81) 2-((4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)amino)ethan-1-ol (Compound 82) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 83) 3-(2,3-Difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 84) 3-(3,5-Difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 85) N-(2-morpholinopyrimidin-4-yl)-3-(3,4,5-trimethoxyphenyl)isoxazol-5-amine (Compound 86) 3-(Benzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 87) 3-(2,5-Difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 88) 3-(2,5-Difluoro-4-methoxyphenyl)-N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 89) N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -Methylpyrimidine-2,4-diamine (Compound 90) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 91) 3-(3-Fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 92) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 93) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 94) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 95) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 96) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetramethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 97) N-(2-(3,3-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 98) N-(2-(2,2-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 99) N-(2-(3,5-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 100) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 101) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 102) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(2-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 103) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 104) N-(2-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 105) N-(2-(7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 106) N-(2-(4,4-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 107) N-(2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 108) N-(2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 109) N-(2-(6-azaspiro[2.5]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 110) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-methyl-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 111) N-(2-(2,2-difluoromorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 112) N-(2-(2-oxa-5-azabicyclo[2.2.2]octan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 113) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 114) N-(2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 115) N-(2-(2-oxa-5-azabicyclo[4.1.0]heptan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 116) N-(2-(4-oxa-7-azaspiro[2.5]octan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 117) N-(2-(7-oxa-4-azaspiro[2.5]octan-4-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 118) N-(2-(2-oxa-8-azaspiro[4.5]decan-8-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 119) N-(2-(2-oxa-6-azaspiro[3.4]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 120) N-(2-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 121) N-(2-(2-oxa-7-azaspiro[4.4]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 122) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetrafluoromorpholino)pyrimidin-4-yl)isoxazol-5-amine (Compound 123) N-(2-(6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 124) 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-methyl-6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 125) N-(2-(3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 126), 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-isopropylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine (Compound 127), and N-(2-(4-(difluoromethyl)piperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 128). N-(2-(6-azaspiro[3.5]nonan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 129) N-(2-(2-azaspiro[3.5]nonan-2-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 130) 3-(2-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 131)

[0041] Each of the parent intermediates of this disclosure is shown below. N-(2-chloropyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (compound 4a) N-(2-chloropyrimidin-4-yl)-3-(3-methoxyphenyl)isoxazol-5-amine (compound 4b) N-(2-chloropyrimidin-4-yl)-3-(2-methoxyphenyl)isoxazol-5-amine (compound 4c) N-(2-chloropyrimidin-4-yl)-3-phenylisoxazol-5-amine (compound 4d) N-(2-chloropyrimidin-4-yl)-3-(4-ethoxyphenyl)isoxazol-5-amine (compound 4e) N-(2-chloropyrimidin-4-yl)-3-(4-propoxyphenyl)isoxazol-5-amine (compound 4f) N-(2-chloropyrimidin-4-yl)-3-(4-fluorophenyl)isoxazol-5-amine (compound 4g) 3-(4-chlorophenyl)-N-(2-chloropyrimidin-4-yl)isoxazol-5-amine (Compound 4h) N-(2-chloropyrimidin-4-yl)-3-(p-tolyl)isoxazol-5-amine (compound 4i) N-(2-chloropyrimidin-4-yl)-3-(4-ethylphenyl)isoxazol-5-amine (compound 4j) N-(2-chloropyrimidin-4-yl)-3-(4-isopropylphenyl)isoxazol-5-amine (compound 4k) N-(2-chloropyrimidin-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazol-5-amine (Compound 4l) N-(2-chloropyrimidin-4-yl)-3-(4-(1,1-difluoroethyl)phenyl)isoxazol-5-amine (Compound 4m) N-(2-chloropyrimidin-4-yl)-3-(4-(difluoromethoxy)phenyl)isoxazol-5-amine (Compound 4n) N-(2-chloropyrimidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazol-5-amine (Compound 4o) N-(2-chloropyrimidin-4-yl)-3-(4-(methylthio)phenyl)isoxazol-5-amine (Compound 4p) N-(2-chloropyrimidin-4-yl)-3-(4-(dimethylamino)phenyl)isoxazol-5-amine (Compound 4r) N-(2-chloropyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 4s) 3-(3-chloro-4-methoxyphenyl)-N-(2-chloropyrimidin-4-yl)isoxazol-5-amine (Compound 4t) N-(2-chloropyrimidin-4-yl)-3-(3,4-dimethoxyphenyl)isoxazol-5-amine (Compound 4u) N-(2-chloropyrimidin-4-yl)-3-(2,3-difluoro-4-methoxyphenyl)isoxazol-5-amine (compound 4v) 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-chloropyrimidin-4-yl)isoxazol-5-amine (compound 4y) N-(2-chloropyrimidin-4-yl)-3-(6-methoxypyridin-3-yl)isoxazol-5-amine (Compound 4z) 3-(4-Methoxyphenyl)-N-(2-(methylthio)pyrimidin-4-yl)isoxazol-5-amine (Compound 28) 4-(5-((2-chloropyrimidin-4-yl)amino)isoxazol-3-yl)phenol (Compound 29) 3-(4-(benzyloxy)phenyl)-N-(2-chloropyrimidin-4-yl)isoxazol-5-amine (Compound 31) N-(2,5-dichloropyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 55) N-(2-chloro-5-fluoropyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 56) N-(2-chloro-6-methylpyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 57) N-(2-chloropyridin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine (Compound 58) N-(2-chloropyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 132) N-(2-chloropyrimidin-4-yl)-3-(2,5-difluoro-4-methoxyphenyl)isoxazol-5-amine (Compound 133)

[0042] Anti-cancer agents of the present disclosure include compounds of general formula (I), which are represented by the following chemical structures of certain final compounds of the present disclosure: [ka] [ka] [ka] [ka]

[0043] In certain embodiments, the compound is [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0044] The compounds shown above are novel compounds synthesized by the present inventors. The following describes the synthesis methods of these novel compounds. Representative compounds of the present disclosure according to the general synthesis methods described below are more specifically shown in the following schemes. Because the schemes are illustrative, the disclosure should not be construed as being limited by the chemical reactions and conditions shown. The preparation of the various starting materials used in the schemes is within the skill of a person skilled in the art. The substituents of the compound of formula (I) or its form, as represented in the following schemes, are as previously defined herein.

[0045] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable excipient.

[0046] In yet another aspect, the present disclosure provides a method for treating a TACC3-mediated disease or disorder in a subject, comprising administering to the subject a compound of any one of claims 1-53, or a pharmaceutically acceptable salt thereof. In certain embodiments, the TACC3-mediated disease or disorder is cancer. In certain embodiments, the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer, or prostate cancer. In certain embodiments, the cancer is a cancer selected from the NCI-60 panel.

[0047] In yet another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject a compound of any one of claims 1 to 53, or a pharmaceutically acceptable salt thereof. In certain embodiments, the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer, or prostate cancer. In certain embodiments, the cancer is a cancer selected from the NCI-60 panel.

[0048] In yet another aspect, the present disclosure provides a method for producing a compound of the present disclosure, wherein the method is represented by Scheme I: [ka] or a pharmaceutically acceptable salt thereof, wherein: X1 is N or CR6, X2 is N or CR3, R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R5 is heterocyclyl, alkyl, or amino; R 51 is a halo, R 52 is heterocyclyl or alkyl, X 10 is a base, X 11 is a noble metal catalyst, X 12 is a phosphine ligand.

[0049] In certain embodiments, the base is a carbonate, an oxide, a tertiary amine, a secondary amine, or a hydride. In certain embodiments, the oxide is an alkoxide (e.g., tert-butoxide). In certain embodiments, the tertiary amine is a tertiary alkylamine (e.g., diisopropylethylamine). In certain embodiments, the hydride is a metal hydride (e.g., sodium hydride). In certain embodiments, the carbonate is a metal carbonate (e.g., cesium carbonate).

[0050] In certain embodiments, the noble metal catalyst is a palladium catalyst (eg, palladium II acetate).

[0051] In certain embodiments, the phosphine catalyst is an arylphosphine (e.g., triphenylphosphine). In certain embodiments, the phosphine catalyst is xantphos.

[0052] In certain embodiments, the method further comprises a solvent, hi certain embodiments, the solvent is tertiary butanol, dimethylacetamide, or dioxane.

[0053] In certain embodiments, the method further comprises heating.

[0054] In certain embodiments, the method is carried out under an inert atmosphere.

[0055] Pharmaceutical Composition The compositions and methods of the present disclosure can be used to treat individuals in need of treatment. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents, or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for human administration, particularly for invasive administration routes (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in the form of a dosage unit such as a tablet, capsule (including a sprinkle capsule and a gelatin capsule), granule, liquid solution for reconstitution, powder, liquid, syrup, suppository, injection, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0056] Pharmaceutically acceptable carriers can include physiologically acceptable agents that stabilize, increase the solubility, or enhance absorption of compounds, such as the compounds of the present disclosure. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be a liposome or other polymer matrix, into which, for example, the compounds of the present disclosure can be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers, and are relatively easy to prepare and administer.

[0057] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0058] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer solution, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0059] The pharmaceutical compositions (preparations) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., as liquid or non-liquid or suspension formulations, tablets, drenches such as capsules (including sprinkle capsules and gelatin capsules), boluses for application to the tongue, powders, granules, pastes); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., as a patch applied to the skin); or topically (e.g., as a cream, ointment, or spray applied to the skin). The compounds can also be prepared for inhalation. In certain embodiments, the compounds can be simply dissolved or suspended in sterile water. Details of suitable administration routes and compositions suitable therefor are described, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0060] The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0061] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0062] Formulations of the present disclosure suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophiles, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as a electuary or syrup, or as a troche (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or mouthwash, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0063] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with sodium citrate or dicalcium phosphate, and / or one or more pharmaceutically acceptable carriers, such as any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0064] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0065] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulation art. They can also be prepared to provide sustained or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various proportions to produce the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents, and can be compositions that release the active ingredient only, or preferentially, in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0066] The liquid dosage form useful for oral administration includes pharmaceutically acceptable emulsion, reconstituted liquid emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form can contain the inert diluent commonly used in the art, such as water or other solvent, cyclodextrin and its derivatives, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed, peanut, corn, germ, olive, castor oil, sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and their mixtures.

[0067] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0068] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, metahydroxyaluminum, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0069] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required.

[0070] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal, vegetable and physical fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0071] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane or propane.

[0072] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present disclosure to the body.Such dosage forms can be prepared by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across the skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0073] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately before use and may contain antioxidants, buffers, bacteriostats, solvents that render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0074] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants.

[0075] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of the injectable dosage form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0076] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to maintain the effect of the drug.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0077] Injectable depot forms are prepared by forming microencapsulated matrices of the target compound in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0078] For use in the methods of the present disclosure, the active compound can be provided per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0079] The method of introduction can also be provided by a rechargeable or biodegradable device. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained-release polymeric devices have been developed and tested in vivo in recent years. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0080] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied to provide an amount of the active ingredient effective to achieve a therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0081] The selected dosage level will depend on a variety of factors, including factors well known in the medical arts, such as the activity of the particular compound or combination of compounds, or esters, salts, or amides thereof, being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or substances being used in combination with the particular compound being used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated.

[0082] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the required therapeutically effective amount of the pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if necessary, other types of therapeutic agents administered together with the compound of the present disclosure. Multiple administrations of the drug can deliver a larger total dose. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0083] In general, a suitable daily dose of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective amount will generally depend on the factors described above.

[0084] If necessary, the effective daily dose of the active compound can be administered as 1, 2, 3, 4, 5, 6 or more subdoses at appropriate intervals throughout the day, optionally administered separately in unit dosage forms. In certain embodiments of the present disclosure, the active compound can be administered two or three times a day. In a preferred embodiment, the active compound is administered once a day.

[0085] Patients receiving this treatment are any animal in need of treatment, including primates, particularly humans, and other mammals such as horses, cows, pigs, sheep, cats, dogs, poultry, and common pets.

[0086] In certain embodiments, the compounds of the present disclosure can be used alone or can be administered in combination with another type of therapeutic agent.

[0087] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present disclosure in the compositions and methods of the present disclosure.In certain embodiments, contemplated salts of the present disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts.In certain embodiments, contemplated salts of the present disclosure include, but are not limited to, L-arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.In certain embodiments, contemplated salts of the present disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present disclosure include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptan-1,2-one ... Acids that may be used include, but are not limited to, carboxylic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0088] Pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0089] Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0090] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0091] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0092] The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, "Principles of Neural Science," McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics," Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", W.H. Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", W.H. Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0093] Chemical terms used herein, unless otherwise defined herein, are used in accordance with conventional usage in the art, as exemplified in "The McGraw-Hill Dictionary of Chemical Terms," Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0094] All of the above, and other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including specific definitions, will control.

[0095] The term "drug" is used herein to refer to a compound (organic or inorganic compound, a mixture of compounds, etc.), a biological macromolecule (nucleic acid, antibody, a portion thereof, and humanized, chimeric and human antibodies and monoclonal antibodies, a protein or a portion thereof, e.g., peptide, lipid, carbohydrate), or an extract made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (e.g., mammals). Drugs include, for example, drugs with known structures and drugs with unknown structures. The ability of such drugs to inhibit AR or promote AR degradation may make them suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0096] The terms "patient," "subject," or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock (including cows, pigs, etc.), companion animals (dogs, cats, etc.), and rodents (mice, rats, etc.).

[0097] "Treating" a condition or patient refers to taking measures to obtain beneficial or desired results, including clinical results. As used herein, and as is well understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stable (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of disease symptoms, and remission (partial or total). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0098] The term "preventing," when used in reference to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or other medical conditions, is art-recognized and well understood in the art and includes administration of a composition that reduces the frequency of symptoms of, or delays the onset of, the medical condition in a subject compared to subjects who do not receive the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population by a statistically and / or clinically significant amount.

[0099] "Administering" or "administration" of a substance, compound, or agent to a subject can be carried out using one of a variety of methods known in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through the skin tract). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations that provide sustained, slow-release, or controlled release of the compound or agent. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0100] The appropriate method of administering a substance, compound, or agent to a subject also depends, for example, on the age and / or physical condition of the subject, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a sustained-release or timed-release formulation or is administered using a device for such sustained-release or timed-release.

[0101] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents in which a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective in a patient simultaneously, and a synergistic effect of the two agents may be involved). For example, the different therapeutic compounds can be administered simultaneously or sequentially, in the same formulation or in separate formulations. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0102] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is the amount of drug or agent that has the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur in a single administration, but may occur only after a series of administrations. Therefore, a therapeutically effective amount can be administered in one or more administrations. The exact effective amount required by a subject depends, for example, on the subject's size, health, and age, and the nature and extent of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective amount for a given situation through routine experimentation.

[0103] As used herein, the term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" refers to cases where the alkyl can be substituted, as well as cases where the alkyl is not substituted.

[0104] The substituents and substitution patterns on the compounds of the present disclosure can be selected by those skilled in the art to result in chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art, as well as by the methods described below. When a substituent is itself substituted with multiple groups, it is understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure results.

[0105] As used herein, the term "optionally substituted" refers to the replacement of 1 to 6 hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH-O-alkyl, -OP(O)(O-alkyl) or -CH-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of 1 to 4 hydrogen radicals in a given structure with the aforementioned substituents. More preferably, 1 to 3 hydrogen radicals are replaced by the aforementioned substituents. It is understood that the substituents can be further substituted.

[0106] As used herein, the term "alkyl" refers to a C1-C 10 Straight chain alkyl group or C1-C 10 "Alkyl" refers to saturated aliphatic groups, including, but not limited to, branched alkyl groups. Preferably, "alkyl" refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, "alkyl" refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. "Alkyl" groups can be optionally substituted.

[0107] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0108] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0109] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0110] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0111] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0112] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C1-C for a straight chain). 30 , for branched chains C3 to C 30 ), more preferably having 20 or fewer carbon atoms.

[0113] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, and includes haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0114] "C x~y " or "C x ~C yThe term "alkyl" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. CO alkyl indicates a hydrogen in the group's terminal position, and if internal, indicates a bond. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0115] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0116] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0117] As used herein, the term "amide" refers to the group [ka] refers to, In the formula, R 9 and R 10 each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0118] The terms "amine" and "amino" are art-recognized and include both unsubstituted and substituted amines, salts thereof, e.g., amines of the formula [ka] and In the formula, R 9 , R 10 , and R 10‘ each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0119] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0120] The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group.

[0121] As used herein, the term "aryl" includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0122] The term "carbamate" is art-recognized and refers to a group [ka] refers to, In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0123] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0124] The term "carbocycle" includes 5- to 7-membered monocyclic and 8- to 12-membered bicyclic rings. Each ring in a bicyclic carbocycle can be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring in a fused carbocycle can be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, can be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings, valence permitting, is included in the definition of carbocycle. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any one or more positions that can retain a hydrogen atom.

[0125] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0126] The term "carbonate" is art-recognized and refers to a group -OCO 2- Refers to...

[0127] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0128] As used herein, the term "ester" refers to the group -C(O)OR 9 In the formula, R 9 represents a hydrocarbyl group.

[0129] The term "ether" as used herein refers to a hydrocarbyl group bonded to another hydrocarbyl group via an oxygen atom. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0130] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo, and iodo.

[0131] The terms "hetarylalkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0132] The terms "heteroaryl" and "hetaryl" refer to substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, in which the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings, in which two or more carbons are common to two adjacent rings, and at least one of the rings is a heteroaromatic ring, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0133] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0134] The term "heterocyclic alkyl," as used herein, refers to an alkyl group substituted with a heterocyclic group.

[0135] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, which ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocycle" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0136] As used herein, the term "hydrocarbyl" refers to a group that is bonded through a carbon atom that does not have an =O or =S substituent, typically has at least one carbon-hydrogen bond, and a primarily carbon backbone, but can optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (which has an =O substituent on the bonded carbon) and ethoxy (which is bonded through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0137] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0138] The term "lower" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer, preferably 6 or fewer atoms in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, an acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituent as defined herein, such as hydroxyalkyl and aralkyl in reference thereto (where, for example, atoms in an aryl group are not counted when counting the carbon atoms of an alkyl substituent), is lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents.

[0139] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings (e.g., the rings are "fused rings"). Each ring of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0140] The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0141] The term "sulfonamide" is art-recognized and can be represented by the general formula [ka] It refers to a group represented by In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0142] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0143] The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0144] The term "sulfone" is art-recognized and refers to the group -S(O)2-.

[0145] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more backbone carbons. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the replacing atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Substituents can include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0146] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0147] As used herein, the term "thioester" refers to the group -C(O)SR 9 or -SC(O)R 9 refers to, In the formula, R 9 represents a hydrocarbyl.

[0148] The term "thioether" as used herein is equivalent to an ether where the oxygen has been replaced with a sulfur.

[0149] The term "urea" is art-recognized and has the general formula [ka] It can be expressed by In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0150] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0151] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, this term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0152] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable or compatible with the treatment of a patient.

[0153] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to any non-toxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids that form suitable salts include hydrochloride, hydrobromide, sulfate, and phosphate salts, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either mono- or di-acid salts can be formed, and such salts can exist in either hydrated, solvated, or substantially anhydrous form. In general, acid addition salts of compounds of Formula I are soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points compared to their free base forms. The selection of an appropriate salt is known to those skilled in the art. Other non-pharmaceutically acceptable salts, such as oxalates, may be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0154] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acidic compound represented by Formula I or any of its intermediates. Exemplary inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of an appropriate salt is known to those skilled in the art.

[0155] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may exist in the R or S configuration, and the R and S designations are used according to the rules set forth in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, including enantiomeric and diastereoisomeric forms, of compounds, salts, prodrugs, or mixtures thereof, including all possible mixtures of stereoisomers. See, for example, WO01 / 062726.

[0156] Furthermore, certain compounds containing alkenyl groups can exist as Z (Zusammen) or E (Entgegen) isomers. In each case, the present disclosure includes both mixtures and the separate individual isomers.

[0157] Some compounds may also exist in tautomeric forms, and such forms, although not explicitly shown in the formulae given herein, are intended to be included within the scope of the present disclosure.

[0158] A "prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized in the host after administration, e.g., hydrolyzed or oxidized, to form a compound of the present disclosure (e.g., a compound of Formula I). Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on the functional portion of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs that use esters or phosphoramidates as the biologically labile or cleavable (protecting) group are disclosed in U.S. Pat. Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0159] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is useful in formulating a drug for medicinal or therapeutic use.

[0160] As used herein, the terms "logarithm of solubility," "LogS," or "logS" are used in the art to quantify the water solubility of a compound. A compound's water solubility significantly affects its absorption and distribution characteristics. Poor solubility often results in poor absorption. The LogS value is the unit stripped logarithm (decimal number) of solubility measured in mol / liter. [Example]

[0161] The invention will now be generally described, which will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the disclosure.

[0162] Example 1: Exemplary synthesis of compounds of the disclosure Derivatives of formula (I) with R1 modification when R2, R3, and R4 are H and R5 is morpholine The synthesis of compounds 5-27 is shown in Scheme 1, which includes the following process steps: i) The appropriate acid derivatives were esterified by refluxing in EtOH (ethanol) in the presence of SOCl2 (thionyl chloride) to give compounds 1a-z. ii) Solutions of compounds 1a-z in toluene were refluxed with acetonitrile in the presence of NaH (sodium hydride; 60% dispersion in mineral oil) to give β-ketonitrile derivatives 2a-z. iii) Compounds 2a-z were reacted with H2N.OH HCl (hydroxylamine hydrochloride) in aqueous NaOH (sodium hydroxide) at 100°C to give isoxazol-5-amine analogs 3a-z. iv) Compounds 3a-z were reacted with 2,4-dichloropyrimidine or 4-chloro-2-(methylthio)pyrimidine (compound 28 only) in t-BuOH (tertiary butanol) at room temperature in the presence of t-BuOK (potassium tertiary butoxide) to give compounds 4a-z and compound 28. v) Compounds 4a-z in n-BuOH (butanol) were refluxed with morpholine to produce compounds 5-27. [ka] Scheme 1. Synthesis of Compounds 5–28. Reagents and Conditions: (A) SOCl2, EtOH, reflux, 3 h, (B) NaH, MeCN, toluene, reflux, 2 h, (C) H2N.OH HCl, NaOH, HO, reflux, 4 h, (D) t-BuOK, 2,4-dichloropyrimidine, t-BuOH, room temperature, 24 h, (E) t-BuOK, 4-chloro-2-(methylthio)pyrimidine, t-BuOH, room temperature, 24 h, (F) morpholine, n-BuOH, reflux, 5 h.

[0163] The synthesis of compound 30 is shown in Scheme 2 and includes the following process steps: i) Demethylation of compound 4a was carried out in the presence of BBr3 (boron tribromide) in DCM (dichloromethane) at 0° C. to give compound 29. ii) Compound 29 in n-BuOH was refluxed with morpholine to give compound 30.

[0164] The synthesis of compound 32 is shown in Scheme 2 and includes the following process steps: i) Compound 31 was produced by reaction of compound 29 with benzyl alcohol in the presence of DIAD (diisopropyl azodicarboxylate) and PPh3 (triphenylphosphine) in dry THF (tetrahydrofuran) at 0°C. ii) Compound 31 in n-BuOH was refluxed with morpholine to give compound 32. [ka] Scheme 2. Synthesis of compounds 30 and 32. Reagents and conditions: (A) BBr3, DCM, 0 °C, 24 h, (B) benzyl alcohol, DIAD, PPh3, THF, 0 °C, 24 h, (C) morpholine, n-BuOH, reflux, 5 h.

[0165] Derivatives with R5 modification where R1 is 4-methoxyphenyl and R2, R3 and R4 are H in formula (I) The synthesis of derivatives bearing R5 modifications utilized the synthetic procedures outlined in Scheme 3. Thus, compound 4a or compound 28 was used as a starting intermediate and then treated with various amines, including but not limited to, morpholine, thiomorpholine, piperazine, piperidine, and pyrrolidine as secondary amine derivatives, or aminomorpholine, aminopiperidine, and aminopyran derivatives as primary amines, to afford the final compounds (33–54) via Methods A, B, or C. When the amine derivative was in the form of an HCl salt, Method B was used (as in compounds 34–39). When the amine derivative was a primary amine, Method C was used (as in compounds 52–54).

[0166] The synthesis of compounds 33-54 is shown in Scheme 3, which includes the following process steps: i) Compound 4a and the appropriate amine derivative, not in the HCl (hydrogen chloride) salt form, were refluxed in BuOH to give compounds 33, 40-51 (Method A). In the case of compound 49, N-Boc protected piperazine was used, followed by hydrolysis of the protecting group with TFA (trifluoroacetic acid) in DCM to give the final compound 49. Compound 50 was also hydrolyzed in THF:H2O at reflux in the presence of LiOH.H2O (lithium hydroxide monohydrate) to give compound 51. ii) If the amine derivative was in the form of a salt, the appropriate HCl salt of the amine was dissolved in BuOH in the presence of DIPEA (N,N-diisopropylethylamine) to give the free amine. iii) This was then reacted with compound 4a under reflux to give final compounds 34-39 (Method B). iv) When the amine derivative is a primary amine, compound 28 was used as the starting material and first treated with m-CPBA (metachloroperbenzoic acid) in DCM, and then the resulting sulfone intermediate was reacted with the corresponding amine derivative to give the final compounds 52-54 (Method C). [ka] Scheme 3. Synthesis of Compounds 33-54. Reagents and Conditions: (A) appropriate amine derivative, n-BuOH, reflux, 5 hours; (B) appropriate amine derivative, DIPEA, n-BuOH, reflux, 5 hours; (C) i) m-CPBA, DCM, 0°C, 2 hours; ii) appropriate amine derivative, n-BuOH, reflux, 5 hours.

[0167] Derivatives having modifications to the pyrimidine ring (X, R3, R4 substitution) when R1 is 4-phenylmethoxy, R2 is H, and R5 is morpholine or 4-fluoropiperidine in formula (I) For derivatives bearing pyrimidine ring modifications, the synthetic procedure outlined in Scheme 4 was utilized. Compound 3a was used as the starting material and underwent nucleophilic aromatic substitution reactions with various pyrimidine derivatives, such as 2,4,5-trichloropyrimidine, 2,4-dichloro-5-fluoropyrimidine, 2,4-dichloro-6-methylpyrimidine, or 2,4-dichloropyridine, to give final compounds 59–62.

[0168] The synthesis of compounds 59-62 is shown in Scheme 4, which includes the following process steps: i) Compound 3a was reacted with an appropriate pyrimidine derivative in t-BuOH in the presence of t-BuOK at room temperature to give compounds 55-57 (Method A). Compound 3a was reacted with 2,4-dichloropyridine in DMA (N,N-dimethylacetamide) in the presence of NaH at room temperature to give compound 58 (Method B). ii) Compounds 55-57 were reacted with 4-fluoropiperidine HCl in n-BuOH in the presence of DIPEA to give compounds 59-61 (Method C). iii) Compound 58 was treated with morpholine in dioxane in the presence of Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthene), CsCO (cesium carbonate) and Pd(OAc) (palladium(II) acetate) in a microwave oven at 100°C for 30 minutes to give compound 62 (Method D). [ka] Scheme 4. Synthesis of Compounds 59–62. Reagents and Conditions: (A) appropriate pyrimidine derivative, t-BuOK, t-BuOH, room temperature, 24 hours; (B) 2,4-dichloropyridine, NaH, DMA, room temperature, 24 hours; (C) 4-fluoropiperidine HCl, DIPEA, n-BuOH, reflux, 5 hours; (D) morpholine, CsCO, xantphos, Pd(OAc), dioxane, 100°C, 30 minutes, microwave.

[0169] "N"-bridged methylation (R2 substitution) where R1 is 4-methoxyphenyl, R3 and R4 are H, and R5 is morpholine The synthesis of compound 63 utilized the synthetic procedure outlined in Scheme 5. Compound 5 was used as the starting material and was alkylated to give the N-methylated derivative 63.

[0170] The synthesis of compound 63 is shown in Scheme 5, which includes the process steps. i) Compound 5 was dissolved in DMF (N,N-dimethylformamide) and reacted with CH3I (methyl iodide) in the presence of Cs2CO3 at room temperature to give compound 63. [ka] Scheme 5. Synthesis of Compound 63. Reagents and Conditions: (A) CH3I, Cs2CO3, DMF, room temperature, 2 hours.

[0171] Synthesis of compounds having different combinations of R1 and R5 in formula (I) when R2, R3 and R4 are H According to the results obtained from cytotoxicity studies, R1 and R2 of some compounds with potent activity were combined with new molecules to obtain new compounds. The synthesis of the hybrid compounds utilized the synthetic procedure outlined in Scheme 6.

[0172] The synthesis of compounds 64-67 is shown in Scheme 6, which includes the following process steps: i) Compound 4r or 4s and the appropriate amine derivative in BuOH were refluxed to give the final compounds. If the amine derivative was in the form of a salt, the appropriate HCl salt of the amine was dissolved in BuOH in the presence of DIPEA (N,N-diisopropylethylamine) to give the free amine, which was reacted with compound 4r or 4s under reflux to give final compounds 64-66 (Method A). ii) Compound 4s was treated with the amine derivative in n-BuOH to give the final compound 67, if the amine derivative was not in the form of the HCl salt (Method B). [ka] Scheme 6. Synthesis of Compounds 64-67. Reagents and Conditions: (A) appropriate amine derivative, DIPEA, n-BuOH, reflux, 5 hours, (B) appropriate amine derivative, n-BuOH, reflux, 5 hours.

[0173] Embodiments of the present disclosure include chemical structures of the parent intermediate compounds that are reacted with the amine derivatives described herein to synthesize compounds of general formula (I), and can be selected from the compounds listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0174] Embodiments of the present disclosure include the following features listed in Table 2: 1 H NMR / 13 Includes compounds with C NMR characteristics. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0175] Example 2: Exemplary Biological Results The ability of compounds of the present disclosure to treat TACC3-mediated cancer, or related symptoms or complications thereof, was determined using the following procedure. Compounds of the present disclosure were tested for their activity in inducing cell death using the JIMT-1 cell line, a cell line with high levels of TACC3. The concentration of each compound required for 50% maximal inhibition of cell proliferation was calculated using GraphPad Prism (GraphPad Software). Table 3 below lists the compounds in order of the strength of their inhibitory effect on cell growth. Compounds 5, 9, 13, 14, 20-24, 26, 33, 34, 37-40, 42-45, 59, 60, and 63-67 of the present disclosure were determined to have significant inhibitory effects on cell growth. [Table 3-1] [Table 3-2]

[0176] These compounds of the present disclosure exhibited cell growth inhibitory effects, and therefore, when used in pharmaceutical compositions as anticancer agents, are expected to inhibit tumor growth. Therefore, the present inventors selected compound 5 for further detailed analysis of its activity on cell growth, particularly its effect on TACC3 and cell division, and its in vivo inhibitory effect on tumor growth in relevant animal models.

[0177] To determine whether compound 5 targets TACC3, we performed a cellular thermal shift assay (CETSA) based on drug target stabilization by temperature elevation (Martinez Molina et al., 2013). To this end, JIMT-1 cells were incubated with vehicle, compound 5, or SPL-B (as a positive control) for 6 hours, and then cell lysates were collected. Treatment of JIMT-1 breast cancer cells with compound 5 significantly stabilized cellular TACC3 at elevated temperatures, indicating that compound 5 can specifically interact with TACC3 in JIMT-1 cells (Figure 4A). The thermal melting curves of the protein also show a thermal shift between the band intensities after treatment with vehicle and compound 5. This interaction was also confirmed by isothermal titration calorimetry (ITC). Titration of TACC3 with compound 5 was performed and monitored by recording the calorimetric and thermal changes. As shown in Figure 4B, the thermodynamic parameters (K ) of compound 5 binding to TACC3 at 25 °C were calculated. d The ΔH: 1.5 nM; ΔH: 4.929E7 cal / mol; ΔS: 1.65E5 cal / mol / deg, N: 0.704) indicate an interaction between these two molecules. The binding of compound 5 to TACC3 was finally confirmed using the Drug Affinity-Responsive Target Stability (DARTS) method. DARTS is a label-free strategy for identifying potential direct protein targets of small molecules, based on the stabilization and protection of the target protein from proteolysis immediately after binding to the small molecule (Lomenick, Jung et al. 2011; Pai, Lomenick et al. 2015). As shown in Figure 6C, TACC3 protein was stabilized upon incubation with compound 5 in the presence of pronase (Figure 4D).

[0178] We then compared the relative effects of the TACC3 inhibitor compound 5 with available TACC3 inhibitors KHS101 and SPL-B on cell viability. JIMT-1, MDA-MB-436, MDA-MB-157, and BT-474 T-DM1R cell lines were tested for their response to these three drugs. Compound 5 had a significantly lower IC than the two available TACC3 inhibitors in all cell lines tested. 50The significant decrease in cell viability of various cancer cell lines treated with compound 5 compared with KHS101 and SPL-B was also confirmed by colony formation assays using JIMT-1 cells. As a result, the average colony number of JIMT-1 cells treated with compound 5 was significantly lower than that of the other two inhibitors at the same dose (Figure 5B). Therefore, the lower cell viability detected by the SRB assay and the lower colony number of JIMT-1 cells indicate that compound 5 is more effective and potent in vitro than the other two TACC3 inhibitors, KHS101 and SPL-B. The induction of mitotic arrest, apoptosis, and DNA damage processes, as in the case of siTACC3 treatment in breast cancer cell lines, was further examined immediately after treatment with compound 5, SPL-B, and KHS101. The analytical results using compound 5 were highly consistent with the experimental results regarding the suppression of TACC3 expression using siRNA, further suggesting that compound 5 targets TACC3. As a result, siTACC3-induced mitotic arrest, DNA damage, and apoptosis could be reproduced by compound 5 in a dose-dependent manner (Figure 5C). On the other hand, similar induction levels of these markers by SPL-B and KHS101 treatment were observed at significantly higher doses compared to cells treated with compound 5. Although a higher KHS101 dose than used by others (Campo & Breuer, 2018) was included, induction of these markers was only observed at very high doses. Clearly, these results are consistent with the cell viability data and IC obtained through treatment with the three inhibitors. 50 This is consistent with the values shown in Figure 5A. Furthermore, treatment of JIMT-1 cells with compound 5 for 72 hours induced a significant increase in the percentage of apoptotic cells (from 4.1% to 45.6%), as assessed by Annexin V / PI staining (Figure 7D). These results further confirm 1) the high potency of compound 5 at working doses in the nM range and 2) its specificity with respect to the similar molecular changes obtained by TACC3 downregulation using siRNA.

[0179] Next, we examined the potential disruption of mitotic spindles as a result of TACC3 inhibition. This has previously been demonstrated to cause severe spindle defects (Schneider, Essmann et al. 2007). Inhibition of TACC3 with compound 5 resulted in the formation of abnormal spindle structures in a dose-dependent manner (Figure 5E). The most striking phenotype observed upon TACC3 inhibition with compound 5 was the formation of multipolar spindles characterized by improperly aligned chromosomes at the metaphase plate (73.4% frequency at the highest dose) in JIMT-1 cells (Figure 5F). The presence of spindle defects activates the spindle assembly checkpoint (SAC), further arresting mitosis and providing the necessary time for spindle defect repair (Musacchio and Salmon 2007). To demonstrate SAC activation upon TACC3 inhibition, we performed immunofluorescence staining for BuBR1, a marker of SAC activation (Chen 2002). In JIMT-1 cells treated with compound 5, we observed increased localization of BuBR1 to chromosomes immediately after compound 5 treatment (Figure 5G). To further examine the contribution of active SAC signaling to compound 5-induced mitotic arrest, DNA damage, and cell death, we inhibited the SAC kinase Mps1 using a specific inhibitor (TC Mps1 (Choi, Min et al. 2017)) in compound 5-treated cells and analyzed the expression of related markers. As shown in Figure 5H, inhibiting Mps1 kinase led to a significant reduction in mitotic arrest, apoptosis, and DNA damage, suggesting that compound 5 functions by activating SAC during the induction of severe spindle defects, which may further lead to prolonged mitosis, apoptotic cell death, and DNA damage.

[0180] The FGFR3-TACC3 oncogenic fusion protein has been detected in numerous solid tumors and has emerged as an attractive therapeutic target, allowing for selective targeting of fusion-harboring cancers (Costa, Carneiro et al. 2016). To test whether compound 5 treatment could inhibit the growth of fusion cell lines, we utilized two human bladder cancer cell lines, RT112 and RT4, known to harbor the FGFR3-TACC3 fusion protein (Williams, Hurst et al. 2013). Western blot analysis of TACC3 revealed high expression in RT112 cells (Figure 6A), which was further accompanied by a strong response to compound 5, as indicated by a low IC50 in RT112 cells (Figure 6B and C). Despite the relatively low sensitivity to TACC3 inhibition in RT4 cells (likely due to low TACC3 levels), compound 5 obtained the lowest IC50 values in both models compared to SPL-B and KHS101, suggesting that compound 5 may represent a highly relevant therapeutic opportunity for targeting tumors harboring the FGFR3-TACC3 fusion (Figure 6B). Furthermore, compound 5 reduced ERK1 / 2 phosphorylation, a marker of activated FGFR signaling, along with potent mitotic arrest (Figure 6D) at doses at least 10-fold lower than other TACC3 inhibitors, suggesting that compound 5 specifically blocks the function of the FGFR3-TACC3 fusion protein.

[0181] Following these promising results in breast cancer cell lines, compound 5 was also tested in other cancer types. Therefore, compound 5 was screened for antiproliferative activity in the NCI-60 human cell line. Analysis of a five-dose screen revealed that almost all cell lines showed 50% growth inhibition (GI) at less than 1 μM. 50) values, suggesting its potential application in other cancer types (Figure 6E). Notably, the GI50 values of the NCI-60 cell line were found to be positively correlated with the TACC3 dependency score obtained from DepMap.org (McFarland, Ho et al. 2018) (Figure 6F). This indicates that cells with a high dependency on TACC3, i.e., a low TACC3 dependency score, are more sensitive to compound 5. Collectively, these data demonstrate the potent anticancer activity of compound 5 and shed light on its potential applications in various cancer types.

[0182] Next, we tested the specificity of compound 5 for cancer cell lines over normal cells. Therefore, we examined the sensitivity of normal breast epithelial cells, MCF-12A, and several other breast cancer cells to compound 5. Surprisingly, treatment with high doses of compound 5 (5, 10 μM) did not achieve 50% cell growth inhibition (Figure 7A). However, compound 5 inhibited the viability of triple-negative (MDA-MB-231, MDA-MB-436, CAL51, and HCC1143) and HER2-positive (JIMT-1 and HCC1954) breast cancer cell lines at low doses compared with luminal (MCF7, T47D, ZR75, and BT-474) breast cancer cell lines (Figure 7B). In other words, compound 5 specifically targets tumor cells but has no effect on normal breast cells. As shown in Figure 6C, breast cancer cell lines that responded better to compound 5 expressed higher TACC3 levels than MCF-12A, further supporting the cancer-specific high expression of TACC3. This indicates that sensitivity to compound 5 correlates with abnormal TACC3 expression levels in cancer cells. In addition, low TACC3 levels may explain why MCF-12A cells did not respond to any of the TACC3 inhibitors at low doses. Furthermore, a trend toward increased TACC3 expression was observed in TNBC and HER2-positive breast cancers, the two most aggressive subtypes, compared with luminal cell lines, suggesting that TACC3 expression may be associated with cancer invasiveness. To test the transforming potential of TACC3 and the cellular dependence on TACC3 for survival, we performed clonogenic growth assays in MCF-12A cells after overexpressing TACC3. As shown in Figures 7D and 9E, overexpression of TACC3 increased the colony-forming ability of a normal breast cell line, MCF-12A. Importantly, overexpression of TACC3 in MCF-12A cells sensitized them to the cytotoxic effects of compound 5, further demonstrating their dependence on TACC3 and the specificity of compound 5 (Figure 7F). To exclude the possibility that the observed differences between cancer and normal cells were due to differences in the proliferation rates of these cells, the cell doubling time was calculated.As shown in Figure 7G, there was no significant difference between the doubling times of MCF-12A and cancer cell lines, suggesting that responsiveness to compound 5 may not be determined by an increased cell division rate.

[0183] The above results indicated that breast cancer cells expressing high levels of TACC3 were more sensitive in vitro to the novel TACC3 inhibitor, Compound 5. Therefore, the effect of Compound 5 compared with SPL-B on tumor growth of the highly tumorigenic breast cancer cell line JIMT-1 (Barok et al., 2007; Tanner et al., 2004) was tested in immunodeficient mice. To this end, female nude mice were injected with JIMT-1 cells into the mammary fat pad (MFP) and subsequently treated with vehicle or 5 mg / kg (oral) Compound 5 or SPL-B every other day for 30 days. It was concluded that Compound 5 showed a significant reduction in tumor growth compared to SPL-B, without a significant effect on mouse body weight (Figure 8).

[0184] Next, we used JIMT-1 xenografts to test various doses and administration routes of low-dose Compound 5. Compound 5 was administered to mice at a dose of 2 mg / kg (oral or intravenous) or 5 mg / kg (oral) every 2 days for 30 days (Figure 9). The tumor growth rates of all three Compound 5-treated groups were reduced compared to the control group and among these groups (Figure 9A). Figure 9B shows that Compound 5 administration did not adversely affect mouse body weight. Furthermore, to test the effect of higher doses of Compound 5 on tumor growth compared to previous experiments to obtain better antitumor efficacy and test its tolerability, female nude mice were injected with JIMT-1 cells into the mammary fat pad (MFP) and subsequently treated with vehicle or Compound 5 (25 mg / kg, oral) for 23 days. Compound 5 showed a highly significant reduction in tumor growth compared to vehicle-treated mice (Figure 9C). Tumor weight was significantly lower compared to that of the vehicle group (Figure 9D). Importantly, compound 5 was well tolerated, as treatment did not affect mouse body weight (Figure 9E). The effects of TACC3 inhibition were also tested in vivo using an immunocompetent mouse model (Figure 10). EMT6, a rapidly growing and highly aggressive mouse triple-negative breast cancer (TNBC) cell line (Yang, Yang et al. 2017), was injected into the MFP of syngeneic Balb / c mice. Significant tumor growth inhibition (Figure 10A) and a 57.1% lifespan extension (Figure 10B) were observed in compound 5-treated mice compared with vehicle-treated mice. Furthermore, compound 5 was again well tolerated in the syngeneic mouse model (Figure 10C).

[0185] In addition to breast cancer xenograft and syngeneic models, the antitumorigenic potential of compound 5 was tested in a colon cancer animal model (Figure 11). Female nude mice and Balb / c mice were injected with the human colon cancer cell line HCT-116 and the murine colon cancer cell line CT26, respectively, into the flank region of the mice. Mice were orally treated daily with either vehicle or 25–50 mg / kg of compound 5. Similar to the breast cancer model, compound 5 significantly impaired tumor growth (Figure 11A) and was well tolerated in both models (Figure 11B).

[0186] Next, to examine the effect of TACC3 inhibition on metastatic growth, immunocompetent female mice were intravenously injected with 4T1-Luc2 (luciferase-labeled), a highly malignant mouse mammary tumor cell line. The objective here was to determine the effect of compound 5 on metastatic growth and lung colonization using a clinically relevant metastasis model. When mice developed metastatic lesions in the lungs, they were treated daily with either vehicle or 50 mg / kg of compound 5. Metastatic growth was monitored with an in vivo imaging system (IVIS) by measuring bioluminescence. Compound 5 was found to impair metastatic growth compared to vehicle (Figure 12A) and significantly improved overall survival of mice (Figure 12B).

[0187] Finally, to determine the maximum tolerated dose, female nude mice were administered 100 mg / kg of compound 5 daily for 7 days, and their body weights were recorded (Figure 13A). Compound 5 did not affect body weight or produce observable toxicity at this high dose. In another experiment, mice were administered 500 mg / kg of compound 5 once and monitored for 1–3 days. Mice administered compound 5 lost 10% of their body weight 24 hours after drug treatment, but their general condition remained stable (Figure 13B). Organs were collected at the end of the experiment, and no organ toxicity was observed, indicating that compound 5 was well tolerated (Figure 13C).

[0188] Based on the overall profile, the physicochemical properties and metabolic stability of compound 5 were evaluated. Compound 5 exhibited a log D 7.4 It has a moderate lipophilicity of 2.3, exhibits low solubility and stability in both human and mouse liver microsomes, and exhibits relatively high plasma protein binding (unbound fraction 1.13%), but exhibits good Caco-2 permeability with a low efflux ratio (AB = 190 × 10 -6 nm / sec, ratio = <2.0) (Table 1). Accordingly, to evaluate potential drug-drug interactions, we also characterized cytochrome P450 inhibition by compound 5 in human liver microsomes (Lin & Lu, 1998) (Table 1). Thus, compound 5 inhibited CYP2C9 (IC50 = 2.63 μM) and CYP3A (testosterone as a substrate; IC 50 = 8.59 μM, but was a moderate inhibitor of CYP2CD6 (IC 50 = 18.46 μM) and CYP3A (IC 50 = midazolam as substrate; 30.04 μM) is a weak or not inhibitor, indicating that compound 5 has low activity at the P450 cytochromes tested.

[0189] Biological Materials and Methods Cell culture and reagents Human breast cancer cell lines MDA-MB-436, MDA-MB-157, MDA-MB-231, BT-474, MCF-7, ZR-75-1, and T-47D, mouse breast cancer cell lines EMT6 and 4T1, human bladder cancer cell lines RT112 and RT4, mouse colon cancer cell line CT-26, and normal human breast epithelial cell line MCF-12A were purchased from ATCC. The T-DM1-resistant HER2-positive breast cancer cell line BT-474T-DM1R was developed and characterized as previously described (Saatci et al., 2018). The human colon cancer cell line HCT-116 was a kind gift from Serkan Goktuna, Bilkent. JIMT-1, HCC1954, CAL51, and HCC1143 were provided by Ali Osmay Gure, Bilkent University. Cells were cultured in Dulbecco's modified Eagle's medium (Lonza, NJ, USA) supplemented with 10% fetal bovine serum (FBS, Lonza), 1% non-essential amino acids (NEAA), 2 mM L-glutamine (Sigma-Aldrich, MO, USA), and 50 U / ml penicillin / streptomycin (P / S). BT-474WT and T-DM1R cells were also supplemented with 0.1% insulin (Sigma-Aldrich). MCF-12A cells were further supplemented with 20 ng / ml epidermal growth factor (EGF) and 500 ng / ml hydrocortisone. T-47D and MCF-7 cells were cultured in phenol red-free DMEM (Gibco, Carlsbad, CA) containing 10% FBS, 1% NEAA, 1% L-glutamine, 50 U / ml P / S, and 0.1% insulin. EMT6, 4T1, CT-26, and RT112 cells were maintained in RPMI-1640 (Biowest, Nuaille, France), whereas RT4 and HCT-116 cells were cultured in McCoy's 5A (modified) (Gibco) medium supplemented with FBS, NEAA, L-glutamine, and P / S. All cell lines were routinely tested using the MycoAlert Mycoplasma Detection Kit (Lonza).

[0190] Cellular thermal shift assay (CETSA) To analyze the interaction between compound 5(5) and TACC3 in intact cells, CETSA was performed as previously described (Martinez Molina et al., 2013) with minor modifications. Briefly, JIMT-1 cells were incubated with vehicle, 1 μM compound 5(5), or SPL-B for 6 h. After treatment, the cell pellet was resuspended in Tris-buffered saline (TBS) containing protease and phosphatase inhibitors. The cell suspension was divided into six PCR tubes and heated to 45, 46, 47, 48, 49, and 50 °C for 5 min. Subsequently, cells were lysed by three freeze-thaw cycles using liquid nitrogen. Soluble proteins were collected by centrifugation at 20,000 g for 20 min at 4 °C and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis.

[0191] Isothermal Titration Calorimeter (ITC) Purified TACC3 recombinant protein (TP310754; Origene, MD, USA) and compound 5 (5) were prepared in 25 mM Tris.HCl, pH 7.3, 100 mM glycine, and 10% glycerol. Compound 5 (5) was loaded into the sample cell and titrated with TACC3 protein (at a 10-fold higher concentration in the syringe) in duplicate experiments. Titrations were performed at 25 °C using a Microcal200 instrument (GE Healthcare, Austria). Ten injections were performed per titration, spaced 6 min apart. The reference output was set at 2 μcal / s, and the sample cell was continuously stirred at 500 rpm. To evaluate the binding efficiency between the drug and protein, background data obtained with protein injected in buffer alone were subtracted from the experimental isotherm. Data were analyzed using Origin7 Software provided with the ITC200, and binding parameters such as the binding constant (Ka), number of binding sites (N), and enthalpy (ΔH) were calculated.

[0192] Drug Affinity Responsive Target Stability (DARTS) DARTS was performed as previously described (26). Briefly, JIMT-1 cells were grown to 70–80% confluence and lysed in RIPA lysis buffer without SDS and sodium deoxycholate. The concentration of the protein extract was determined using a BCA Protein Assay Reagent Kit (Thermo Scientific, IL, USA) and diluted to 4 μg / μl in lysis buffer. The cell lysate was divided into 99 μl aliquots and mixed separately with 1 μl of 100× concentrated solutions of 10 μM compound 5 and SPL-B. The cell lysate and drug mixtures were incubated on a shaker at room temperature for 20 min to allow binding. 20 μl of each sample was then mixed with 2 μl of 8 ng / μl pronase solution (Sigma-Aldrich) or buffer alone (undigested) and incubated at room temperature for 12 min. Protein digestion was stopped by adding 2 μl of 20× protease inhibitor (Roche, Switzerland) and incubating on ice for 10 min. The lysates were then mixed with 8 μl of 4×SDS supplement and heated for 10 min at 70° C. SDS-PAGE was performed using anti-TACC3 and anti-CDK4 antibodies as negative controls.

[0193] Inhibitor treatment, cell viability assay, and Annexin V / PI staining KHS101 (Sigma Aldrich) and SPL-B (Axon MedChem, VA, USA) were dissolved in 100% DMSO to obtain a stock concentration of 50 mM. Newly synthesized molecules were dissolved in 100% DMSO to obtain a stock concentration of 10 mM. For cell viability assays, JIMT-1 (3 × 10 3 cells / well), BT-474WT and T-DM1R (6 × 10 3 ), MDA-MB-436(4×10 3 ), MDA-MB-157(3×10 3 ), HCC1954(5×10 3 ), CAL51(5×10 3 ), HCC1143(4.5×10 3 ), MDA-MB-231(4.5×10 3 ), MCF-7(7×103 ), T-47D(6×10 3 ), RT112(6×10 3 ), RT4(6×10 3 ), and MCF-12A (5 × 10 3 ) cells were seeded in 96-well plates and treated with various concentrations of inhibitors 24 hours after cell seeding. Cell viability was measured 72 hours after treatment using sulforhodamine B (SRB, Sigma-Aldrich) assay as recommended by the manufacturer. Western blotting showed that different concentrations of KHS101, SPL-B, or compound 5 were inhibited by JIMT-1 (1.5 × 10 5 ) and RT112(2×10 5 ) cells were treated for 24 hours. Annexin V / PI staining (Biolegend, USA) was performed according to the manufacturer's instructions using JIMT-1 cells treated with 500 nM of Compound 5 for 48 hours.

[0194] Transient transfection with siRNA and overexpression vectors In the cell viability assay, JIMT-1 (3 × 10 3 cells / well), BT-474 T-DM1R (6 × 10 3 ), MDA-MB-436(4×10 3 ) and MDA-MB-157 (3 × 10 3 ) cells were seeded into 96-well plates in P / S-free growth medium. 24 hours after seeding, two different siRNAs targeting TACC3 (Dharmacon, CO, USA) were transfected into the cells at a final concentration of 20 nM (siTACC3#1: D-004155-03 and siTACC3#2: D-004155-02) using Lipofectamine 2000™ (Invitrogen, CA) transfection reagent as previously described (Mutlu et al., 2016). 72 hours after transfection, cell viability was measured using an SRB assay. To assess the level of TACC3 knockdown immediately after siRNA transfection, JIMT-1 (1.5 × 10 5 ), BT-474 T-DM1R (2 x 10 5), MDA-MB-436(1.5×10 5 ) and MDA-MB-157 (1.5 × 10 5 ) cells were transfected with two different TACC3 siRNAs for 48 hours. Knockdown efficiency at the mRNA and protein levels was analyzed by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively. For transient TACC3 overexpression, MCF-12A cells were transfected with 250 ng of empty or TACC3 vector (OHu21751; Genscript, NJ, USA) for 48 hours.

[0195] Colony formation assay For monolayer culture, a single cell suspension of JIMT-1 cells (3 × 10 3 10 cells / well) were plated in a 12-well plate. After 6 hours of incubation, the cells were treated with different doses of compound 5 (5), SPL-B, and KHS101. To test the colony-forming ability of MCF-12A cells during TACC3 overexpression, MCF-12A cells (2 × 10 5 ) were seeded into 6-well plates and TACC3 transfection was performed the following day. 48 hours after transfection, cells were counted and 1 × 10 3 Cells were plated per well in 12-well plates. In both experimental setups, the medium was refreshed every 4 days and the cells were incubated for 12 days. Cells were then fixed with 2% paraformaldehyde for 15 minutes and stained with 1% crystal violet (Merck, Darmstadt, Germany) for 15 minutes at room temperature. Viable colonies (consisting of at least 50 cells) were counted using ImageJ software (NIH).

[0196] Doubling time assessment To assess doubling time, normal breast epithelial cell line MCF-12A and breast cancer cell lines were plated in 6-well plates (3 × 10 4 Cells were harvested by trypsinization and counted every 24 hours for one week. Growth curves of these cells were plotted as the number of cells / cm versus the number of days after seeding.2 The doubling time was calculated using the following formula:

number

[0197] Immunofluorescence Immunofluorescence staining of JIMT-1 cells was performed as previously described (Cizmecioglu, Arnold et al. 2010). Essentially, 1.5 × 10 cells were plated onto glass coverslips in 6-well plates. 5 JIMT-1 cells were seeded per well. The next day, cells were treated with either vehicle, 200 nM, or 500 nM Compound 5 for 12 hours. Next, cells were fixed with ice-cold methanol at -20°C for 10 minutes. Next, cells were blocked with 3% BSA in PBS solution at room temperature for 1 hour and then incubated with primary and secondary antibodies at room temperature for 1 hour. Cells were counterstained with DAPI (0.01 μg / ml) for 5 minutes. Finally, cover slides were mounted using ImmunoHistomount (Santa Cruz). Images were taken with an upright fluorescence microscope equipped with a DIC prism (upright).

[0198] NCI-60 Cancer Cell Line Panel Screening Compound 5 (5) was submitted to the National Cancer Institute for screening in the NCI-60 panel of human tumor cell lines, consisting of 60 human cancer cell lines derived from nine different cancer types (NCI number S807620). Compound 5 (5) was initially tested in a single-dose screen at a concentration of 10 μM in each cell line. After obtaining the results of the single-dose assay, a Development Therapeutics Program (DTP) analysis was conducted, and compound 5 meeting the predetermined threshold inhibition criteria was selected for the NCI full panel five-dose assay. Compound 5 was then tested twice in a five-dose NCI-60 screen at doses ranging from 10 nM to 100 μM, demonstrating its GI activity across the 60 cell lines. 50(50% growth inhibition) value, TGI (total growth inhibition) value, and LC 50 The lethal concentration (lethal dose concentration inducing 50% cell death) value was determined. The detailed screening method can be accessed at https: / / dtp.cancer.gov / discovery_development / nci-60 / methodology.htm webpage. Briefly, 24 hours after seeding cells in 96-well plates, cells were treated with compounds over a 5 logM concentration range for 2 days. Cytotoxicity was assessed using the SRB assay. Data shown in the figures are the average of both experiments.

[0199] Mouse experiments Six- to eight-week-old female athymic nude or Balb / c mice were housed in a temperature-controlled environment with a 12-hour light / 12-hour dark cycle. This study was conducted in accordance with the Institutional Animal Care and Use Committee of Bilkent University and in accordance with institutional guidelines and principles for animal research. For in vivo breast cancer tumor growth in nude mice, 4 × 10 6 JIMT-1 cells were prepared in 150 μl of 1:1 DMEM and Trigel (Corning, NY, USA), v / v, and injected into the mammary fat pad (MFP) of female nude mice. Mouse weight and tumor volume were measured daily using calipers. Tumor volume was calculated as length × width. 2 The calculation was made as ×0.5. The tumor volume was approximately 90 to 100 mm 3Once the xenografts reached a mass index (MGR), they were randomized and divided into groups. Animals were treated with vehicle (0.05% HPMC (hydroxypropyl methylcellulose) and 2% Tween-80 in ddHO) or compound 5 (2 or 5 mg / kg, orally or intravenously every other day (qod.)). In a separate experiment, animals were also tested with a higher dose of compound 5 (25 mg / kg). The effect of compound 5 (5 mg / kg, qod., po.) on tumor growth was also compared with SPL-B (5 mg / kg, every other day, orally) using JIMT-1 cells. Mice were sacrificed 20–30 days after the start of treatment, and tumors were collected and stored for subsequent analysis. To test the effect of compound 5 in an immunocompetent female Balb / c mouse model, a highly aggressive murine breast cancer cell line was used. 1 × 10 6 EMT-6 cells were prepared in PBS and injected into the MFP of mice. 3 Once the mice reached 1500 mm, they were randomized into two groups and administered either vehicle or 25 mg / kg of Compound 5 orally daily. 3 was calculated using a predefined tumor volume cutoff of .

[0200] Additionally, compound 5 was tested immediately after the induction of lung metastases. Highly aggressive and metastatic 4T1-Luc2 (luciferase-labeled) cells were cultured in PBS at 1 × 10 6 The cells were prepared as 1000 cells / mouse and injected intravenously into Balb / c female mice. Metastatic development was monitored using an in vivo imaging system (IVIS), and bioluminescence was periodically quantified. Once lung metastases developed, mice were randomized into two groups and orally administered either vehicle or 50 mg / kg of Compound 5 daily. Survival was calculated when mice died.

[0201] In addition to the breast cancer model, the antitumorigenic effect of compound 5 was tested in both immunodeficient nude mice (HCT-116) and syngeneic immunocompetent Balb / c mice (CT-26) colon cancer animal models. 6 HCT-116 and 1 × 10 6Cells were prepared in either Matrigel:PBS or PBS, respectively, and injected subcutaneously into the flank region of mice. 3 Once the mice reached maturity, they were randomized into two groups and orally administered vehicle or 25–50 mg / kg of Compound 5 daily for 20–25 days. Mice were weighed periodically.

[0202] For toxicity analysis, nude female mice were dosed with 100 mg / kg of Compound 5 for 7 days or orally administered a single dose of vehicle or 500 mg / kg of Compound 5. Mice were weighed periodically, and organs were harvested to assess potential toxicity.

[0203] Bioinformatics analysis TACC3 differential plots between different tumor and normal tissues were generated using The Cancer Genome Atlas (TCGA) patient data (Akbani et al., 2014), and the data were downloaded from http: / / firebrowse.org / . For survival analysis and prognostic significance of TACC3, various independent publicly available cancer datasets were used. One was the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset (Curtis, Shah et al., 2012). TACC3 expression levels in the METABRIC Discovery and Validation set were used to estimate overall survival in breast cancer patients. Patients in the 25th and 75th quartiles of TACC3 levels were used, defined as low TACC3 and high TACC3, respectively. The association between TACC3 expression and overall survival in gastric cancer patients was analyzed using the Kaplan-Meier plotter database, which contains information on overall survival in 876 gastric cancer patients (Szasz et al., 2016). Disease-free survival data for 122 prostate cancer patients were obtained from The Cancer Genome Atlas (TCGA) database using https: / / www.cancer.gov / tcga. Patients were separated based on the 25th and 75th percentiles. Finally, recurrence-free survival data for lung cancer patients was obtained from the GSE31210 dataset. Similarly, the 25th and 75th percentiles of patients were used for this analysis (Okayama, Kohno et al. 2012). Gene set enrichment analysis (GSEA) of mitosis- and DNA repair-related gene sets available on the Broad Institute website (http: / / software.broadinstitute.org / gsea / index.jsp) was performed using breast cancer. In the METABRIC Validation dataset (n = 995), patients were separated into two groups (high vs. low) based on TACC3 expression levels.For the analysis of TACC3 dependency in the NCI-60 cell line, we used combined dependency data from the Broad Institute, Novartis, and Marcotte et al. (13) RNAi screens available at https: / / depmap.org / portal / . Multivariate Cox regression analysis was performed using the METABRIC dataset in SPSS software. TACC3 levels, tumor grade, tumor stage, ER, PR, and HER2 status were selected as covariates. TACC3 expression was separated based on the 25th percentile.

[0204] statistical analysis Data were analyzed using GraphPad Prism software (GraphPad Software, Inc.) and are presented as the mean ± standard deviation from three independent experiments unless otherwise specified. Statistical significance of two-group comparisons was determined by a two-tailed Student's t-test. One-way analysis of variance was used to compare doubling time curves of different cell lines. Multiple t-tests were used to determine pairwise significance between treatment groups for tumor volume of EMT6 xenografts. p and corrected p(q) values of less than 0.05 were considered statistically significant. Kaplan-Meier survival curve analysis was performed using the log-rank (Mantel-Cox) test.

[0205] Example 3: Further exemplary biological results [Table 4-1] [Table 4-2]

[0206] Incorporation by Reference All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including definitions herein, will control.

[0207] equivalent While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. The present invention includes, for example, the following aspects. [Section 1] A compound of formula (I), [ka] or a pharmaceutically acceptable salt thereof, wherein: X1 is N or CR6; X2 is N or CR3; R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; A compound wherein R5 is heterocyclyl, alkyl, or amino. [Section 2] The compound is [ka] The compound according to item 1, which is not [Section 3] The compound according to item 1 or 2, wherein R1 is aryl (eg, phenyl). [Section 4] The compound according to paragraph 1 or 2, wherein R1 is heteroaryl (eg, benzofuran, or pyrimidinyl). [Section 5] The compound of paragraph 1 or 2, wherein R1 is heterocyclyl (eg, benzodioxole or dihydrobenzofuran). [Section 6] The compound according to any one of items 1 to 5, wherein R1 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl, or sulfonamido. [Section 7] The compound according to any one of items 1 to 5, wherein R1 is substituted with alkyl (e.g., methyl, ethyl, isopropyl, fluoroethyl, or trifluoromethyl), alkyloxy (e.g., methoxy, trifluoromethyloxy, difluoromethyloxy, ethoxy, or propyloxy), alkylthio (e.g., methylthio), aralkyloxy (e.g., benzyloxy), hydroxyl, halo (e.g., fluoro or chloro), or amino (e.g., dimethylaminoalkyl). [Section 8] 8. The compound according to any one of clauses 1 to 7, wherein R1 is substituted with halo (eg, fluoro). [Section 9] The compound according to any one of items 1 to 8, wherein R1 is substituted with alkyloxy (eg, methoxy). [Section 10] The compound according to any one of items 1 to 9, wherein R1 is substituted with alkyl (eg, methyl, ethyl, or trifluoromethyl). [Section 11] The compound according to any one of items 1 to 7, wherein R1 is substituted with one halo (eg, F). [Section 12] 12. The compound of clause 11, wherein the halo (eg, F) is para to the isoxazole. [Section 13] 12. The compound of clause 11, wherein the halo (eg, F) is ortho to the isoxazole. [Section 14] 12. The compound of clause 11, wherein the halo (eg, F) is meta to the isoxazole. [Section 15] The compound according to any one of items 1 to 7, wherein R1 is substituted with two halo (eg, F). [Section 16] 16. The compound of clause 15, wherein one halo (eg, F) is meta to the isoxazole and one halo (eg, F) is ortho to the isoxazole. [Section 17] Item 8. The compound according to any one of items 1 to 7, wherein R1 is substituted with alkoxy (eg, methoxy). [Section 18] 18. The compound according to clause 17, wherein the alkoxy (eg, methoxy) is para to the isoxazole. [Section 19] 18. The compound according to clause 17, wherein the alkoxy (eg, methoxy) is ortho to the isoxazole. [Section 20] 18. The compound according to clause 17, wherein the alkoxy (eg, methoxy) is meta to the isoxazole. [Section 21] The compound according to any one of items 1 to 7, wherein R1 is substituted with one alkoxy (eg, methoxy) and one halo (eg, F). [Section 22] 22. The compound according to clause 21, wherein the alkoxy (eg, methoxy) is para to the isoxazole and the F is meta to the isoxazole. [Section 23] 22. The compound of clause 21, wherein the alkoxy (eg, methoxy) is para to the isoxazole and the F is ortho to the isoxazole. [Section 24] 22. The compound of clause 21, wherein the halo (eg, F) is para to the isoxazole and the alkoxy (eg, methoxy) is meta to the isoxazole. [Section 25] The compound according to any one of items 1 to 7, wherein R1 is substituted with alkoxy (eg, methoxy) and two halo (eg, F). [Section 26] 26. The compound of clause 25, wherein the alkoxy (eg, methoxy) is para to the isoxazole and both halo (eg, F) are meta to the isoxazole. [Section 27] 26. The compound of clause 25, wherein the alkoxy (e.g., methoxy) is para to the isoxazole, one halo (e.g., F) is meta to the isoxazole, and one halo (e.g., F) is ortho to the isoxazole. [Section 28] 28. The compound according to any one of items 1 to 27, wherein R2 is alkyl (eg, methyl or ethyl). [Section 29] 29. The compound according to any one of clauses 1 to 28, wherein R2 is substituted with amino (eg, dimethylamino or diethylamino), or nitrile. [Section 30] Item 28. The compound according to any one of items 1 to 27, wherein R2 is H. [Section 31] 31. The compound according to any one of items 1 to 30, wherein X1 is N. [Section 32] Item 31. The compound according to any one of items 1 to 30, wherein X1 is CR6. [Section 33] 33. The compound according to item 32, wherein R6 is H. [Section 34] Item 34. The compound according to any one of items 1 to 33, wherein X2 is N. [Section 35] Item 34. The compound according to any one of items 1 to 33, wherein X2 is CR3. [Section 36] 36. The compound of clause 35, wherein R3 is H or halo (eg, fluoro or chloro). [Section 37] Item 37. The compound according to any one of items 1 to 36, wherein R4 is alkyl (eg, methyl). [Section 38] 38. The compound according to any one of items 1 to 37, wherein R5 is heterocyclyl (e.g., azetidinyl, morpholino, pyrrolidinyl, piperazinyl, piperidinyl, oxaazabicyclooctanyl, oxaazabicycloheptnyl, thiomorpholino, thiomorpholino dioxide, hexahydrofuropyrrolyl, or azabicyclohexanyl). [Section 39] Item 39. The compound according to any one of items 1 to 38, wherein R5 is a 6-membered heterocyclyl and the backbone of the cycle contains one nitrogen. [Section 40] Item 39. The compound according to any one of items 1 to 38, wherein R5 is a 6-membered heterocyclyl and the backbone of the cycle contains one nitrogen and one oxygen. [Section 41] Item 39. The compound according to any one of items 1 to 38, wherein R5 is a 7-membered heterocyclyl and the backbone of the cycle contains one nitrogen. [Section 42] 39. The compound according to any one of items 1 to 38, wherein R5 is a 7-membered heterocyclyl, the backbone of the cycle containing one nitrogen and one oxygen. [Section 43] Item 39. The compound according to any one of items 1 to 38, wherein R5 is an 8-membered heterocyclyl and the backbone of the cycle contains one nitrogen. [Section 44] 39. The compound according to any one of items 1 to 38, wherein R5 is an 8-membered heterocyclyl, the backbone of the cycle containing one nitrogen and one oxygen. [Section 45] [Item 46] The compound according to any one of Items 1 to 38, wherein R5 is a nitrogen-containing heterocyclyl, and the nitrogen is directly bonded to an aryl or heteroaryl ring bearing an R4 substituent. 46. The compound according to any one of items 1 to 45, wherein R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or sulfonamido. [Section 47] 47. The compound according to any one of the preceding paragraphs, wherein R5 is substituted with an ester (e.g., ethyl ester), carboxyl, alkyl (e.g., methyl or trifluoromethyl), hydroxyalkyl (e.g., hydroxyethyl), halo (e.g., fluoro), cycloalkyl (e.g., cyclopropyl or cyclobutyl), or heterocyclyl (e.g., oxetnyl or tetrahydrofuranyl). [Section 48] 48. The compound according to any one of clauses 1 to 47, wherein R5 is substituted with halo (eg, fluoro). [Section 49] 49. The compound of any one of clauses 1-48, wherein R5 is substituted with two alkyl moieties (eg, two methyl moieties). [Section 50] Item 39. The compound according to any one of items 1 to 38, wherein R5 is 2,6-dimethylmopholine, 4-methylpiperidine, or 4-(trifluoromethyl)piperidine. [Section 51] Item 38. The compound according to any one of items 1 to 37, wherein R5 is amino. [Section 52] 52. The compound of claim 51, wherein R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, sulfonamido, cycloalkyl, or heterocyclyl. [Section 53] 53. The compound according to clause 51 or 52, wherein R5 is substituted with alkyl (e.g., difluoroethyl, or isobutyl), alkyloxyalkyl (e.g., methyloxyethyl), hydroxyalkyl (e.g., hydroxyethyl), cycloalkyl (e.g., cyclopropyl), or heterocyclyl (e.g., pyranyl). [Section 54] the compound has a structure represented by Formula II: [ka] During the ceremony, R1 is unsubstituted phenyl or a ring selected from the group consisting of o-, m-, p-CH3, C2H5, CH(CH3)2, OCH3, OC2H5, OC3H7, SCH3, CF2CH3, CF3, OCF3, OCHF2, N(CH3)2, F, Cl, OH mono- or di-substituted phenyl, pyridyl, benzyloxy or piperonyl; R2 is selected from the group consisting of H or CH3; R3 is selected from the group consisting of H, F, and Cl; R4 is selected from the group consisting of H or CH3; R5 is morpholine, 2,6-dimethylmorpholine, thiomorpholine, thiomorpholine 1,1-dioxide, morpholin-4-amine, piperidine, tetrahydro-2H-pyran-4-amine, piperidin-1-amine, 4-fluoropiperidine, 4,4-difluoropiperidine, 4-methylpiperidine, 4-(trifluoromethyl)piperidine, piperazine, N-methylpiperazine, pyrrolidine, 2-(4-piperidinyl)ethoxyethanol, an amine selected from the group consisting of alcohol, 2-(1-piperazinyl)ethanol, 4-piperidinecarboxylic acid, ethyl 4-piperidinecarboxylate, (1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane, (1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane, 3-oxa-8-azabicyclo[3.2.1]octane, or 8-oxa-3-azabicyclo[3.2.1]octane; X1 is CH or N; Item 1. The compound according to item 1, and / or a pharmaceutically acceptable salt and / or solvate thereof. [Section 55] 55. The compound according to item 54, wherein R1 is phenyl. [Section 56] 55. The compound according to item 54, wherein R1 is pyridyl. [Section 57] 55. The compound according to item 54, wherein R1 is benzyloxy. [Section 58] 55. The compound according to item 54, wherein R1 is piperonyl. [Section 59] 59. The compound according to any one of items 54 to 58, wherein R1 is substituted with CH3, C2H5, CH(CH3)2, OCH3, OC2H5, OC3H7, SCH3, CF2CH3, CF3, OCF3, OCHF2, N(CH3)2, F, Cl, or OH. [Section 60] 59. The compound according to any one of items 54 to 58, wherein R1 is substituted with one F. [Section 61] 61. The compound according to clause 60, wherein F is para to the isoxazole. [Section 62] 61. The compound according to clause 60, wherein F is ortho to the isoxazole. [Section 63] 61. The compound according to clause 60, wherein F is meta to the isoxazole. [Section 64] 59. The compound according to any one of items 54 to 58, wherein R1 is substituted with two F. [Section 65] 65. The compound of paragraph 64, wherein the first F is meta to the isoxazole and the second F is ortho to the isoxazole. [Section 66] Item 66. The compound according to any one of items 54 to 65, wherein R1 is substituted with OCH3. [Section 67] 67. The compound according to claim 66, wherein the OCH3 is para to the isoxazole. [Section 68] 67. The compound according to claim 66, wherein the OCH3 is ortho to the isoxazole. [Section 69] 67. The compound according to claim 66, wherein the OCH3 is meta to the isoxazole. [Section 70] 59. The compound according to any one of items 54 to 58, wherein R1 is substituted with one OCH3 and one F. [Section 71] 71. The compound of claim 70, wherein said OCH3 is para to said isoxazole and said F is meta to said isoxazole. [Section 72] 71. The compound of claim 70, wherein said OCH3 is para to said isoxazole and said F is ortho to said isoxazole. [Section 73] 71. The compound according to clause 70, wherein said F is para to the isoxazole and said OCH3 is meta to the isoxazole. [Section 74] 59. The compound according to any one of items 53 to 58, wherein R1 is substituted with OCH3 and two F. [Section 75] 75. The compound of clause 74, wherein the OCH3 is para to the isoxazole and both F are meta to the isoxazole. [Section 76] 75. The compound of claim 74, wherein said OCH3 is para to said isoxazole, one F is meta to said isoxazole, and one F is ortho to said isoxazole. [Section 77] 77. The compound according to any one of items 53 to 76, wherein R2 is H. [Section 78] 77. The compound according to any one of items 53 to 76, wherein R2 is CH3. [Section 79] 77. The compound according to any one of items 53 to 76, wherein R3 is H. [Section 80] 77. The compound according to any one of items 53 to 76, wherein R3 is F. [Section 81] 77. The compound according to any one of items 53 to 76, wherein R3 is Cl. [Section 82] Item 82. The compound according to any one of items 53 to 81, wherein R5 is morpholine. [Section 83] 82. The compound according to any one of items 53 to 81, wherein R5 is piperidine. [Section 84] 82. The compound according to any one of items 53 to 81, wherein R5 is 4-fluoropiperidine. [Section 85] 82. The compound according to any one of items 53 to 81, wherein R5 is 4,4-difluoropiperidine. [Section 86] 82. The compound according to any one of items 53 to 81, wherein R5 is 3-oxa-8-azabicyclo[3.2.1]octane. [Section 87] 82. The compound according to any one of items 53 to 81, wherein R5 is 8-oxa-3-azabicyclo[3.2.1]octane. [Section 88] 88. The compound according to any one of items 53 to 87, wherein X1 is C. [Section 89] Item 88. The compound according to any one of items 53 to 87, wherein X1 is N. [Section 90] The compound is 3-(4-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-phenylisoxazol-5-amine 3-(4-ethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-propoxyphenyl)isoxazol-5-amine 3-(4-Fluorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-chlorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(p-tolyl)isoxazol-5-amine 3-(4-ethylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-Isopropylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazol-5-amine 3-(4-(1,1-difluoroethyl)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(difluoromethoxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazol-5-amine 3-(4-(methylthio)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3,4-dimethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(6-Methoxypyridin-3-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 4-(5-((2-morpholinopyrimidin-4-yl)amino)isoxazol-3-yl)phenol 3-(4-(benzyloxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-thiomorpholinopyrimidin-4-yl)isoxazol-5-amine 4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)thiomorpholine 1,1-dioxide 3-(4-Methoxyphenyl)-N-(2-(piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 2-(1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidin-4-yl)ethan-1-ol 2-(4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethan-1-ol 3-(4-Methoxyphenyl)-N-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(piperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine Ethyl 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylate 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid N4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -Morpholinopyrimidine-2,4-diamine N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(Piperidin-1-yl)pyrimidine-2,4-diamine N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(Tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine N-(5-chloro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(5-fluoro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4-fluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-methyl-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N 4 -(3-(3-fluoro-4-methoxyphenyl)isoxazol-5-yl)-N 2 -(Tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine 3-(2,3-Dihydrobenzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N 2 -cyclopropyl-N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N 2 -Isobutyl-N 4 -(3-(4 - (Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N 2 -(2-Methoxyethyl)-N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N 1 ,N 1 -Diethyl-N 2 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine N 1 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 ,N 2 -dimethyl-N1-(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine 2-((3-(4-methoxyphenyl)isoxazol-5-yl)(2-morpholinopyrimidin-4-yl)amino)acetonitrile 3-(5-Methoxypyridin-2-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(4-morpholino-1,3,5-triazin-2-yl)isoxazol-5-amine N 2 -(2,2-difluoroethyl)-N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine 2-((4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)amino)ethan-1-ol 3-(2-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(3,4,5-trimethoxyphenyl)isoxazol-5-amine 3-(Benzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -Methylpyrimidine-2,4-diamine 3-(3-Fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetramethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3,3-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2,2-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(3,5-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(2-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(4,4-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(6-azaspiro[2.5]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-methyl-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(2,2-difluoromorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-5-azabicyclo[2.2.2]octan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-5-azabicyclo[4.1.0]heptan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(4-oxa-7-azaspiro[2.5]octan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(7-oxa-4-azaspiro[2.5]octan-4-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-8-azaspiro[4.5]decan-8-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-6-azaspiro[3.4]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-7-azaspiro[4.4]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetrafluoromorpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-Fluoro-4-methoxyphenyl)-N-(2-(3-methyl-6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-isopropylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(4-(difluoromethyl)piperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(6-azaspiro[3.5]nonan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-azaspiro[3.5]nonan-2-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine and / or a pharmaceutically acceptable salt and / or solvate thereof. [Section 91] The compound is [ka] [ka] [ka] Item 55. The compound according to item 1 or 54, selected from the group consisting of: [Section 92] The compound is [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. [Section 93] A pharmaceutical composition comprising the compound according to any one of items 1 to 92 and a pharmaceutically acceptable excipient. [Section 94] A method for treating a TACC3-mediated disease or disorder in a subject, comprising administering to the subject a compound according to any one of items 1 to 92 or a pharmaceutically acceptable salt thereof. [Section 95] 95. The method of paragraph 94, wherein the TACC3-mediated disease or disorder is cancer. [Section 96] 96. The method of paragraph 94 or 95, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, kidney cancer or prostate cancer. [Section 97] Item 97. The method of any one of items 94 to 96, wherein the cancer is a cancer selected from the NCI-60 panel. [Section 98] 98. The method of any one of paragraphs 94 to 97, wherein the compound is orally administered to the subject. [Section 99] 93. The compound of any one of paragraphs 1 to 92 for use in the treatment of a TACC3-mediated disease or disorder. [Section 100] 99. The compound for use according to paragraph 99, wherein the TACC3-mediated disease or disorder is cancer. [Section 101] 101. The compound for use according to paragraph 99 or 100, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, kidney cancer or prostate cancer. [Section 102] 101. The compound for use according to paragraph 99 or 100, wherein the cancer is a cancer selected from the NCI-60 panel. [Section 103] Item 99. The compound for use according to any one of items 99 to 102, wherein the compound is for oral administration. [Section 104] Item 93. The compound according to any one of items 1 to 92, for use in treating cancer. [Section 105] Item 105. The anticancer agent according to Item 104, wherein the anticancer agent targets cancers that express TACC3. [Section 106] Item 106. The compound according to item 104 or 105, wherein the anticancer agent is a TACC3 protein inhibitor. [Section 107] Item 107. The anticancer agent of item 106, wherein the targeted cancers expressing TACC3 are breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer, and prostate cancer cell lines listed in the NCI-60 panel. [Section 108] Item 108. The anticancer agent according to any one of Items 104 to 107, wherein the anticancer agent is for oral administration. [Section 109] Item 110: The compound according to Item 104, wherein the compound is 3-(4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (Compound 5). Item 109. Compound for use according to item 109, characterized in that said compound has a structure that induces mitotic arrest, apoptosis and DNA damage. [Section 111] The compound described in paragraph 110 functions by activating the SAC, inducing severe spindle defects that further result in prolonged mitosis, apoptotic cell death, and DNA damage. [Section 112] The compound according to paragraph 111 inhibits the growth of cancer cells harboring the FGFR3-TACC3 oncogenic fusion. [Section 113] A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of items 1 to 92 and / or a pharmaceutically acceptable salt and / or solvate thereof, and at least one pharmaceutically acceptable carrier. [Section 114] Item 114. A composition comprising the compound of item 113, wherein the composition further comprises a pharmaceutically acceptable chemotherapeutic or targeted therapeutic agent. [Section 115] A therapeutic combination of a chemotherapeutic or targeted therapy agent or drug for an individual in need of cancer treatment, wherein the anticancer agent according to any one of items 1 to 92 is part of the therapeutic combination. [Section 116] Item 93. The compound according to any one of items 1 to 92 for use as a medicament. [Section 117] 93. A compound according to any one of clauses 1 to 92 for use in inhibiting and / or preventing tumor growth and metastasis. [Section 118] 118. The compound of paragraph 117, wherein the cancer is a cancer that responds to inhibition of TACC3. [Section 119] Item 119. The compound according to item 118, wherein the targeted cancer is selected from the group consisting of breast cancer, colon cancer, melanoma cancer, lung cancer, and central nervous system cancer. [Section 120] A method for synthesizing a compound described in any one of paragraphs 1 to 90, comprising reacting an amine derivative described herein, including but not limited to, with an intermediate compound from Table 1. [Section 121] The method comprises the steps represented by Scheme I: [ka] or a pharmaceutically acceptable salt thereof, wherein: X1 is N or CR6; X2 is N or CR3; R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R5 is heterocyclyl, alkyl, or amino; R 51 But it's a halo, R 52 is heterocyclyl or alkyl; X 10 But there is a base, X 11 is a noble metal catalyst, X 12 121. The method of claim 120, wherein is a phosphine ligand. [Section 122] The compound according to any one of items 1 to 92, represented by Scheme I: [ka] or a pharmaceutically acceptable salt thereof, comprising the steps of: X1 is N or CR6; X2 is N or CR3; R1 is aryl or heteroaryl; R2 is H or alkyl; R3, R4 and R6 are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R5 is heterocyclyl, alkyl, or amino; R 51But it's a halo, R 52 is heterocyclyl or alkyl; X 10 is a base, X 11 is a noble metal catalyst, X 12 is a phosphine ligand. [Section 123] 123. The method of claim 121 or 122, wherein the base is a carbonate, an oxide, a tertiary amine, a secondary amine, or a hydride. [Section 124] Item 124. The method of item 123, wherein the oxide is an alkoxide (e.g., tert-butoxide). [Section 125] Item 124. The method of item 123, wherein the tertiary amine is a tertiary alkylamine (e.g., diisopropylethylamine). [Section 126] Item 124. The method of item 123, wherein the hydride is a metal hydride (e.g., sodium hydride). [Section 127] Item 128. The method according to Item 123, wherein the carbonate is a metal carbonate (e.g., cesium carbonate). Item 128. The method according to any one of Items 121 to 127, wherein the noble metal catalyst is a palladium catalyst (for example, palladium II acetate). [Section 129] 129. The method of any one of paragraphs 121 to 128, wherein the phosphine catalyst is an aryl phosphine (for example, triphenylphosphine). [Section 130] 129. The method of any one of items 121 to 128, wherein the phosphine catalyst is Xantphos. [Section 131] Item 131. The method of any one of items 121 to 130, wherein the method further comprises a solvent. [Section 132] Item 132. The method of item 131, wherein the solvent is tertiary butanol, dimethylacetamide, or dioxane. [Section 133] Item 133. The method of any one of items 121 to 132, wherein the method further comprises heating. [Section 134] Item 134. The method according to any one of items 121 to 133, wherein the method is carried out under an inert atmosphere.

Claims

1. A compound of formula (I), 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: X １ But N or CR ６ and X ２ But N or CR ３ and R １ is aryl or heteroaryl; R ２ is H or alkyl, R ３ , R ４ and R ６ are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R ５ is heterocyclyl, alkyl, or amino.

2. The compound is 【Chemistry 2】 The compound of claim 1 which is not

3. R １ 3. The compound of claim 1 or 2, wherein is aryl (e.g., phenyl).

4. R １ 3. The compound of claim 1 or 2, wherein is heteroaryl (e.g., benzofuran, or pyrimidinyl).

5. R １ 3. The compound of claim 1 or 2, wherein is heterocyclyl (e.g., benzodioxole or dihydrobenzofuran).

6. R １ is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl, or sulfonamido.

7. R １ is substituted with alkyl (e.g., methyl, ethyl, isopropyl, fluoroethyl, or trifluoromethyl), alkyloxy (e.g., methoxy, trifluoromethyloxy, difluoromethyloxy, ethoxy, or propyloxy), alkylthio (e.g., methylthio), aralkyloxy (e.g., benzyloxy), hydroxyl, halo (e.g., fluoro or chloro), or amino (e.g., dimethylaminoalkyl).

8. R １ The compound of any one of claims 1 to 7, wherein is substituted with halo (eg fluoro).

9. R １ A compound according to any one of claims 1 to 8, wherein is substituted with alkyloxy (e.g. methoxy).

10. R １ 10. The compound of any one of claims 1 to 9, wherein is substituted with alkyl (e.g., methyl, ethyl, or trifluoromethyl).

11. R １ A compound according to any one of claims 1 to 7, wherein is substituted with one halo (eg F).

12. 12. The compound of claim 11, wherein the halo (e.g., F) is para to the isoxazole.

13. 12. The compound of claim 11, wherein the halo (e.g., F) is ortho to the isoxazole.

14. 12. The compound of claim 11, wherein the halo (e.g., F) is meta to the isoxazole.

15. R １ A compound according to any one of claims 1 to 7, wherein is substituted with two halo (eg F).

16. 16. The compound of claim 15, wherein one halo (e.g., F) is meta to the isoxazole and one halo (e.g., F) is ortho to the isoxazole.

17. R １ 8. The compound of any one of claims 1 to 7, wherein is substituted with alkoxy (e.g. methoxy).

18. 18. The compound of claim 17, wherein the alkoxy (e.g., methoxy) is para to the isoxazole.

19. 18. The compound of claim 17, wherein the alkoxy (e.g., methoxy) is ortho to the isoxazole.

20. 18. The compound of claim 17, wherein the alkoxy (e.g., methoxy) is meta to the isoxazole.

21. R １ 8. The compound of any one of claims 1 to 7, wherein is substituted with one alkoxy (eg methoxy) and one halo (eg F).

22. 22. The compound of claim 21, wherein the alkoxy (e.g., methoxy) is para to the isoxazole and the F is meta to the isoxazole.

23. 22. The compound of claim 21, wherein the alkoxy (e.g., methoxy) is para to the isoxazole and the F is ortho to the isoxazole.

24. 22. The compound of claim 21, wherein the halo (e.g., F) is para to the isoxazole and the alkoxy (e.g., methoxy) is meta to the isoxazole.

25. R １ 8. The compound of any one of claims 1 to 7, wherein is substituted with alkoxy (eg methoxy) and two halo (eg F).

26. 26. The compound of claim 25, wherein the alkoxy (e.g., methoxy) is para to the isoxazole and both halo (e.g., F) are meta to the isoxazole.

27. 26. The compound of claim 25, wherein the alkoxy (e.g., methoxy) is para to the isoxazole, one halo (e.g., F) is meta to the isoxazole, and one halo (e.g., F) is ortho to the isoxazole.

28. R ２ The compound of any one of claims 1 to 27, wherein is alkyl (for example methyl or ethyl).

29. R ２ 29. The compound of any one of claims 1 to 28, wherein is substituted with amino (for example dimethylamino or diethylamino), or nitrile.

30. R ２ The compound of any one of claims 1 to 27, wherein is H.

31. X １ The compound according to any one of claims 1 to 30, wherein is N.

32. X １ But, CR ６ The compound according to any one of claims 1 to 30,

33. R ６ 33. The compound of claim 32, wherein is H.

34. X ２ The compound according to any one of claims 1 to 33, wherein is N.

35. X ２ But, CR ３ The compound according to any one of claims 1 to 33,

36. R ３ 36. The compound of claim 35, wherein is H or halo (e.g., fluoro or chloro).

37. R ４ A compound according to any one of claims 1 to 36, wherein is alkyl (e.g. methyl).

38. R ５ is heterocyclyl (e.g., azetidinyl, morpholino, pyrrolidinyl, piperazinyl, piperidinyl, oxaazabicyclooctanyl, oxaazabicycloheptnyl, thiomorpholino, thiomorpholino dioxide, hexahydrofuropyrrolyl, or azabicyclohexanyl).

39. R ５ A compound according to any one of claims 1 to 38, wherein is a six-membered heterocyclyl and the backbone of the cycle contains one nitrogen.

40. R ５ A compound according to any one of claims 1 to 38, wherein is a six-membered heterocyclyl and the backbone of the cycle contains one nitrogen and one oxygen.

41. R ５ A compound according to any one of claims 1 to 38, wherein is a seven-membered heterocyclyl and the backbone of the cycle contains one nitrogen.

42. R ５ A compound according to any one of claims 1 to 38, wherein is a seven-membered heterocyclyl and the backbone of the cycle contains one nitrogen and one oxygen.

43. R ５ A compound according to any one of claims 1 to 38, wherein is an 8-membered heterocyclyl and the backbone of the cycle contains one nitrogen.

44. R ５ A compound according to any one of claims 1 to 38, wherein is an 8-membered heterocyclyl and the backbone of the cycle contains one nitrogen and one oxygen.

45. R ５ is a nitrogen-containing heterocyclyl, said nitrogen being R ４ 39. The compound of any one of claims 1 to 38, directly attached to a substituted aryl or heteroaryl ring.

46. R ５ is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or sulfonamido.

47. R ５ is substituted with an ester (e.g., ethyl ester), carboxyl, alkyl (e.g., methyl or trifluoromethyl), hydroxyalkyl (e.g., hydroxyethyl), halo (e.g., fluoro), cycloalkyl (e.g., cyclopropyl or cyclobutyl), or heterocyclyl (e.g., oxetnyl or tetrahydrofuranyl).

48. R ５ 48. The compound of any one of claims 1 to 47, wherein is substituted with halo (eg fluoro).

49. R ５ 49. The compound of any one of claims 1 to 48, wherein is substituted with two alkyl moieties (eg, two methyl moieties).

50. R ５ 39. The compound of any one of claims 1 to 38, wherein is 2,6-dimethylmopholine, 4-methylpiperidine, or 4-(trifluoromethyl)piperidine.

51. R ５ The compound of any one of claims 1 to 37, wherein is amino.

52. R ５ is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, sulfonamido, cycloalkyl, or heterocyclyl).

53. R ５ is substituted with alkyl (e.g., difluoroethyl, or isobutyl), alkyloxyalkyl (e.g., methyloxyethyl), hydroxyalkyl (e.g., hydroxyethyl), cycloalkyl (e.g., cyclopropyl), or heterocyclyl (e.g., pyranyl).

54. The compound has a structure represented by Formula II: 【Chemistry 3】 During the ceremony, R １ is unsubstituted phenyl or o-, m-, p-CH ３ , C ２ H ５ , CH(CH ３ ) ２ , OCH ３ , O.C. ２ H ５ , O.C. ３ H ７ , S.C.H. ３ , C.F. ２ CH ３ , C.F. ３ , OCF ３ , OCHF ２ , N(CH ３ ) ２ , F, Cl, OH, mono- or di-substituted phenyl, pyridyl, benzyloxy or piperonyl; R ２ is H or CH ３ is selected from the group consisting of R ３ is selected from the group consisting of H, F, and Cl; R ４ is H or CH ３ is selected from the group consisting of R ５ However, morpholine, 2,6-dimethylmorpholine, thiomorpholine, thiomorpholine 1,1-dioxide, morpholin-4-amine, piperidine, tetrahydro-2H-pyran-4-amine, piperidin-1-amine, 4-fluoropiperidine, 4,4-difluoropiperidine, 4-methylpiperidine, 4-(trifluoromethyl)piperidine, piperazine, N-methylpiperazine, pyrrolidine, 2-(4-piperidinyl)ethanoic acid an amine selected from the group consisting of 4-piperidinecarboxylic acid, ethyl 4-piperidinecarboxylate, (1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane, (1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane, 3-oxa-8-azabicyclo[3.2.1]octane, or 8-oxa-3-azabicyclo[3.2.1]octane; X １ is CH or N; 10. The compound of claim 1, and / or a pharmaceutically acceptable salt and / or solvate thereof.

55. R １ 55. The compound of claim 54, wherein is phenyl.

56. R １ 55. The compound of claim 54, wherein is pyridyl.

57. R １ is benzyloxy.

58. R １ is piperonyl.

59. R １ But CH ３ , C ２ H ５ , CH(CH ３ ) ２ , OCH ３ , O.C. ２ H ５ , O.C. ３ H ７ , S.C.H. ３ , C.F. ２ CH ３ , C.F. ３ , OCF ３ , OCHF ２ , N(CH ３ ) ２ 59. The compound of any one of claims 54 to 58, wherein the compound is substituted with , F, Cl, or OH.

60. R １ is substituted with one F.

61. 61. The compound of claim 60, wherein F is para to the isoxazole.

62. 61. The compound of claim 60, wherein F is ortho to the isoxazole.

63. 61. The compound of claim 60, wherein F is meta to the isoxazole.

64. R １ is substituted with two F.

65. 65. The compound of claim 64, wherein a first F is meta to the isoxazole and a second F is ortho to the isoxazole.

66. R １ But OCH ３ 66. The compound of any one of claims 54 to 65, substituted with:

67. The OCH ３ is para to the isoxazole.

68. The OCH ３ is ortho to the isoxazole.

69. The OCH ３ is meta to the isoxazole.

70. R １ But one OCH ３ and one F.

71. The OCH ３ 71. The compound of claim 70, wherein is para to the isoxazole and said F is meta to said isoxazole.

72. The OCH ３ 71. The compound of claim 70, wherein said is para to said isoxazole and said F is ortho to said isoxazole.

73. the F is para to the isoxazole, and the OCH ３ is meta to said isoxazole.

74. R １ But OCH ３ and two F.

75. The OCH ３ 75. The compound of claim 74, wherein is para to the isoxazole and both F are meta to said isoxazole.

76. The OCH ３ 75. The compound of claim 74, wherein: is para to the isoxazole; one F is meta to said isoxazole; and one F is ortho to said isoxazole.

77. R ２ The compound of any one of claims 53 to 76, wherein is H.

78. R ２ But CH ３ 77. The compound according to any one of claims 53 to 76, wherein

79. R ３ The compound of any one of claims 53 to 76, wherein is H.

80. R ３ The compound of any one of claims 53 to 76, wherein is F.

81. R ３ The compound of any one of claims 53 to 76, wherein is Cl.

82. R ５ The compound of any one of claims 53 to 81, wherein is morpholine.

83. R ５ 82. The compound of any one of claims 53 to 81, wherein is piperidine.

84. R ５ 82. The compound of any one of claims 53 to 81, wherein is 4-fluoropiperidine.

85. R ５ The compound of any one of claims 53 to 81, wherein is 4,4-difluoropiperidine.

86. R ５ 82. The compound of any one of claims 53 to 81, wherein is 3-oxa-8-azabicyclo[3.2.1]octane.

87. R ５ The compound of any one of claims 53 to 81, wherein is 8-oxa-3-azabicyclo[3.2.1]octane.

88. X １ The compound of any one of claims 53 to 87, wherein is C.

89. X １ The compound of any one of claims 53 to 87, wherein is N.

90. The compound is 3-(4-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2-Methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-phenylisoxazol-5-amine 3-(4-ethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-propoxyphenyl)isoxazol-5-amine 3-(4-fluorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-chlorophenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(p-tolyl)isoxazol-5-amine 3-(4-ethylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-isopropylphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazol-5-amine 3-(4-(1,1-difluoroethyl)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(difluoromethoxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazol-5-amine 3-(4-(methylthio)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3,4-dimethoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(6-methoxypyridin-3-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 4-(5-((2-morpholinopyrimidin-4-yl)amino)isoxazol-3-yl)phenol 3-(4-(benzyloxy)phenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4,4-difluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-thiomorpholinopyrimidin-4-yl)isoxazol-5-amine 4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)thiomorpholine 1,1-dioxide 3-(4-Methoxyphenyl)-N-(2-(piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 2-(1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidin-4-yl)ethan-1-ol 2-(4-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperazin-1-yl)ethan-1-ol 3-(4-Methoxyphenyl)-N-(2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-(piperazin-1-yl)pyrimidin-4-yl)isoxazol-5-amine Ethyl 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylate 1-(4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ -morpholinopyrimidine-2,4-diamine N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ -(piperidin-1-yl)pyrimidine-2,4-diamine N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ -(tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine N-(5-chloro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(5-fluoro-2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine N-(2-(4-fluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-3-(4-methoxyphenyl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-methyl-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-fluoropiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(pyrrolidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N ４ -(3-(3-fluoro-4-methoxyphenyl)isoxazol-5-yl)-N ２ -(tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine 3-(2,3-dihydrobenzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N ２ -cyclopropyl-N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N ２ -isobutyl-N ４ -(3-(4 － (Methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N ２ -(2-methoxyethyl)-N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine N １ , N １ -Diethyl-N ２ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ -(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine N １ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ , N ２ -dimethyl-N1-(2-morpholinopyrimidin-4-yl)ethane-1,2-diamine 2-((3-(4-methoxyphenyl)isoxazol-5-yl)(2-morpholinopyrimidin-4-yl)amino)acetonitrile 3-(5-methoxypyridin-2-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyridin-4-yl)isoxazol-5-amine 3-(4-Methoxyphenyl)-N-(4-morpholino-1,3,5-triazin-2-yl)isoxazol-5-amine N ２ -(2,2-difluoroethyl)-N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)pyrimidine-2,4-diamine 2-((4-((3-(4-methoxyphenyl)isoxazol-5-yl)amino)pyrimidin-2-yl)amino)ethan-1-ol 3-(2-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(3,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine N-(2-morpholinopyrimidin-4-yl)-3-(3,4,5-trimethoxyphenyl)isoxazol-5-amine 3-(benzofuran-5-yl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine N ４ -(3-(4-methoxyphenyl)isoxazol-5-yl)-N ２ -methylpyrimidine-2,4-diamine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetramethylmorpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3,3-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2,2-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(3,5-dimethylmorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methylmorpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(tetrahydro-1H-furo[3,4-c]pyrrol-5(3H)-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(4,4-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(6-azaspiro[2.5]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-8-azabicyclo[3.2.1]octan-8-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(2,2-difluoromorpholino)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-5-azabicyclo[2.2.2]octan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-(trifluoromethyl)morpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-5-azabicyclo[4.1.0]heptan-5-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(4-oxa-7-azaspiro[2.5]octan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(7-oxa-4-azaspiro[2.5]octan-4-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-8-azaspiro[4.5]decan-8-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-6-azaspiro[3.4]octan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-oxa-7-azaspiro[4.4]nonan-7-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetrafluoromorpholino)pyrimidin-4-yl)isoxazol-5-amine N-(2-(6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-6-azabicyclo[3.1.1]heptan-6-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-isopropylpiperidin-1-yl)pyrimidin-4-yl)isoxazol-5-amine N-(2-(4-(difluoromethyl)piperidin-1-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(6-azaspiro[3.5]nonan-6-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine N-(2-(2-azaspiro[3.5]nonan-2-yl)pyrimidin-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazol-5-amine 3-(2-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine and / or a pharmaceutically acceptable salt and / or solvate thereof.

91. The compound is 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 55. The compound of claim 1 or 54, selected from the group consisting of:

92. The compound is 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 or a pharmaceutically acceptable salt thereof.

93. A pharmaceutical composition comprising a compound according to any one of claims 1 to 92 and a pharmaceutically acceptable excipient.

94. 93. A method of treating a TACC3-mediated disease or disorder in a subject, comprising administering to the subject a compound of any one of claims 1 to 92, or a pharmaceutically acceptable salt thereof.

95. 95. The method of claim 94, wherein the TACC3-mediated disease or disorder is cancer.

96. 96. The method of claim 94 or 95, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer or prostate cancer.

97. 97. The method of any one of claims 94 to 96, wherein the cancer is a cancer selected from the NCI-60 panel.

98. 98. The method of any one of claims 94 to 97, wherein the compound is administered orally to the subject.

99. 93. A compound according to any one of claims 1 to 92 for use in the treatment of a TACC3 mediated disease or disorder.

100. 100. The compound for use according to claim 99, wherein the TACC3-mediated disease or disorder is cancer.

101. 101. The compound for use according to claim 99 or 100, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer or prostate cancer.

102. 101. The compound for use according to claim 99 or 100, wherein the cancer is a cancer selected from the NCI-60 panel.

103. 103. The compound for use according to any one of claims 99 to 102, wherein the compound is for oral administration.

104. A compound according to any one of claims 1 to 92 for use in the treatment of cancer.

105. The anticancer agent of claim 104, wherein the anticancer agent targets a cancer that expresses TACC3.

106. The compound of claim 104 or 105, wherein the anti-cancer agent is a TACC3 protein inhibitor.

107. The anticancer agent of claim 106, wherein the targeted cancers expressing TACC3 are breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia cancer, renal cancer, and prostate cancer cell lines listed in the NCI-60 panel.

108. The anticancer agent according to any one of claims 104 to 107, wherein the anticancer agent is for oral administration.

109. The compound of claim 104, wherein the compound is 3-(4-methoxyphenyl)-N-(2-morpholinopyrimidin-4-yl)isoxazol-5-amine (compound 5).

110. 110. The compound for use according to claim 109, characterized in that the compound has a structure that induces mitotic arrest, apoptosis and DNA damage.

111. The compound of claim 110 functions by activating SAC, inducing severe spindle defects that further lead to prolonged mitosis, apoptotic cell death, and DNA damage.

112. The compound of claim 111 inhibits the growth of cancer cells harboring the FGFR3-TACC3 oncogenic fusion.

113. 93. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claims 1 to 92 and / or a pharmaceutically acceptable salt and / or solvate thereof and at least one pharmaceutically acceptable carrier.

114. 114. A composition comprising the compound of claim 113, wherein the composition further comprises a pharmaceutically acceptable chemotherapeutic or targeted therapy agent.

115. A therapeutic combination of chemotherapeutic or targeted therapy agents or drugs for an individual in need of cancer treatment, wherein the anti-cancer agent of any one of claims 1 to 92 is part of said therapeutic combination.

116. A compound according to any one of claims 1 to 92 for use as a medicament.

117. A compound according to any one of claims 1 to 92 for use in inhibiting and / or preventing tumor growth and metastasis.

118. 118. The compound of claim 117, wherein the cancer is a cancer that responds to inhibition of TACC3.

119. The compound of claim 118, wherein the targeted cancer is selected from the group consisting of breast cancer, colon cancer, melanoma cancer, lung cancer, and central nervous system cancer.

120. 91. A method for synthesizing a compound according to any one of claims 1 to 90, comprising reacting an amine derivative, including but not limited to, as described herein, with an intermediate compound, including but not limited to, from Table 1.

121. The method comprises the steps represented by Scheme I: 【Chemistry 6】 or a pharmaceutically acceptable salt thereof, wherein: X １ But N or CR ６ and X ２ But N or CR ３ and R １ is aryl or heteroaryl; R ２ is H or alkyl, R ３ , R ４ and R ６ are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R ５ is heterocyclyl, alkyl, or amino; R ５１ But it's a halo, R ５２ is heterocyclyl or alkyl; X １０ But there is a base, X １１ is a noble metal catalyst, X １２ is a phosphine ligand.

122. A compound according to any one of claims 1 to 92, represented by Scheme I: 【Chemistry 7】 or a pharmaceutically acceptable salt thereof, comprising the steps of: X １ But N or CR ６ and X ２ But N or CR ３ and R １ is aryl or heteroaryl; R ２ is H or alkyl, R ３ , R ４ and R ６ are each independently H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamido; R ５ is heterocyclyl, alkyl, or amino; R ５１ But it's a halo, R ５２ is heterocyclyl or alkyl; X １０ is a base, X １１ is a noble metal catalyst, X １２ is a phosphine ligand.

123. 123. The method of claim 121 or 122, wherein the base is a carbonate, an oxide, a tertiary amine, a secondary amine, or a hydride.

124. 124. The method of claim 123, wherein the oxide is an alkoxide (e.g., tert-butoxide).

125. 124. The method of claim 123, wherein the tertiary amine is a tertiary alkylamine (e.g., diisopropylethylamine).

126. 124. The method of claim 123, wherein the hydride is a metal hydride (e.g., sodium hydride).

127. 124. The method of claim 123, wherein the carbonate is a metal carbonate (e.g., cesium carbonate).

128. 128. The method of any one of claims 121 to 127, wherein the noble metal catalyst is a palladium catalyst (e.g., palladium II acetate).

129. 129. The method of any one of claims 121 to 128, wherein the phosphine catalyst is an arylphosphine (e.g., triphenylphosphine).

130. 129. The method of any one of claims 121 to 128, wherein the phosphine catalyst is Xantphos.

131. The method of any one of claims 121 to 130, wherein the method further comprises a solvent.

132. 132. The method of claim 131, wherein the solvent is tertiary butanol, dimethylacetamide, or dioxane.

133. 133. The method of any one of claims 121 to 132, wherein the method further comprises heating.

134. 134. The method of any one of claims 121 to 133, wherein the method is carried out under an inert atmosphere.

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