Isoxazole derivatives that target 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 interests of EP Patent Application No. 19209120.5, filed on November 14, 2019, the contents of the aforementioned application being incorporated herein by reference in their entirety.

[0002] This disclosure relates to substituted compounds of formula (I) that are useful as transformations of acid-coiled-coil protein 3 (TACC3) inhibitors, pharmaceutical compositions of such compounds, methods for preparing the same, and uses thereof. More specifically, TACC3 inhibitors are useful for treating or reversing TACC3-mediated cancers, including breast cancer, leukemia, lung cancer, colon cancer, melanoma, prostate cancer, ovarian cancer, kidney cancer, and CNS cancers. [ka] [Background technology]

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

[0004] The Food and Drug Administration (FDA) has approved approximately two dozen drugs for the treatment of breast cancer, yet 500,000 people worldwide still die from breast cancer each year. In particular, given the side effects of currently available chemotherapy drugs, the development of less toxic targeted therapies has become a major focus in recent years. Because cancer is characterized as abnormal and uncontrolled 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 certain macromolecules involved in the growth and survival of tumor cells are used in targeted treatments for breast cancer.

[0005] Microtubule rearrangement is a critical step during cell division, making drugs that disrupt this process a major focus of cancer research. Cell division inhibitors disrupt microtubule polymerization dynamics by activating spindle formation checkpoints (SACs), which prevent the transition from metaphase to anaphase. As a result, cells halt division, and these mitotically arrested cells eventually die. Ongoing investigation of the mechanisms of mitotic events may lead to new target protein candidates and / or pathways, which is crucial for providing more effective treatment options for cancer patients. Microtubule inhibitors such as vinca alkaloids, mytansinoids, and taxanes are examples of such drugs and are widely used as chemotherapeutic agents for various tumors (Marzo & Naval, 2013). However, a major concern with these drugs is their toxicity to non-tumor-forming cells, which can cause serious side effects.

[0006] Drug resistance is another major problem that makes patient responses to these drugs highly unpredictable (Gascoigne & Taylor, 2009). To overcome these problems and improve the response to chemotherapy, anti-mitotic, cancer-specific therapies targeting mitosis-specific kinases and microtubule motor proteins have been identified (Dominguez-Brauer et al., 2015). Importantly, since phosphorylation is a crucial step in cell cycle regulation and spindle formation, kinases that play a role in these processes have long been studied as potential targets. Among these, specific inhibitors of cyclin-dependent kinases (CDKs), aurora kinases, and polo-like kinases (PLKs) have been developed and are being clinically tested (Sanchez-Martinez, Gelbert, Lallena, & de Dios, 2015; Strebhardt & Ullrich, 2006; Tang et al., 2017). Compared to microtubule inhibitors, none of these antimitotic agents demonstrated remarkable clinical outcomes and limited clinical efficacy, despite having lower toxicity profiles (Chan, Koh, & Li, 2012). Therefore, alternative target molecules that selectively and effectively target mitotic cancer cells remain unidentified and undeveloped.

[0007] TACC3, one of the TACC members, is a non-kinase microtubule-binding protein that plays a crucial role in centrosome regulation and ensures microtubule stability (Singh, Thomas, Gireesh, & Manna, 2014). The TACC3 gene also plays a vital role in centrosome microtubule nucleation. Elevated levels of TACC3 have been observed in many cancer types, including prostate cancer, hepatocellular carcinoma, non-small cell lung cancer, and breast cancer. Therefore, knockdown of TACC3 suppresses tumorigenesis and cell growth in renal cell carcinoma (RCC) (Guo & Liu, 2018). Disruption of TACC3 function also leads to a range of different cellular outcomes, including multipolar spindle formation leading to mitotic arrest (Yao et al., 2012), chromosomal misalignment resulting in caspase-dependent apoptosis (Schneider et al., 2007), and, in some cases, senescence (Schmidt et al., 2010). These studies have shown that TACC3 is a key molecule involved in spindle formation in cancer cells, making it an important and potential target for cancer-targeted therapies.

[0008] The small molecule TACC3 inhibitor KHS101 was first identified as promoting neuronal differentiation in rats (Wurdak et al., 2010). Tumor growth in glioblastoma (GBM) xenografts was suppressed by KHS101 treatment (Polson et al., 2018), but its low systemic stability and high workload necessitate pharmacological optimization before clinical application (Wurdak et al., 2010). Another TACC3 inhibitor, SPL-B, has been shown to inhibit centrosome microtubule nucleation in ovarian cancer cells and suppress tumor growth in ovarian cancer xenografts (Yao et al., 2014). In conclusion, the two currently available TACC3 inhibitors, KHS101 and SPL-B, have shown reductions in tumor growth in glioblastoma and ovarian cancer xenografts, respectively. However, both of these inhibitors have not yet reached the clinical stage due to low efficacy or poor systemic stability.

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

[0010] In one embodiment, the present disclosure relates to a compound of formula (I), [ka] or with respect to a pharmaceutically acceptable salt thereof, in the formula, X1 is N or CR6. X2 is either N or CR3. R1 is either an aryl or heteroaryl. R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. R5 is a heterocycline, alkyl, or amino compound.

[0011] In another embodiment, this disclosure relates to a method for treating TACC3-mediated diseases and disorders using compounds disclosed herein. In certain embodiments, the TACC3-mediated disease or disorder is cancer.

[0012] In one embodiment, the object of the present disclosure is to reduce undesirable side effects by using a small amount of a TACC3 inhibitor that has higher efficacy as a mitotic blocker than certain inhibitors available in cancer treatment.

[0013] As an example of all compounds, compound 5 had a slight effect on normal mammary gland cell lines but showed superior antiproliferative effects compared to known TACC3 inhibitors in various breast cancer cell lines of various subtypes. In addition to breast cancer cells, compound 5 showed 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 Compound 5 showed (possessing a value). Furthermore, compound 5 exhibited a significant anticancer effect against cells possessing the FGFR3-TACC3 fusion protein, and its activity correlated with the TACC3 levels of 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 low doses compared to the other two TACC3 inhibitors. Compound 5 also induced abnormal 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 with highly aggressive breast cancer metastases. Similar to breast cancer tumor models, compound 5 significantly suppressed tumor growth in colon cancer xenograft and immunocompetent syngeneic models. Thus, this disclosure provides a novel TACC3 inhibitor with high efficacy as a mitotic blocker for the treatment of both primary and metastatic breast cancer, and potentially other cancers.

[0015] In certain embodiments, the Disclosure provides (i) compounds selected from the group represented by General Formula I as TACC3 inhibitors, (ii) compounds selected from the group represented by General Formula I as anticancer agents responsive to TACC3 inhibition, (iii) a comprehensive analysis of Compound 5 as an example of all compounds against breast cancer cell lines, (iv) evidence that the compound exhibits superior effects on various cellular processes such as mitotic arrest, DNA damage, and apoptosis in response to other available TACC3 inhibitors, (v) in vivo antitumor effects of Compound 5 without observable toxicity when administered orally to animal models of breast and colon cancer, and (vi) its ability to impair metastatic growth and improve overall survival in mice with metastases, suggesting its use as a mitotic blocker for the treatment of breast and other cancers responsive to TACC3 inhibition. [Brief explanation of the drawing]

[0016] [Figure 1A] In general, Figure 1 shows that TACC3 is upregulated in several different cancer types, and its high levels are associated with poorer overall survival. Figure 1A is a differential mRNA expression plot of TACC3 between tumor and normal tissues in 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: panrenal cohort (KICH+KIRC+KIRP); KIRC: renal clear cell carcinoma; LIHC: hepatocellular carcinoma of the liver; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; STAD: gastric adenocarcinoma; STES: gastric and esophageal cancer; UCEC: endometrial cancer). Figure 1B plots showing the effect of TACC3 levels on overall survival for breast cancer (B-1) and gastric cancer (B-2), recurrence-free survival for lung cancer (B-3), and disease-free survival for prostate cancer (B-4), retrieved from the METABRIC, KM Plotter database, GSE31210, and TCGA datasets, respectively. Log-rank tests were used for statistical analysis. [Figure 1B] In general, Figure 1 shows that TACC3 is upregulated in several different cancer types, and its high levels are associated with poorer overall survival. Figure 1A is a differential mRNA expression plot of TACC3 between tumor and normal tissues in 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: panrenal cohort (KICH+KIRC+KIRP); KIRC: renal clear cell carcinoma; LIHC: hepatocellular carcinoma of the liver; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; STAD: gastric adenocarcinoma; STES: gastric and esophageal cancer; UCEC: endometrial cancer). Figure 1B plots showing the effect of TACC3 levels on overall survival for breast cancer (B-1) and gastric cancer (B-2), recurrence-free survival for lung cancer (B-3), and disease-free survival for prostate cancer (B-4), retrieved from the METABRIC, KM Plotter database, GSE31210, and TCGA datasets, respectively. Log-rank tests were used for statistical analysis. [Figure 2] This figure shows a multivariate analysis performed in METABRIC patients, with TACC3 level, tumor grade, tumor stage, ER, PR, and HER2 status selected as covariates. TACC3 expression is separated based on the 25th percentile. [Figure 3A]This figure shows that, in general, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A is an enrichment plot from GSEA performed using a set of mitotic and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. The significance of the data is expressed as normalized enrichment score (NES) and FDR(q) values. N indicates the total number of genes used for analysis. Figure 3B shows a qRT-PCR test demonstrating the knockdown efficiency of TACC3-specific siRNAs in breast cancer cell lines. Cells were transfected with two different siRNAs against TACC3 (20 nM), and TACC3 mRNA levels were examined 48 hours after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C is a graph showing the inhibition of breast cancer cell growth during 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 during TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3B]This figure shows that, in general, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A is an enrichment plot from GSEA performed using a set of mitotic and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. The significance of the data is expressed as normalized enrichment score (NES) and FDR(q) values. N indicates the total number of genes used for analysis. Figure 3B shows a qRT-PCR test demonstrating the knockdown efficiency of TACC3-specific siRNAs in breast cancer cell lines. Cells were transfected with two different siRNAs against TACC3 (20 nM), and TACC3 mRNA levels were examined 48 hours after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C is a graph showing the inhibition of breast cancer cell growth during 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 during TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3C]This figure shows that, in general, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A is an enrichment plot from GSEA performed using a set of mitotic and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. The significance of the data is expressed as normalized enrichment score (NES) and FDR(q) values. N indicates the total number of genes used for analysis. Figure 3B shows a qRT-PCR test demonstrating the knockdown efficiency of TACC3-specific siRNAs in breast cancer cell lines. Cells were transfected with two different siRNAs against TACC3 (20 nM), and TACC3 mRNA levels were examined 48 hours after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C is a graph showing the inhibition of breast cancer cell growth during 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 during TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 3D]This figure shows that, in general, TACC3 inhibition induces mitotic arrest, apoptosis, and DNA damage. Figure 3A is an enrichment plot from GSEA performed using a set of mitotic and DNA repair-related genes in METABRIC patients, separated according to TACC3 expression levels. The significance of the data is expressed as normalized enrichment score (NES) and FDR(q) values. N indicates the total number of genes used for analysis. Figure 3B shows a qRT-PCR test demonstrating the knockdown efficiency of TACC3-specific siRNAs in breast cancer cell lines. Cells were transfected with two different siRNAs against TACC3 (20 nM), and TACC3 mRNA levels were examined 48 hours after transfection. Percentages on the graph indicate knockdown efficiency. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3C is a graph showing the inhibition of breast cancer cell growth during 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 during TACC3 knockdown. GAPDH was used as a protein loading control. [Figure 4A]Generally, this figure shows that compound 5, a novel TACC3 inhibitor, binds to TACC3. Figure 4A shows the targeted engagement of compound 5 to the TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with the vehicle, compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, the cells were collected, heated at the indicated temperature, and then lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. The CETSA curve shows the TACC3% relative band intensity and the shift between treatment groups. Figure 4B is a graph showing the determination of the binding constant of the interaction between TACC3 and compound 5 using an isothermal titration calorimeter (ITC). The upper trace shows the raw data, while the lower trace shows the integrated data from the titration of TACC3 to compound 5. Model fitting of the 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 extract 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 the drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing the quantification of TACC3 relative band intensity between treatment groups, normalized to β-actin, representing two independent experiments. Data are expressed as mean ± SD. **p<0.01 [Figure 4B]Generally, this figure shows that compound 5, a novel TACC3 inhibitor, binds to TACC3. Figure 4A shows the targeted engagement of compound 5 to the TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with the vehicle, compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, the cells were collected, heated at the indicated temperature, and then lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. The CETSA curve shows the TACC3% relative band intensity and the shift between treatment groups. Figure 4B is a graph showing the determination of the binding constant of the interaction between TACC3 and compound 5 using an isothermal titration calorimeter (ITC). The upper trace shows the raw data, while the lower trace shows the integrated data from the titration of TACC3 to compound 5. Model fitting of the 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 extract 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 the drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing the quantification of TACC3 relative band intensity between treatment groups, normalized to β-actin, representing two independent experiments. Data are expressed as mean ± SD. **p<0.01 [Figure 4C]Generally, this figure shows that compound 5, a novel TACC3 inhibitor, binds to TACC3. Figure 4A shows the targeted engagement of compound 5 to the TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with the vehicle, compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, the cells were collected, heated at the indicated temperature, and then lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. The CETSA curve shows the TACC3% relative band intensity and the shift between treatment groups. Figure 4B is a graph showing the determination of the binding constant of the interaction between TACC3 and compound 5 using an isothermal titration calorimeter (ITC). The upper trace shows the raw data, while the lower trace shows the integrated data from the titration of TACC3 to compound 5. Model fitting of the 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 extract 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 the drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing the quantification of TACC3 relative band intensity between treatment groups, normalized to β-actin, representing two independent experiments. Data are expressed as mean ± SD. **p<0.01 [Figure 4D]Generally, this figure shows that compound 5, a novel TACC3 inhibitor, binds to TACC3. Figure 4A shows the targeted engagement of compound 5 to the TACC3 protein in intact JIMT-1 cells. JIMT-1 cells were treated with the vehicle, compound 5, or SPL-B (as a positive control) (1 μM) for 6 hours, the cells were collected, heated at the indicated temperature, and then lysed. Soluble proteins in the supernatant were subjected to Western blotting to detect TACC3 protein levels. Ponceau staining was used as a loading control. The CETSA curve shows the TACC3% relative band intensity and the shift between treatment groups. Figure 4B is a graph showing the determination of the binding constant of the interaction between TACC3 and compound 5 using an isothermal titration calorimeter (ITC). The upper trace shows the raw data, while the lower trace shows the integrated data from the titration of TACC3 to compound 5. Model fitting of the 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 extract 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 the drug was used. SPL-B was used as a positive control for binding. Figure 4D is a graph showing the quantification of TACC3 relative band intensity between treatment groups, normalized to β-actin, representing two independent experiments. Data are expressed as mean ± SD. **p<0.01 [Figure 5A]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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]In general, 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 the cell viability of breast cancer cell lines (IC50: 50% inhibitory concentration). Cell viability was measured in triplicate by sulforhodamine B (SRB) assay in all cell viability experiments below. 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 (bottom panel). Data are expressed 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 test the dose-response effects on mitotic arrest, DNA damage, and apoptosis markers. Western blot experiments compared the effects of the three drugs on these markers using the same amount of protein and the same 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 the quantification of spindle abnormalities as seen in D. The data represent the mean and standard deviation (SD) of three independent experiments (t-tests). *: p<0.05; **: p<0.01; ***: p<0.001. Significance was determined by comparison with the vehicle group. Figure 5G shows immunofluorescence staining of the 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 after 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] In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 6B]In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 6C]In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 6D]In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 6E]In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 6F]In general, Figure 6 shows compound 5 exhibiting significant anticancer activity in cell lines possessing the FGFR3-TACC3 fusion protein and in NCI-60 cancer cell lines. 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 in FGFR3-TACC3 fusion-expressing cell lines, RT112 and RT4. Cell viability was tested after a 3-day drug incubation and measured by SRB assay. Figure 6C is an image showing representative wells stained by SRB after treatment with the three inhibitors. Figure 6D shows Western blot analysis of mitotic arrest markers, p-histone H3 and ERK phosphorylation (Thr202 / Tyr204) in RT112 cells immediately after 24-hour treatment with different doses of TACC3 inhibitors. Figure 6E is a graph showing the mean GI50 value (M) determined from NCI-60 5 dose screening for compound 5. The black dotted line represents the 1 μM threshold. Figure 6F is a graph showing the correlation between the GI50 value of compound 5 and the TACC3 dependency score obtained from DepMap.org. A low score indicates a high probability that TACC3 is dependent on a given cell line. [Figure 7A]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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]This figure shows that, in general, TACC3 levels are important for the reaction of compound 5. Figure 7A shows the pharmacological inhibition of TACC3 by three different inhibitors and their effects on the cell viability of a normal mammary cell line, MCF-12A. Figure 7B shows that normal mammary epithelial cell lines, MCF-12A, and cancer cell lines with compound 5 IC50 levels were separated according to their subtype. Figure 7C shows the TACC3 protein levels of these cell lines. TACC3 protein levels were analyzed by Western blotting. GAPDH was used as a loading control. Figure 7D shows the colony formation assay of MCF-12A cells. Cells were transiently transfected for 48 hours with either the control or the TACC3 vector (250 ng). Transfected cells were later seeded in 12-well plates. After 12 days, cells were stained with crystal violet. Figure 7E shows that the number of colonies was counted and analyzed using ImageJ software. Data are expressed 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 the cell viability assay of MCF-12A cells overexpressing TACC3 after treatment with compound 5. Figure 7G shows the doubling time evaluation 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] This figure shows that, in general, compound 5 significantly impairs tumor growth more than SPL-B. Figure 8A shows tumor volume changes in JIMT-1 xenografts treated orally with the vehicle, 5 mg / kg of compound 5, or SPL-B for 30 days. Treatment was initiated when the tumor reached 90–100 mm3. Figure 8B shows percentage body weight changes in JIMT-1 xenografts followed up for 30 days. *: p<0.05; **: p<0.01; ***: p<0.001; ns: not significant. [Figure 8B]This figure shows that, in general, compound 5 significantly impairs tumor growth more than SPL-B. Figure 8A shows tumor volume changes in JIMT-1 xenografts treated orally with the vehicle, 5 mg / kg of compound 5, or SPL-B for 30 days. Treatment was initiated when the tumor reached 90–100 mm3. Figure 8B shows percentage body weight changes in JIMT-1 xenografts followed up for 30 days. *: p<0.05; **: p<0.01; ***: p<0.001; ns: not significant. [Figure 9A] In general, the figures show that different doses and routes of administration of compound 5 inhibit tumor growth in in vivo JIMT-1 xenografts without affecting mouse body weight. Figure 9A shows the change in tumor volume after application of all three groups of compound 5 at different doses and routes of administration. Figure 9B shows the change in mouse body weight during treatment at different doses and methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts after treatment with vehicle or 25 mg / kg of compound 5 in female nude mice. Treatment was started when the tumor 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] In general, the figures show that different doses and routes of administration of compound 5 inhibit tumor growth in in vivo JIMT-1 xenografts without affecting mouse body weight. Figure 9A shows the change in tumor volume after application of all three groups of compound 5 at different doses and routes of administration. Figure 9B shows the change in mouse body weight during treatment at different doses and methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts after treatment with vehicle or 25 mg / kg of compound 5 in female nude mice. Treatment was started when the tumor 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]In general, the figures show that different doses and routes of administration of compound 5 inhibit tumor growth in in vivo JIMT-1 xenografts without affecting mouse body weight. Figure 9A shows the change in tumor volume after application of all three groups of compound 5 at different doses and routes of administration. Figure 9B shows the change in mouse body weight during treatment at different doses and methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts after treatment with vehicle or 25 mg / kg of compound 5 in female nude mice. Treatment was started when the tumor 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] In general, the figures show that different doses and routes of administration of compound 5 inhibit tumor growth in in vivo JIMT-1 xenografts without affecting mouse body weight. Figure 9A shows the change in tumor volume after application of all three groups of compound 5 at different doses and routes of administration. Figure 9B shows the change in mouse body weight during treatment at different doses and methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts after treatment with vehicle or 25 mg / kg of compound 5 in female nude mice. Treatment was started when the tumor 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] In general, the figures show that different doses and routes of administration of compound 5 inhibit tumor growth in in vivo JIMT-1 xenografts without affecting mouse body weight. Figure 9A shows the change in tumor volume after application of all three groups of compound 5 at different doses and routes of administration. Figure 9B shows the change in mouse body weight during treatment at different doses and methods. Figure 9C shows the change in tumor volume of JIMT-1 xenografts after treatment with vehicle or 25 mg / kg of compound 5 in female nude mice. Treatment was started when the tumor 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] In general, the figures show that compound 5 significantly impairs tumor growth and improves the survival of immunocompetent mice. Figure 10A shows the tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly malignant syngeneic mouse mammary cancer model. Significance was calculated using multiple t-tests for each data point at each day compared to the vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows the Kaplan-Meier survival curves for EMT6 xenografts treated with the vehicle (median survival time 14 days) or compound 5 (median survival time 22 days). P-values ​​were calculated using the log-rank test. Figure 10C shows the percentage body weight change of EMT6 xenografts after treatment. [Figure 10B] In general, the figures show that compound 5 significantly impairs tumor growth and improves the survival of immunocompetent mice. Figure 10A shows the tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly malignant syngeneic mouse mammary cancer model. Significance was calculated using multiple t-tests for each data point at each day compared to the vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows the Kaplan-Meier survival curves for EMT6 xenografts treated with the vehicle (median survival time 14 days) or compound 5 (median survival time 22 days). P-values ​​were calculated using the log-rank test. Figure 10C shows the percentage body weight change of EMT6 xenografts after treatment. [Figure 10C]In general, the figures show that compound 5 significantly impairs tumor growth and improves the survival of immunocompetent mice. Figure 10A shows the tumor volume assessment of the effect of compound 5 on EMT6 xenografts, a highly malignant syngeneic mouse mammary cancer model. Significance was calculated using multiple t-tests for each data point at each day compared to the vehicle control. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 10B shows the Kaplan-Meier survival curves for EMT6 xenografts treated with the vehicle (median survival time 14 days) or compound 5 (median survival time 22 days). P-values ​​were calculated using the log-rank test. Figure 10C shows the percentage body weight change of EMT6 xenografts after treatment. [Figure 11A] In general, the figure shows that targeting TACC3 with compound 5 significantly impairs tumor growth in a mouse model of colorectal cancer. Figure 11A shows the tumor volume assessment of the effect of compound 5 on colorectal cancer xenografts in immunonatal (HCT-116) and syngeneically immunocompetent (CT-26) mouse models. Mice were orally administered daily with vehicle or compound 5 (25 and / or 50 mg / kg). Treatment was initiated when the tumor volume reached 90–100 mm3. Significance was calculated using Student's t-test. *: p<0.05; **: p<0.01. Figure 11B shows the percentage body weight change of HCT-116 and CT-26 xenografts after treatment. [Figure 11B] In general, the figure shows that targeting TACC3 with compound 5 significantly impairs tumor growth in a mouse model of colorectal cancer. Figure 11A shows the tumor volume assessment of the effect of compound 5 on colorectal cancer xenografts in immunonatal (HCT-116) and syngeneically immunocompetent (CT-26) mouse models. Mice were orally administered daily with vehicle or compound 5 (25 and / or 50 mg / kg). Treatment was initiated when the tumor volume reached 90–100 mm3. Significance was calculated using Student's t-test. *: p<0.05; **: p<0.01. Figure 11B shows the percentage body weight change of HCT-116 and CT-26 xenografts after treatment. [Figure 12A]This figure shows the general effect of compound 5 on TACC3 targeting of metastatic growth in the 4T1.luc2 model. Figure 12A shows bioluminescence (BLI) images of mice injected with 4T1.luc2 cells into the tail vein. After metastasis was established in the lungs, the mice were treated orally daily with either the vehicle or compound 5 (50 mg / kg). Metastatic growth was monitored weekly by BLI using IVIS (in vivo imaging system). Figure 12B shows Kaplan-Meier survival curves of 4T1.luc2 syngeneic models treated with the vehicle (median survival time 19 days) or compound 5 (median survival time 28 days). P-values ​​were calculated using the log-rank test. [Figure 12B] This figure shows the general effect of compound 5 on TACC3 targeting of metastatic growth in the 4T1.luc2 model. Figure 12A shows bioluminescence (BLI) images of mice injected with 4T1.luc2 cells into the tail vein. After metastasis was established in the lungs, the mice were treated orally daily with either the vehicle or compound 5 (50 mg / kg). Metastatic growth was monitored weekly by BLI using IVIS (in vivo imaging system). Figure 12B shows Kaplan-Meier survival curves of 4T1.luc2 syngeneic models treated with the vehicle (median survival time 19 days) or compound 5 (median survival time 28 days). P-values ​​were calculated using the log-rank test. [Figure 13A] This figure shows the general effects of high-dose compound 5 on the body weight and internal organs of mice. Figure 13A shows the body weight (g) of mice after daily oral administration of 100 mg / kg of compound 5 for 7 days. 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 is a photograph of mice treated with either the vehicle or 500 mg / kg of compound 5 and their organs (bottom panel). Mice were sacrificed 48 hours after drug treatment. [Figure 13B]This figure shows the general effects of high-dose compound 5 on the body weight and internal organs of mice. Figure 13A shows the body weight (g) of mice after daily oral administration of 100 mg / kg of compound 5 for 7 days. 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 is a photograph of mice treated with either the vehicle or 500 mg / kg of compound 5 and their organs (bottom panel). Mice were sacrificed 48 hours after drug treatment. [Figure 13C] This figure shows the general effects of high-dose compound 5 on the body weight and internal organs of mice. Figure 13A shows the body weight (g) of mice after daily oral administration of 100 mg / kg of compound 5 for 7 days. 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 is a photograph of mice treated with either the vehicle or 500 mg / kg of compound 5 and their organs (bottom panel). 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 hours (Buttar et al., 2010), b: partition coefficient in octanol / sodium phosphate buffer (50 mM, pH 7.4) (Unger et al., 1978), c: plasma protein binding evaluated by equilibrium dialysis in humans at 37°C (Buttar et al., 2010), dT1 / 2: half-life of mouse and human liver microsomes (MLM and HLM, respectively) (Kalvass et al., 2001), eClint: intrinsic clearance in MLM and HLM (Kalvass et al., 2001), fCYP: cytochrome P450 isozyme inhibition by compound 5 evaluated in HLM (Bourrie et al., 1996), gPapp: apparent permeability coefficient in Caco-2 cell monolayer (Camenisch et al., 1998). [Modes for carrying out the invention]

[0017] Elevated TACC3 levels have been observed in many different cancer types, making them a very attractive target for cancer therapy. TACC3 plays a crucial role in regulating microtubules and centrosomes and maintaining 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. Next, we analyzed different patient survival datasets and datasets (GSE31210 and TCGA) using different databases (METABRIC and KM Plotter). We found that high TACC3 levels were significantly correlated with worse overall survival in breast cancer (Figure 1B-1) and gastric cancer (Figure 1B-2). Furthermore, high TACC3 levels were associated with worse 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 showed that TACC3 expression is an independent prognostic factor in breast cancer (Figure 2). In summary, these results indicate that TACC3 is a clinically relevant target, associated with a strong prognosis in several different cancers, and that its expression level is a key factor defining disease severity and patient survival. Therefore, inhibiting TACC3 function is a promising therapeutic strategy for improving survival in breast cancer and other cancer patients.

[0019] Decreased TACC3 levels in Hela cells have been shown to induce mitotic arrest (Schneider et al., 2007) and caspase-dependent apoptosis (Kimura et al., 2013). In this disclosure, breast cancer patients expressing high TACC3 levels were found to have enhanced mitotic progression and DNA repair gene activity, supporting the oncogenic role of TACC3 in breast cancer progression (Figure 3A). Next, the inventors investigated the effect of TACC3 deficiency on cell viability in different breast cancer cell lines. Four different breast cancer cell line models with different molecular subtypes were used. The HER2-positive breast cancer cell line JIMT-1 and the luminal B subtype breast cancer cell line BT-474 T-DM1R (T-DM1 resistant) were shown to have high TACC3 levels (Saatci et al., 2018). Meanwhile, MDA-MB-436 and MDA-MB-157 are both trinegative breast cancer (TNBC) cell lines. In this disclosure, we first used small interfering RNA (siRNA) to reduce TACC3 levels in these cell lines. Figure 3B shows that overall TACC3 levels decreased 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 inhibition of breast cancer cell growth (Figure 3C). Next, we investigated the relevant mechanisms that may be triggered by 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] The inventors conducted an in-house screening of a series of small molecules by testing their antiproliferative effects in breast cancer cells that abnormally express TACC3 (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 tumorigenic potential (Saatci et al., 2018, Tanner et al., 2004) (explained in Figures 7 and 8-9, respectively). In experiments using cultured cells, the inventors found that compound 5 inhibited cell growth (at 190 nM IC50). 50 ), and it was revealed that it exhibits an antitumor effect. Therefore, by using this compound as a pharmacokinetic agent (API) for anticancer drugs, effective anticancer drugs can be developed. Accordingly, in order to obtain a novel compound that has anticancer activity against cancer cells expressing high levels of TACC3, a compound having the basic structure of formula (I) was synthesized.

[0021] In one embodiment, the present disclosure relates to a compound of formula (I), [ka] or with respect to a pharmaceutically acceptable salt thereof, in the formula, X1 is N or CR6. X2 is either N or CR3. R1 is either an aryl or heteroaryl. R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. R5 is a heterocycline, alkyl, or amino compound.

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

[0023] In certain embodiments, R1 is an aryl (e.g., phenyl). In other embodiments, R1 is a 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, amide, cyano, nitro, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl (e.g., methylsulfonyl), or sulfonamide. 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 relative to isoxazole. In other embodiments, the halo (e.g., F) is ortho relative to isoxazole. In other embodiments, the halo (e.g., F) is meta relative to isoxazole. In certain preferred embodiments, R1 is substituted with two halos (e.g., F). In certain embodiments, one halo (e.g., F) is meta relative to isoxazole, and one halo (e.g., F) is ortho relative to isoxazole. In other preferred embodiments, R1 is substituted with an alkyloxy (e.g., methoxy). In certain embodiments, the alkoxy (e.g., methoxy) is para relative to isoxazole. In other embodiments, the alkoxy (e.g., methoxy) is ortho relative to isoxazole. In certain embodiments, the alkoxy (e.g., methoxy) is meta relative to isoxazole. In yet another preferred embodiment, R1 is substituted with an alkyl (e.g., methyl, ethyl, or trifluoromethyl). In yet another preferred embodiment, R1 is substituted with a halo (e.g., fluoro) and an alkyloxy (e.g., methoxy).In a more preferred embodiment, R1 is substituted with a methoxy moiety and one or two fluoro moieties. In the most preferred embodiment, R1 is substituted with a methoxy moiety and two fluoro moieties. In certain embodiments, the alkoxy (e.g., methoxy) is para relative to the isoxazole, and F is meta relative to the isoxazole. In yet other modifications, the alkoxy (e.g., methoxy) is para relative to the isoxazole, and F is ortho relative to the isoxazole. In other embodiments, the halo (e.g., F) is para relative to the isoxazole, and the alkoxy (e.g., methoxy) is meta relative to the isoxazole. In yet another embodiment, the alkoxy (e.g., methoxy) is para relative to the isoxazole, one halo (e.g., F) is meta relative to the isoxazole, and one halo (e.g., F) is ortho relative 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 a halo (e.g., fluoro or chloro).

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

[0029] In certain embodiments, R4 is an alkyl group (e.g., methyl).

[0030] In certain embodiments, R5 is a heterocycline (e.g., azetidinyl, morpholino, pyrrolidinyl, piperazinyl, piperidinyl, oxaazabicyclooctanyl, oxaazabicycloheptnyl, thiomorpholino, thiomorpholino dioxide, hexahydrophyrrolyl, or azabicyclohexanyl). In certain embodiments, R5 is a 6-membered heterocycline, and the cycle skeleton contains one nitrogen. In certain embodiments, R5 is a 6-membered heterocycline, and the cycle skeleton contains one nitrogen and one oxygen. In other embodiments, R5 is a 7-membered heterocycline, and the cycle skeleton contains one nitrogen. In certain embodiments, R5 is a 7-membered heterocycline, and the cycle skeleton contains one nitrogen and one oxygen. In other embodiments, R5 is an 8-membered heterocycline, and the cycle skeleton contains one nitrogen. In specific embodiments, R5 is an 8-membered heterocycline, and the cycle skeleton contains one nitrogen and one oxygen. In certain preferred embodiments, R5 is a heterocycline-containing nitrogen, and the nitrogen is directly bonded to an aryl or heteroaryl ring having 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, amide, cyano, nitro, azide, 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, amide, cyano, nitro, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, sulfonamide, 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] The anticancer agents of this 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 monosubstituted or disubstituted 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, morpholine-4-amine, piperidine, tetrahydro-2H-pyran-4-amine, piperidine-1-amine N, 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 another embodiment, R1 is benzyloxy. In yet another embodiment, 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 relative to isoxazole. In certain embodiments, OCH3 is ortho relative to isoxazole. In certain embodiments, OCH3 is meta relative to isoxazole. In yet another preferred embodiment, R1 is substituted with F. In certain embodiments, R1 is substituted with one F. In certain embodiments, F is para relative to isoxazole. In certain embodiments, F is ortho relative to isoxazole. In certain embodiments, F is meta relative to isoxazole. In certain embodiments, R1 is substituted with two Fs. In certain embodiments, the first F is meta relative to isoxazole, and the second F is ortho relative to isoxazole. In a more preferred embodiment, R1 is substituted with OCH3 and F. In certain embodiments, OCH3 is para relative to isoxazole, and F is meta relative to isoxazole. In certain embodiments, OCH3 is para relative to isoxazole, and F is ortho relative to isoxazole. In certain embodiments, F is para relative to isoxazole, and OCH3 is meta relative to isoxazole. In a more preferred embodiment, R1 is substituted with OCH3 and two Fs. In certain embodiments, OCH3 is para relative to isoxazole, and both Fs are meta relative to isoxazole. In certain embodiments, OCH3 is para relative to the isoxazole, one F is meta relative to the isoxazole, and one F is ortho relative 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 another embodiment, R3 is Cl.

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

[0039] In certain embodiments, X1 is C. In other embodiments, X1 is N.

[0040] The specific final components of this disclosure are listed below. 3-(3-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 6) 3-(2-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 7) N-(2-morpholinopyrimidine-4-yl)-3-phenylisoxazole-5-amine (compound 8) 3-(4-ethoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 9) N-(2-Molfolinopyrimidine-4-yl)-3-(4-propoxyphenyl)isoxazole-5-amine (Compound 10) 3-(4-fluorophenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 11) 3-(4-chlorophenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 12) N-(2-morpholinopyrimidine-4-yl)-3-(p-tolyl)isoxazole-5-amine (compound 13) 3-(4-ethylphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 14) 3-(4-isopropylphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 15) N-(2-Molfolinopyrimidine-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazole-5-amine (Compound 16) 3-(4-(1,1-difluoroethyl)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 17) 3-(4-(difluoromethoxy)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 18) N-(2-Molfolinopyrimidine-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazole-5-amine (Compound 19) 3-(4-(methylthio)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 20) 3-(4-(dimethylamino)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 21) 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 22) 3-(3-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 23) 3-(3,4-dimethoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 24) 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 25) 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 26) 3-(6-methoxypyridine-3-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 27) 4-(5-((2-Molfolinopyrimidine-4-yl)amino)isoxazole-3-yl)phenol (compound 30) 3-(4-(benzyloxy)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 32) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (Compound 33) N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 34) N-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 35) N-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 36) N-(2-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 37) N-(2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 38) N-(2-(4,4-difluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 39) 3-(4-methoxyphenyl)-N-(2-thiomorpholinopyrimidine-4-yl)isoxazole-5-amine (compound 40) 4-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)thiomorpholine 1,1-dioxide (compound 41) 3-(4-methoxyphenyl)-N-(2-(piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 42) 3-(4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 43) 3-(4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 44) 3-(4-methoxyphenyl)-N-(2-(pyrrolidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 45) 2-(1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperidine-4-yl)ethane-1-ol (compound 46) 2-(4-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperazine-1-yl)ethane-1-ol (compound 47) 3-(4-methoxyphenyl)-N-(2-(4-methylpiperazine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 48) 3-(4-methoxyphenyl)-N-(2-(piperazin-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 49) Ethyl 1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperidine-4-carboxylate (compound 50) 1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-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 -(piperidine-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-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 59) N-(5-fluoro-2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 60) N-(2-(4-fluoropiperidine-1-yl)-6-methylpyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 61) 3-(4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine (compound 62) 3-(4-methoxyphenyl)-N-methyl-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 63) 3-(4-(dimethylamino)phenyl)-N-(2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)isoxazole-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-morpholinopyrimidine-4-yl)ethane-1,2-diamine (Compound 75) 2-((3-(4-methoxyphenyl)isoxazole-5-yl)(2-morpholinopyrimidine-4-yl)amino)acetonitrile (compound 76) 3-(5-methoxypyridine-2-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 77) 3-(4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine (compound 78) 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine (compound 79) 3-(4-methoxyphenyl)-N-(4-morpholino-1,3,5-triazine-2-yl)isoxazole-5-amine (compound 80) N 2 -(2,2-difluoroethyl)-N 4 -(3-(4-methoxyphenyl)isoxazole-5-yl)pyrimidine-2,4-diamine (compound 81) 2-((4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)amino)ethane-1-ol (Compound 82) 3-(2-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 83) 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 84) 3-(3,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 85) N-(2-Molfolinopyrimidine-4-yl)-3-(3,4,5-trimethoxyphenyl)isoxazole-5-amine (Compound 86) 3-(benzofuran-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 87) 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (Compound 88) 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)isoxazole-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)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 91) 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 92) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 93) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 94) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 95) N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (Compound 96) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetramethylmorpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 97) N-(2-(3,3-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 98) N-(2-(2,2-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 99) N-(2-(3,5-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 100) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-methylmorpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 101) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methylmorpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 102) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-(trifluoromethyl)morpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 103) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(tetrahydro-1H-fluoro[3,4-c]pyrrole-5(3H)-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 104) N-(2-(3-azabicyclo[3.1.0]hexane-3-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 105) N-(2-(7-azaspiro[3.5]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 106) N-(2-(4,4-dimethylpiperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 107) N-(2-(2-oxa-6-azaspiro[3,3]heptan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 108) N-(2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 109) N-(2-(6-azaspiro[2.5]octan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 110) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-8-azabicyclo[3.2.1]octan-8-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 111) N-(2-(2,2-difluoromorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 112) N-(2-(2-oxa-5-azabicyclo[2.2.2]octan-5-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 113) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-(trifluoromethyl)morpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 114) N-(2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 115) N-(2-(2-oxa-5-azabicyclo[4.1.0]heptan-5-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 116) N-(2-(4-oxa-7-azaspiro[2.5]octan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 117) N-(2-(7-oxa-4-azaspiro[2.5]octan-4-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 118) N-(2-(2-oxa-8-azaspiro[4.5]decane-8-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 119) N-(2-(2-oxa-6-azaspiro[3,4]octan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 120) N-(2-(2-oxa-7-azaspiro[3.5]nonanane-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 121) N-(2-(2-oxa-7-azaspiro[4,4]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 122) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetrafluoromorpholino)pyrimidine-4-yl)isoxazole-5-amine (compound 123) N-(2-(6-azabicyclo[3.1.1]heptan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 124) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-6-azabicyclo[3.1.1]heptan-6-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 125) N-(2-(3,5-dimethylpiperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 126) 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-isopropylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine (compound 127) N-(2-(4-(difluoromethyl)piperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 128) N-(2-(6-azaspiro[3.5]nonan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 129) N-(2-(2-azaspiro[3.5]nonanane-2-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 130) 3-(2-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 131)

[0041] The intermediates of each element in this disclosure are shown below. N-(2-chloropyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 4a) N-(2-chloropyrimidine-4-yl)-3-(3-methoxyphenyl)isoxazole-5-amine (compound 4b) N-(2-chloropyrimidine-4-yl)-3-(2-methoxyphenyl)isoxazole-5-amine (compound 4c) N-(2-chloropyrimidine-4-yl)-3-phenylisoxazole-5-amine (compound 4d) N-(2-chloropyrimidine-4-yl)-3-(4-ethoxyphenyl)isoxazole-5-amine (compound 4e) N-(2-chloropyrimidine-4-yl)-3-(4-propoxyphenyl)isoxazole-5-amine (compound 4f) N-(2-chloropyrimidine-4-yl)-3-(4-fluorophenyl)isoxazole-5-amine (compound 4g) 3-(4-chlorophenyl)-N-(2-chloropyrimidine-4-yl)isoxazole-5-amine (compound 4h) N-(2-chloropyrimidine-4-yl)-3-(p-tolyl)isoxazole-5-amine (compound 4i) N-(2-chloropyrimidine-4-yl)-3-(4-ethylphenyl)isoxazole-5-amine (compound 4j) N-(2-chloropyrimidine-4-yl)-3-(4-isopropylphenyl)isoxazole-5-amine (compound 4k) N-(2-chloropyrimidine-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazole-5-amine (compound 4l) N-(2-chloropyrimidine-4-yl)-3-(4-(1,1-difluoroethyl)phenyl)isoxazole-5-amine (compound 4m) N-(2-chloropyrimidine-4-yl)-3-(4-(difluoromethoxy)phenyl)isoxazole-5-amine (compound 4n) N-(2-chloropyrimidine-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazole-5-amine (compound 4o) N-(2-chloropyrimidine-4-yl)-3-(4-(methylthio)phenyl)isoxazole-5-amine (compound 4p) N-(2-chloropyrimidine-4-yl)-3-(4-(dimethylamino)phenyl)isoxazole-5-amine (compound 4r) N-(2-chloropyrimidine-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 4s) 3-(3-chloro-4-methoxyphenyl)-N-(2-chloropyrimidine-4-yl)isoxazole-5-amine (compound 4t) N-(2-chloropyrimidine-4-yl)-3-(3,4-dimethoxyphenyl)isoxazole-5-amine (compound 4u) N-(2-chloropyrimidine-4-yl)-3-(2,3-difluoro-4-methoxyphenyl)isoxazole-5-amine (compound 4v) 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-chloropyrimidine-4-yl)isoxazole-5-amine (compound 4y) N-(2-chloropyrimidine-4-yl)-3-(6-methoxypyridine-3-yl)isoxazole-5-amine (compound 4z) 3-(4-methoxyphenyl)-N-(2-(methylthio)pyrimidine-4-yl)isoxazole-5-amine (compound 28) 4-(5-((2-chloropyrimidine-4-yl)amino)isoxazole-3-yl)phenol (compound 29) 3-(4-(benzyloxy)phenyl)-N-(2-chloropyrimidine-4-yl)isoxazole-5-amine (compound 31) N-(2,5-dichloropyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 55) N-(2-chloro-5-fluoropyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 56) N-(2-chloro-6-methylpyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 57) N-(2-chloropyridine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine (compound 58) N-(2-chloropyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine (compound 132) N-(2-chloropyrimidine-4-yl)-3-(2,5-difluoro-4-methoxyphenyl)isoxazole-5-amine (compound 133)

[0042] The anticancer agents of this disclosure include compounds of general formula (I) represented by the following chemical structures of the specific final compounds of this disclosure. [ka] [ka] [ka] [ka]

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

[0044] The compounds shown above are novel compounds synthesized by the present inventors. The synthesis methods for these novel compounds are described below. Representative compounds of this disclosure synthesized by the general synthesis methods described below are shown more specifically in the following scheme. Since the scheme is illustrative, the disclosure should not be construed as being limited by the chemical reactions and conditions expressed. The preparation of the various starting materials used in the scheme is within the scope of the skills of a person familiar with the art. The compounds of formula (I) or substituents in that form, as shown in the following scheme, are as already defined herein.

[0045] In another aspect, the Disclosure provides a pharmaceutical composition comprising the compounds disclosed herein and pharmaceutically acceptable excipients.

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

[0047] In yet another embodiment, the Disclosure provides a method for treating a target cancer, comprising administering a compound or a pharmaceutically acceptable salt thereof described in any one of claims 1 to 53 to the target. In certain embodiments, the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia, kidney 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 relates to the compound of the present disclosure represented by Scheme I, [ka] or a method for preparing a pharmaceutically acceptable salt thereof, in the formula, X1 is N or CR6. X2 is either N or CR3. R1 is either an aryl or heteroaryl. R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. R5 is a heterocyclyl, alkyl, or amino compound. R 51 It is a halo, R 52 It is a heterocycline or alkyl group, X 10 It is a base, X 11 It is a precious metal catalyst, X 12 It is a phosphine ligand.

[0049] In certain embodiments, the base is a carbonate, oxide, tertiary amine, secondary amine, or 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 precious metal catalyst is a palladium catalyst (e.g., palladium-II acetate).

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

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

[0053] In certain embodiments, this method further includes heating.

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

[0055] Pharmaceutical composition The compositions and methods of this disclosure can be used to treat individuals in need of treatment. In certain embodiments, the individuals are mammals such as humans, or non-human mammals. When administered to animals such as humans, the compositions or compounds are preferably administered as a pharmaceutical composition comprising, for example, the compounds of this 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 or glycerols, oils such as olive oil, or injectable organic esters. In preferred embodiments, if such a pharmaceutical composition is for human administration, particularly for invasive administration routes (i.e., routes such as injection or transplantation that avoid transport or diffusion across the epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to result in delayed release of the drug or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions may be in the form of dosage units such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, reconstituted solutions, powders, liquids, syrups, suppositories, and injections. Compositions may also be present in transdermal delivery systems, such as skin patches. Compositions may also be present in solutions suitable for topical administration, such as lotions, creams, or ointments.

[0056] A pharmaceutically acceptable carrier may include, for example, a physiologically acceptable agent that acts to stabilize, increase the solubility of, or increase the absorption of a compound, such as the compounds of this 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 selection 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 may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, for example, in which the compounds of this disclosure may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to manufacture and administer.

[0057] The phrase “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems and complications, and that is commensurate with a reasonable benefit / risk ratio, within the bounds of sound medical judgment.

[0058] 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. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that may 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. Examples include (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, suitable substances used in pharmaceutical formulations.

[0059] Pharmaceutical compositions (preparations) can be administered to a target by any of several routes of administration, including, for example, oral (e.g., liquids or non-liquids or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses for application to the tongue, powders, granules, pastes); absorption via the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment or spray applied to the skin). Compounds may also be prepared for inhalation. In certain embodiments, compounds may be simply dissolved or suspended in sterile water. Details of suitable routes of administration and suitable compositions are described, for example, in U.S. Patents 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 forms and can be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is the amount of the compound that produces the therapeutic effect. Generally, out of 100 percent, this amount is in the range of about 1 percent to about 99 percent of the active ingredient, preferably about 5 percent to about 70 percent, and most preferably about 10 percent to about 30 percent.

[0061] Methods for preparing these formulations or compositions include the step of binding an active compound, such as the compounds of the Disclosure, to a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by uniformly and closely binding the compounds of the Disclosure to a liquid carrier or a finely divided solid carrier, or both, and then shaping the product as needed.

[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 flavoring base, usually sucrose and acacia or tragacanth), liquids, powders, granules, or as liquids or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions, or as licks or syrups, or as lozenges (using inert bases such as gelatin and glycerin, or sucrose and acacia) and / or mouthwashes, each containing a predetermined amount of the compound of the present disclosure as an active ingredient. Compositions or compounds may also be administered as boluses, licks or pastes.

[0063] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient is mixed with sodium citrate or dicalcium phosphate and / or one or more pharmaceutically acceptable carriers, such as: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol. (4) Disintegrants such as 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) Colorants. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain buffers. Similar types of solid compositions can also be used as fillers for soft and hard-filled gelatin capsules with the use of excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0064] Tablets can be prepared by compression or molding, using one or more optional auxiliary components. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose sodium), surfactants, or dispersants. Wet tablets can be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent using appropriate machinery.

[0065] Tablets, and other solid dosage forms of pharmaceutical compositions such as sugar-coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be prepared with scoring or coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulation field. They may also be prepared to provide sustained or controlled release of the active ingredient therein, for example, using oxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various ratios to yield a desired release profile. They may be sterilized, for example, by filtration with a bacterial-retaining filter or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injection medium immediately before use. These compositions may also optionally contain opacifiers, which may optionally release the active ingredient only, or preferentially, in a delayed manner in a specific part of the gastrointestinal tract. Examples of usable embedding compositions include polymers and waxes. The active ingredient may also be in microencapsulated form, together with one or more of the excipients described above, where appropriate.

[0066] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, parental solutions for reconstitution, microemulsifies, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor oil, sesame oil), glycerol, tetrahydrofuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof.

[0067] In addition to inert diluents, oral compositions may also contain auxiliary agents such as humectants, emulsifiers and suspending agents, sweeteners, flavorings, colorants, fragrances and preservatives.

[0068] In addition to the active compound, the suspension may include suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, metahydroxyaluminum, bentonite, 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 as needed.

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

[0071] Powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof, in addition to the active compound. Sprays may also contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

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

[0073] As used herein, the phrases “parenteral administration” and “administered parenterally” typically refer to modes of administration other than enteral and topical administration by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intrathecal, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Pharmaceutical compositions suitable for parenteral administration include one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or one or more active compounds in combination with a sterile powder, which may be reconstituted into a sterile injection solution or dispersion immediately before use, and may contain antioxidants, buffers, bacteriostatic agents, or solvents that make the formulation isotonic with the blood or suspension or thickener of the recipient of the intended purpose.

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

[0075] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, sustained absorption of injectable dosage forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0076] In some cases, it is desirable to slow down the absorption of a drug from subcutaneous or intramuscular injection in order to maintain the drug's effect. This can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. Then, the absorption rate of the drug depends on its dissolution rate, which in turn may depend on the crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oily vehicle.

[0077] Injectable depot formulations are prepared by forming a microencapsulation matrix of the target compound with 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. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot-injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsifies that are compatible with body tissues.

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

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

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

[0081] The selected dosage level depends on a variety of factors, including those well known in the medical field, such as the activity of the specific compound or combination of compounds used, or their esters, salts, or amides; the route of administration; the time of administration; the elimination rate of the specific compound used; the duration of treatment; other drugs, compounds, and / or substances used in combination with the specific compound used; and the age, sex, weight, condition, general health status, and prior medical history of the patient being treated.

[0082] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the required therapeutically effective dose of a pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the pharmaceutical composition or compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. "Therapeutically effective dose" means a concentration of the compound sufficient to produce the desired therapeutic effect. It is generally understood that the effective dose of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that may affect the effective dose 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 with the compound of this disclosure. Multiple doses of a drug can deliver many total doses. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th ed., 1814-1882, incorporated herein by reference).

[0083] Generally, the appropriate daily dose of the active compound used in the compositions and methods of this disclosure is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose generally depends on the factors described above.

[0084] If necessary, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more subdoses, optionally administered separately in unit dosage forms at appropriate intervals throughout the day. In certain embodiments of this disclosure, the active compound may be administered twice or three times daily. In preferred embodiments, the active compound is administered once daily.

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

[0086] In certain embodiments, the compounds of the present disclosure may be used alone or administered in combination with other types of therapeutic agents.

[0087] This disclosure includes the use of pharmaceutically acceptable salts of the compounds of this disclosure in the compositions and methods of this disclosure. In certain embodiments, the salts intended for this disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, the salts intended for this disclosure include, but are not limited to, L-arginine, venentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydravamin, 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, the salts intended for this disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, the salts intended for use in this 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, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, and d-glucoheptoic acid. Examples include, but are not limited to, nic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphate, glycolic acid, hypric 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 with water, methanol, ethanol, dimethylformamide, and others. Mixtures of such solvates can also be prepared. The source of such solvates may be from the crystallization solvent, specific to the preparation or crystallization solvent, or indeterminate to such solvent.

[0089] Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may 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 meanings generally understood by those of an ordinary skill in the art. Generally, the nomenclature and techniques used in relation to chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry as described herein are well known and commonly used in the art.

[0092] The methods and techniques described herein are generally known in the art unless otherwise indicated, and are carried out in accordance with the conventional methods described in various general and more specific references cited and discussed throughout this specification. 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.”, WH Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, WH 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 their 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 foregoing, and other publications, patents, and published patent applications referenced in this application, are incorporated herein by reference. In case of any conflict, this specification, including specific definitions, shall prevail.

[0095] The term “agent” is used herein to refer to compounds (such as organic or inorganic compounds, mixtures of compounds), biological macromolecules (such as nucleic acids, antibodies, parts thereof, as well as humanized, chimeric and human antibodies and monoclonal antibodies, proteins or parts thereof, e.g., peptides, lipids, carbohydrates), or extracts made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals). Agents include, for example, agents with known structures and agents with unknown structures. The ability of such agents to inhibit or promote AR degradation may make them suitable as “therapeutic agents” in the methods and compositions of this disclosure.

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

[0097] "To treat" a condition or patient means to take measures to obtain a beneficial or desirable outcome, including clinical outcomes. As used herein, "treatment," as is well understood in the art, is an approach to obtain a beneficial or desirable outcome, including clinical outcomes. Beneficial or desirable clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of the disease; a stable (i.e., non-worsening) state of the disease; prevention of disease progression; delay or slowing of disease progression; improvement or temporary relief of the condition; and remission (partial or total). "Treatment" may also mean extending survival compared to the survival expected without treatment.

[0098] The term “prevent” is recognized in the art and is well understood in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, complex syndromes such as heart failure, or other medical conditions, and includes the administration of a composition that reduces the frequency of symptoms of a medical condition in a subject or delays the onset of the condition compared to a subject who does not receive the composition. Thus, cancer prevention 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 the treated population by a statistically and / or clinically significant amount compared to an untreated control population.

[0099] The “administration” or “dosage” of a substance, compound, or drug to a target can be carried out using one of the various methods known in the art. For example, a compound or drug can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, orally, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through the skin canal). The compound or drug can also be appropriately introduced by rechargeable or biodegradable polymer devices or other devices, e.g., patches and pumps, or formulations that provide sustained, sustained-release, or controlled release of the compound or drug. Dosage can also be carried out, for example, once, multiple times, and / or over a period of time longer than one.

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

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

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

[0103] Where used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may or may not occur, and the descriptions include both cases in which the events or circumstances occur and cases in which they do not. For example, “optionally substituted alkyl” means both cases in which the alkyl can be substituted and cases in which the alkyl is not substituted.

[0104] The substituents and substitution patterns on the compounds of this 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 known techniques in the art and by the methods described below. If the substituent itself is substituted with multiple groups, it is understood that these multiple groups may be on the same carbon or different carbons, as long as a stable structure is obtained.

[0105] As used herein, the term “optionally substituted” means substituting 1 to 6 hydrogen radicals in a given structure with radicals of specific substituents, including but not limited to hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” means substituting 1 to 4 hydrogen radicals in a given structure with the above substituents. More preferably, 1 to 3 hydrogen radicals are replaced by the above substituents. It is understood that further substitutions of substituents may be possible.

[0106] As used herein, the term "alkyl" refers to C1-C 10 Linear alkyl groups or C1-C 10 This refers to saturated aliphatic groups, including but not limited to branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 linear alkyl group or a C1-C6 branched alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 linear alkyl group or a C1-C4 branched 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. The "alkyl" group can be optionally substituted.

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

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

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

[0110] The term "alkoxy" refers to an alkyl group to which oxygen is bonded. Typical alkoxyl groups include methoxy, ethoxy, propoxy, and tert-butoxy.

[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 linear alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, linear or branched alkyl groups have 30 or fewer (for example, C1-C1 for linear groups) in their skeleton. 30 Regarding the branching chain, C3~C 30 ), more preferably having 20 or fewer carbon atoms.

[0113] Furthermore, as used herein, in the examples and throughout the claims, the term “alkyl” is intended to include both unsubstituted and substituted alkyl groups, the latter referring to alkyl moieties having substituents that substitute hydrogens on one or more carbons of a hydrocarbon skeleton, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0114] "C x~y " or "C x ~C yThe term "alkyl" means a group containing x to y carbon atoms in a chain, when used in combination with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. C0 alkyl indicates a hydrogen atom at a terminal position, while internal hydrogen atoms indicate a bond. For example, C 1~6 Alkyl groups contain 1 to 6 carbon atoms in their chain.

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

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

[0117] The term "amide" as used herein refers to the base [ka] It refers to, In the formula, R 9 and R 10 Each of these independently represents either a hydrogen atom or a hydrocarbyl group, or R 9 and R 10 These atoms, together with the N atoms to which they are bonded, complete a heterocycle with 4 to 8 atoms in the ring structure.

[0118] The terms "amine" and "amino" are recognized in the art, and include both unsubstituted and substituted amines, their salts, for example, formula [ka] This refers to the part that can be represented by, It refers to, and in the formula, R 9 , R 10 , and R 10‘ Each of these independently represents either a hydrogen atom or a hydrocarbyl group, or R 9 and R 10These atoms, together with the N atoms to which they are bonded, complete a heterocycle with 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 recognized in the art and refers to an alkyl group substituted with an aryl group.

[0121] As used herein, the term “aryl” includes substituted or unsubstituted monocyclic 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, in which two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of aryl groups include benzene, naphthalene, phenanthrene, phenol, and aniline.

[0122] The term "carbamate" is recognized in the art, and is based on [ka] It refers to, In the formula, R 9 and R 10 This independently represents a hydrogen atom or a hydrocarbyl group.

[0123] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0124] The term "carbocyclic ring" includes monocyclic rings with 5 to 7 members and bicyclic rings with 8 to 12 members. Each ring in a bicyclic carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. Carbocyclic rings include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "condensed carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring in a condensed carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. In exemplary embodiments, an aromatic ring, e.g., phenyl, may be condensed to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings, as long as the valence allows, is included in the definition of a carbocyclic ring. Exemplary “carbocyclic” compounds include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octa-3-ene, naphthalene, and adamantane. Exemplary condensed carbocyclic compounds 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 “carbocyclic” compound can be substituted at any one or more positions that can retain a hydrogen atom.

[0125] As used herein, the term "carbocykrylalkyl" refers to an alkyl group substituted with a carbocyclic group.

[0126] The term "carbonate" is recognized in the art, and the base -OCO 2- It refers to.

[0127] As used herein, the term "carboxyl" refers to the group represented by the formula -CO2H.

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

[0129] As used herein, the term "ether" refers to a hydrocarbyl group bonded to another hydrocarbyl group through oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether can be either symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocyclic-O-heterocyclic and aryl-O-heterocyclic. The ether includes an "alkoxyalkyl" group, which can be represented by the general formula alkyl-O-alkyl.

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

[0131] As used herein, the terms "heteroalkyl" and "heteroarylalkyl" refer to an alkyl group substituted with a heteroaryl group.

[0132] The terms "heteroaryl" and "heteroaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, and the ring structure includes at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "heteroaryl" 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 a heteroaromatic ring. For example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocylyl. Examples of heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

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

[0134] As used herein, the term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group.

[0135] The terms “heterocyclyl,” “heterocyclic,” and “heterocyclic formula” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, the ring structure containing at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms “heterocyclyl” and “heterocyclic” 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, for example, the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like.

[0136] As used herein, the term "hydrocarbyl" refers to a group that is bonded via a carbon atom without an =O or =S substituent, and typically has at least one carbon-hydrogen bond and is primarily a carbon skeleton, but can optionally include a heteroatom. Therefore, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but not substituents such as acetyl (which has an =O substituent on the bonded carbon) and ethoxy (which is bonded via oxygen rather than carbon). 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 hydroxyl group.

[0138] When used in combination with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term “lower” means that the substituent has 10 or fewer atoms, preferably 6 or fewer. For example, “lower alkyl” refers to an alkyl group having 10 or fewer carbon atoms, preferably 6 or fewer. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are, for example, hydroxyalkyl and aralkyl in the reference (in this case, for example, atoms in the aryl group are not counted when counting the carbon atoms of the alkyl substituent), lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, 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 in a polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring in a polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms.

[0140] The term "sulfate" is recognized in the art and refers to the group -OSO3H or its pharmaceutically acceptable salts.

[0141] The term "sulfonamide" is recognized in the art and has a general formula. [ka] This refers to the base represented by, In the formula, R 9 and R 10 This independently represents hydrogen or hydrocarbyl.

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

[0143] The term "sulfonate" is recognized in the art and refers to the SO3H group or its pharmaceutically acceptable salts.

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

[0145] The term “substituted” refers to a portion having substituents that substitute for hydrogens on one or more carbons of a skeleton. “Substituted” or “substituted with ~” will be understood to imply the implicit condition that such substitutions conform to the allowable valencies of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformations such as rearrangement, cyclization, or elimination. As used herein, the term “substituted” is intended to include all allowable substituents of an organic compound. In broad embodiments, allowable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. There may be one or more allowable substituents, which may be the same or different for a given organic compound. For the purposes of this disclosure, a heteroatom such as nitrogen may have hydrogen substituents and / or any allowable substituents of the organic compounds described herein that satisfy the valency of the heteroatom. Substituents may include any substituents described herein, such as halogens, hydroxyls, carbonyls (carboxyls, alkoxycarbonyls, formyls, or acyls, etc.), thiocarbonyls (thioesters, thioacetates, or thioformates, etc.), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that any substituted moieties on a hydrocarbon chain may, where appropriate, be substituted by themselves.

[0146] As used herein, the term "thioalkyl" 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 and refers to wherein R 9 represents a hydrocarbyl.

[0148] As used herein, the term "thioether" is equivalent to an ether in which oxygen is replaced by sulfur.

[0149] The term "urea" is recognized in the art and can be represented by the general formula

Chemical formula

[0150] As used herein, the term "modulate" includes inhibition or suppression of a function or activity (such as cell proliferation), as well as enhancement of a function or activity.

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

[0152] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to acid addition salts or basic addition salts that are suitable or compatible for the treatment of patients.

[0153] As used herein, the term “pharmaceutically acceptable acid addition salt” means any non-toxic organic or inorganic salt of any base compound represented by formula I. Exemplary inorganic acids that form suitable salts include hydrochlorides, hydrobroms, sulfates, and phosphates, as well as metal salts such as sodium monohydrogen orthophosphate and potassium bisulfate. Exemplary organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, 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. They can form either mono or dicarboxylic acids, and such salts may exist in hydrate, solvated, or substantially anhydrous forms. Generally, 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 appropriate salts 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 pharmaceutically acceptable acid addition salts.

[0154] As used herein, the term “pharmaceutically acceptable basic addition salt” means 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 methylamine, trimethylamine, and aliphatic, alicyclic, or aromatic organic amines such as picoline or ammonia. The selection of suitable salts is known to those skilled in the art.

[0155] Many of the compounds useful for the methods and compositions of this disclosure have at least one stereocenter in their structure. This stereocenter may be present in an R or S configuration, and the R and S notations are used in accordance with the rules set out in Pure Appl. Chem. (1976), 45, 11-30. This disclosure intends all stereoisomeric forms, such as enantiomers and diastereoisomers, 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 may exist as Z (tuzamen) or E (entgegen) isomers. In each case, this disclosure includes both mixtures and distinct individual isomers.

[0157] Some compounds may also exist in tautomer form. Such forms, though not explicitly shown in the formulas described herein, are intended to be included within the scope of this disclosure.

[0158] A “prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that, after administration, is metabolized in the host, for example, by hydrolysis or oxidation, to form the compounds of the Disclosure (e.g., compounds of formula I). ​​Typical examples of prodrugs include compounds having a biologically unstable 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 using esters or phosphoramic acids as biologically unstable or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, which are incorporated herein by reference. The prodrugs of the Disclosure are metabolized to produce compounds of formula I. The Disclosure, to its extent, includes prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of appropriate 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 filter, diluent, excipient, solvent, or encapsulating material, that is useful for preparing 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. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility often results in insufficient absorption. The LogS value is the unit strip logarithm (decimal) of solubility measured in mol / liter. [Examples]

[0161] The present invention is described in general terms here, but will be more readily understood by referring to the following examples, which are included solely for illustrative purposes of specific aspects and embodiments of the invention and are not intended to limit the present disclosure.

[0162] Example 1: Exemplary synthesis of the disclosed compound Derivatives with R1 modification in formula (I) where R2, R3, and R4 are H and R5 is morpholine. The synthesis methods for compounds 5-27 are shown in Scheme 1. These include the following process steps. i) Compounds 1a to z were produced by esterifying a suitable acid derivative under reflux in EtOH (ethanol) in the presence of SOCl2 (thionyl chloride). ii) Solutions of compounds 1a-z in toluene were refluxed with acetonitrile in the presence of NaH (sodium hydride; dispersed in 60% mineral oil) to obtain β-ketonitrile derivatives 2a-z. iii) Compounds 2a-z were reacted with H2N.OH HCl (hydroxylamine hydrochloride) in an aqueous solution of NaOH (sodium hydroxide) at 100°C to obtain isoxazole-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 obtain 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 hours, (B) NaH, MeCN, toluene, reflux, 2 hours, (C) H2N.OH HCl, NaOH, H2O, reflux, 4 hours, (D) t-BuOK, 2,4-dichloropyrimidine, t-BuOH, room temperature, 24 hours, (E) t-BuOK, 4-chloro-2-(methylthio)pyrimidine, t-BuOH, room temperature, 24 hours, (F) Morpholine, n-BuOH, reflux, 5 hours.

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

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

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

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

[0167] In formula (I), derivatives having pyrimidine ring (X, R3, R4 substitution) modification where R1 is 4-phenylmethoxy, R2 is H, and R5 is morpholine or 4-fluoropiperidine. For derivatives having pyrimidine ring modifications, the synthetic procedure outlined in Scheme 4 was used. Using compound 3a as a starting material, nucleophilic aromatic substitution reactions were carried out 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 obtain final compounds 59-62.

[0168] The synthesis methods for compounds 59-62 are shown in Scheme 4. These include the following process steps. i) Compound 3a was reacted with a suitable pyrimidine derivative in t-BuOH at room temperature in the presence of t-BuOK to obtain compounds 55-57 (Method A). Compound 3a was reacted with 2,4-dichloropyridine in DMA (N,N-dimethylacetamide) at room temperature in the presence of NaH to obtain compound 58 (Method B). ii) Compounds 55-57 were reacted with 4-fluoropiperidine HCl in n-BuOH in the presence of DIPEA to produce compounds 59-61 (Method C). iii) Compound 58 was treated with morpholine in dioxane in the presence of xanthophos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthene), Cs2CO3 (cesium carbonate), and Pd(OAc)2 (palladium(II) acetate) for 30 minutes in a microwave oven at 100°C to obtain compound 62 (Method D). [ka] Scheme 4. Synthesis of compounds 59-62. Reagents and conditions: (A) Suitable 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, Cs2CO3, xanthophos, Pd(OAc)2, dioxane, 100°C, 30 minutes, microwave oven.

[0169] N-bridge methylation (R2 substitution) when 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 alkylated to obtain the N-methylated derivative 63.

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

[0171] Synthesis of compounds with different combinations of R1 and R5 in formula (I) when R2, R3 and R4 are H Results from cytotoxicity tests showed that R1 and R2 of several compounds with potent activity were bound to a new molecule, resulting in a novel compound. The synthesis of the hybrid compound utilized the synthetic procedure outlined in Scheme 6.

[0172] The synthesis methods for compounds 64-67 are shown in Scheme 6. These include the following process steps. i) Compound 4r or 4s and a suitable amine derivative in BuOH were refluxed to obtain the final compound. If the amine derivative was in the form of a salt, a suitable HCl salt of the amine was dissolved in BuOH in the presence of DIPEA (N,N-diisopropylethylamine) to obtain the free amine, which was then reacted with compound 4r or 4s under reflux to obtain the final compounds 64-66 (Method A). ii) If the amine derivative is not in the form of an HCl salt, compound 4s is treated with the amine derivative in n-BuOH to obtain the final compound 67 (Method B). [ka] Scheme 6. Synthesis of the compound 64-67. Reagents and conditions: (A) Suitable amine derivative, DIPEA, n-BuOH, reflux, 5 hours; (B) Suitable amine derivative, n-BuOH, reflux, 5 hours.

[0173] Embodiments of the present disclosure include the chemical structures of original intermediate compounds that react with amine derivatives described herein for the synthesis of 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 this disclosure are characterized by the features listed in Table 2. 1 H NMR / 13 Contains compounds with 13C NMR properties. [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 the compounds in this disclosure to treat TACC3-mediated cancer, or related conditions or complications thereof, was determined using the following procedure. The activity of the compounds in this disclosure towards inducing cell death was tested using the JIMT-1 cell line, a cell line with high levels of TACC3. The concentration of each compound required for a maximum 50% 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 this disclosure were determined to have a significant inhibitory effect on cell growth. [Table 3-1] [Table 3-2]

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

[0177] To determine whether compound 5 targets TACC3, this disclosure performed a cell thermal shift assay (CETSA) based on drug target stabilization by temperature increase (Martinez Molina et al., 2013). For this purpose, JIMT-1 cells containing a vehicle, compound 5, or SPL-B (as a positive control) were incubated for 6 hours, and then cell lysates were collected. Treatment of JIMT-1 breast cancer cells with compound 5 significantly stabilized cellular TACC3 upon temperature increase, indicating that compound 5 may specifically interact with TACC3 in JIMT-1 cells (Figure 4A). The thermal fusion curves of the protein also show the thermal shift between band intensities treated with the vehicle and compound 5. This interaction was also confirmed by isothermal titration calorimeter (ITC). Titration of TACC3 to compound 5 was performed and monitored by recording the calorimetry and thermal changes. As shown in Figure 4B, the thermodynamic parameters of compound 5 binding to TACC3 at 25°C (K d The compound (1.5nM; ΔH: 4.929E7cal / mol; ΔS: 1.65E5cal / mol / deg, N: 0.704) indicates the interaction between these two molecules. Binding of compound 5 to TACC3 was finally confirmed by drug affinity-responsive target stability (DARTS). DARTS is a label-free strategy for identifying potential direct protein targets of small molecules, based on immediate stabilization of the small molecule after binding and protection of the target protein from proteolysis (Lomenick, Jung et al. 2011, Pai, Lomenick et al. 2015). As shown in Figure 6C, the TACC3 protein was stabilized during incubation with compound 5 in the presence of pronase (Figure 4D).

[0178] Subsequently, the relative effects of compound 5, a TACC3 inhibitor, on cell viability were compared with those of the available TACC3 inhibitors KHS101 and SPL-B. 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 showed significantly lower IC50 than the two available TACC3 inhibitors in all tested cell lines. 50A value was found to be present (Figure 5A). This significant decrease in cell viability of various cancer cell lines treated with compound 5 compared to KHS101 and SPL-B was also confirmed in a colony formation assay using JIMT-1 cells. As a result, the mean number of colonies of JIMT-1 cells treated with compound 5 was significantly lower than that of cells treated with the other two inhibitors at the same dose (Figure 5B). Therefore, the lower cell viability detected in the SRB assay and the lower number of colonies 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 tested immediately after treatment with compound 5, SPL-B, and KHS101. The analytical results using compound 5 showed high agreement with experimental results regarding the suppression of TACC3 expression using siRNA, further suggesting that compound 5 targets TACC3. As a result, mitotic arrest, DNA damage, and apoptosis induced by siTACC3 can be reproduced in a dose-dependent manner by compound 5 (Figure 5C). On the other hand, similar induction levels of these markers by SPL-B and KHS101 treatment were observed at considerably higher doses compared to cells treated with compound 5. Although this included higher doses of KHS101 than used by others (Campo & Breuer, 2018), induction of these markers was only observed at very high doses. Clearly, these results are in line with the cell viability data and ICs obtained through treatment with the three inhibitors. 50 The values ​​are consistent (see 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 in the nM working dose range, and 2) its specificity for similar molecular changes obtained by TACC3 downregulation using siRNA.

[0179] Next, the inventors tested the potential disruption of the mitotic spindle as a result of TACC3 inhibition. This has been previously 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 prominent phenotype observed during TACC3 inhibition with compound 5 was the formation of multipolar spindles characterized by improperly aligned chromosomes (73.4% frequency with the highest dose treatment) in metaphase plates in JIMT-1 cells (Figure 5F). In the presence of spindle defects, the spindle formation checkpoint (SAC) is activated, and mitosis is further arrested, providing time necessary to repair the spindle defects (Musacchio and Salmon 2007). To demonstrate SAC activation during TACC3 inhibition, immunofluorescence staining for BuBR1 (Chen 2002), a marker of SAC activation, was performed. In JIMT-1 cells treated with compound 5, increased localization of BuBR1 to chromosomes was observed immediately after compound 5 treatment (Figure 5G). To further investigate the contribution of active SAC signaling to compound 5-induced mitotic arrest, DNA damage, and cell death, SAC kinase Mps1 was inhibited using a specific inhibitor (TC Mps1 (Choi, Min et al. 2017)) in cells treated with compound 5, and the expression of relevant markers was analyzed. As shown in Figure 5H, inhibition of Mps1 kinase resulted in a significant reduction in mitotic arrest, apoptosis, and DNA damage, suggesting that compound 5 functions by activating SAC upon induction of severe spindle defect, potentially leading 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 enabling selective targeting of fusion-containing 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 possess the FGFR3-TACC3 fusion protein (Williams, Hurst et al. 2013). Western blot analysis of TACC3 revealed high expression in RT112 cells (Figure 6A), further accompanied by a strong response to compound 5, as indicated by a low IC50 in RT112 cells (Figures 6B and C). Despite relatively low sensitivity to TACC3 inhibition in RT4 cells (presumably 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 be a highly relevant therapeutic opportunity targeting tumors with FGFR3-TACC3 fusions (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 FGFR3-TACC3 fusion proteins.

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

[0182] Next, we tested the specificity of compound 5 to cancer cell lines rather than normal cells. Therefore, we investigated the sensitivity of normal mammary epithelial cells, MCF-12A, and several other breast cancer cells to compound 5. Surprisingly, even high doses of compound 5 (5, 10 μM) did not achieve a 50% inhibition of cell growth (Figure 7A), but compound 5 inhibited the survival 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 to 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 mammary cells. As shown in Figure 6C, breast cancer cell lines that responded more to compound 5 expressed higher TACC3 levels than MCF-12A, further supporting cancer-specific hyperexpression of TACC3. This indicates that sensitivity to compound 5 correlates with abnormal TACC3 expression levels in cancer cells. In addition, lower TACC3 levels may explain why MCF-12A cells did not respond to any TACC3 inhibitors at low doses. Furthermore, compared to luminal cell lines, a tendency for increased TACC3 expression was observed in the two most aggressive breast cancer subtypes, TNBC and HER2-positive, suggesting that TACC3 expression may be associated with cancer invasiveness. To test the transformative potential of TACC3 and the cell's dependence on TACC3 for survival, clonal growth assays were performed in MCF-12A cells after overexpression of TACC3. As shown in Figures 7D and 9E, TACC3 overexpression increased the colony-forming ability of normal breast cell lines, MCF-12A. Importantly, overexpression of TACC3 in MCF-12A cells made these cells sensitive to the cytotoxic effects of compound 5, further demonstrating the cells' dependence on TACC3 and the specificity of compound 5 (Figure 7F). To rule out the possibility that the observed differences between cancer cells and normal cells were due to differences in their proliferation rates, the cell doubling times were calculated.As shown in Figure 7G, there was no significant difference in doubling time between MCF-12A and cancer cell lines, suggesting that the response to compound 5 may not be determined by an increase in cell division rate.

[0183] The results above indicate that breast cancer cells expressing high levels of TACC3 are more sensitive in vitro to compound 5, a novel TACC3 inhibitor disclosed herein. Therefore, the effect of compound 5 on tumor growth in the highly tumorigenic breast cancer cell line JIMT-1 (Barok et al., 2007, Tanner et al., 2004) was tested in immunodeficient mice compared to SPL-B. For this purpose, female nude mice were injected with JIMT-1 cells into mammary fat pads (MFPs) and subsequently treated with a vehicle, or administered 5 mg / kg (oral) of compound 5 or SPL-B every other day for 30 days. Compound 5 showed a significant reduction in tumor growth compared to SPL-B and was concluded to have no significant effect on mouse body weight (Figure 8).

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

[0185] In addition to xenograft and syngeneic models of breast cancer, the antitumor activity of compound 5 was tested in an animal model of colon cancer (Figure 11). Female nude mice and Balb / c mice were injected into the lateral region of the mouse with human colon cancer cell line HCT-116 and mouse colon cancer cell line CT26, respectively. Mice were orally treated daily with either a vehicle or compound 5 at 25-50 mg / kg. 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 investigate the effect of TACC3 inhibition on metastatic growth, highly malignant mouse mammary tumor cells, 4T1-Luc2 (luciferase-labeled), were intravenously injected into immunocompetent female mice. The objective here was to determine the effect of compound 5 on metastatic growth and lung colonization using a clinically relevant metastatic model. When mice showed metastatic lesions in the lungs, they were treated daily with either the vehicle or 50 mg / kg of compound 5. Metastatic growth was monitored in vivo using an imaging system (IVIS) by measuring bioluminescence. Compound 5 was found to impair metastatic growth compared to the vehicle (Figure 12A) and significantly improved the overall survival rate 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 weight was recorded (Figure 13A). Compound 5 did not affect body weight and did not produce observable toxicity at this high dose. In another experiment, mice were administered a single dose of 500 mg / kg of compound 5 and monitored for 1–3 days. Mice administered compound 5 experienced a 10% decrease in body weight 24 hours after drug treatment, but their overall 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 is log D 7.4 It has a moderate lipophilicity of 2.3, exhibits low solubility and low stability in both human and mouse liver microsomes, has relatively high plasma protein binding (unbound rate 1.13%), but good Caco-2 permeability with a low efflux ratio (AB = 190 × 10⁻¹⁰). -6 The results showed a ratio of nm / sec, ratio = <2.0) (Table 1). Accordingly, to evaluate potential drug-drug interactions, cytochrome P450 inhibition by compound 5 in human liver microsomes was also characterized (Lin & Lu, 1998) (Table 1). Therefore, compound 5 inhibits CYP2C9 (IC50).50 =2.63μM) and CYP3A (testosterone as substrate; IC50) 50 It was a moderate inhibitor of CYP2CD6 (IC1 = 8.59 μM), but 50 =18.46μM) and CYP3A(IC) 50 =Midazolam as a substrate (30.04 μM) indicates that compound 5 has low activity at the P450 cytochrome tested, and is not a weak inhibitor.

[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 cancer 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 of Bilkent. JIMT-1, HCC1954, CAL51, and HCC1143 were provided by Ali Osmay Gure of Bilkent University. Cells were cultured in Dulbecco's modified Eagle 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). Furthermore, MCF-12A cells were supplemented with medium containing 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), while RT4 and HCT-116 cells were cultured in McCoy5A (modified) (Gibco) medium supplemented with FBS, NEAA, L-glutamine, and P / S. All cell lines were regularly 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 with slight modifications as previously described (Martinez Molina et al., 2013). Briefly, JIMT-1 cells were incubated with vehicle, 1 μM compound 5(5), or SPL-B for 6 hours. 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 at 45, 46, 47, 48, 49, and 50°C for 5 minutes. Subsequently, the cells were lysed by three freeze-thaw cycles using liquid nitrogen. Soluble proteins were collected by centrifugation at 20,000 g at 4°C for 20 minutes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting.

[0191] Isothermal Titration Calorimeter (ITC) Purified TACC3 recombinant protein (TP310754; Origene, MD, USA) and compound 5(5) were prepared in a 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol solution. Compound 5(5) was packed into sample cells and titrated with TACC3 protein (at a 10-fold higher concentration in a syringe) in a two-run cycle. Titration was performed at 25°C using a Microcal200 instrument (GE Healthcare, Austria). Ten injections were performed at 6-minute intervals between titrations. The sample cells were continuously agitated at 500 rpm with a baseline output of 2 μcal / second. To evaluate the binding efficiency between the drug and protein, background data obtained by protein injected into buffer only was subtracted from the experimental isotherm. Data were analyzed using Origin7 Software provided with the ITC200 to calculate binding parameters such as binding constant (Ka), number of binding sites (N), and enthalpy (ΔH).

[0192] Drug affinity-responsive target stability (DARTS) DARTS was performed as described above (26). Briefly, JIMT-1 cells were grown to a concentration of 70-80% and dissolved in RIPA lysis buffer without SDS and sodium deoxycholate. The concentration of the protein extract was determined using the BCA Protein Assay Reagent Kit (Thermo Scientific, IL, USA) and diluted to 4 μg / μl in lysis buffer. The cell lysates were divided into 99 μl aliquots and mixed separately with 1 μl of 10 μM compound 5 and 100× concentrated solution of SPL-B. The cell lysates and drug mixtures were incubated on a shaker at room temperature for 20 minutes to conjugate. Then, 20 μl of each sample was mixed with 2 μl of 8 ng / μl protease solution (Sigma Aldrich) or buffer only (undigested) and incubated at room temperature for 12 minutes. Protein digestion was stopped by adding 2 μl of 20× protease inhibitor (Roche, Switzerland) and incubating on ice for 10 minutes. Next, the lysate was mixed with 8 μl of 4×SDS-added solution and heated at 70°C for 10 minutes. 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. A newly synthesized molecule was dissolved in 100% DMSO to obtain a stock concentration of 10 mM. In the cell viability assay, 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 x 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 inhibitor treatment was performed at various concentrations 24 hours after cell seeding. Cell viability was measured 72 hours after treatment with the sulforhodamine B (SRB, Sigma Aldrich) assay recommended by the manufacturer. In Western blotting, different concentrations of KHS101, SPL-B, or compound 5 were given to JIMT-1(1.5×10 5 ) and RT112(2×10 5 ) cells 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 in 96-well plates with 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 the Lipofectamine 2000 (trademark) (Invitrogen, CA) transfection reagent as previously described (Mutlu et al., 2016). 72 hours after transfection, cell viability was measured using the SRB assay. To evaluate the TACC3 knockdown level immediately after siRNA transfection, JIMT-1(1.5×10 5 ), BT-474 T-DM1R(2×10 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. The knockdown efficiencies at the mRNA and protein levels were 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 In monolayer culture, single-cell suspensions of JIMT-1 cells (3×10 3 cells / well) were plated in 12-well plates. 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 in 6-well plates and transfected with TACC3 the next day. 48 hours after transfection, the cells were counted and 1×10 3 cells / well were plated in 12-well plates. In both experimental settings, the medium was refreshed every 4 days and the cells were incubated for 12 days. Next, the cells were fixed with 2% paraformaldehyde for 15 minutes and stained with 1% crystal violet (Merck, Darmstadt, Germany) for 15 minutes at room temperature. Viable colonies (composed of at least 50 cells) were counted using ImageJ software (NIH).

[0196] Doubling time evaluation To evaluate the doubling time, normal breast epithelial cell line MCF-12A and breast cancer cell lines were plated in 6-well plates (3×10 4 cells / well). The cells were collected by trypsinization and the cell numbers were counted every 24 hours for 1 week. The growth curves of these cells were plotted as the cell number / cm2 It was drawn as follows. 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). Basically, 1.5 × 10⁶ cells were placed on glass coverslips in a 6-well plate. 5 JIMT-1 cells / well were seeded. The following day, the cells were treated with either a vehicle, 200 nM, or 500 nM compound 5 for 12 hours. Next, the cells were fixed with ice-cold methanol at -20°C for 10 minutes. Then, the cells were blocked with 3% BSA in PBS solution at room temperature for 1 hour and incubated with primary and secondary antibodies at room temperature for 1 hour. The cells were counterstained with DAPI for 5 minutes (0.01 μg / ml). Finally, coverslides were mounted using ImmunoHistomounth (Santa Cruz). Images were taken with an upright fluorescence microscope (upright) equipped with a DIC prism.

[0198] NCI-60 Cancer Cell Line Panel Screening Compound 5(5) was submitted to the National Cancer Institute (NCI number S807620) for screening of human tumor cell lines in the NCI-60 panel, consisting of 60 human cancer cell lines derived from 9 different cancer types. Initially, compound 5(5) was tested in each cell line in a single-dose screening at a concentration of 10 μM. After obtaining the results of the single-dose assay, an analysis by the Development Therapeutics Program (DTP) was performed, and compounds 5 meeting the specified threshold inhibition criteria were selected for the NCI full-panel 5-dose assay. Next, compound 5 was tested twice in the 5-dose NCI-60 screening at doses ranging from 10 nM to 100 μM, and the GI of the 60 cell lines was evaluated. 50(50% growth inhibition) value, TGI (total growth inhibition) value, and LC 50 The lethal dose (the concentration that induces 50% cell death) is determined. Detailed screening methods can be accessed from the webpage https: / / dtp.cancer.gov / discovery_development / nci-60 / methodology.htm. Briefly, cells were seeded in a 96-well plate and treated with compounds in the 5 logM concentration range for 2 days, 24 hours later. Cytotoxicity was evaluated using an SRB assay. The data shown in the figure are the mean values ​​from both experiments.

[0199] Mouse experiment Six-to-eight-week-old female athymoid nude or Balb / c mice were housed in a temperature-controlled 12-hour light / 12-hour dark cycle environment. This study was conducted according to the Institutional Animal Care and Use Committee of Bilkent University and in accordance with institutional guidelines and animal research principles. For in vivo mammary cancer tumor growth in nude mice, 4 × 10⁶ mice were used. 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 pads (MFPs) of female nude mice. Mouse body weight and tumor volume were measured daily using calipers. Tumor volume was measured as length × width 2 The calculation was performed using a multiplier of 0.5. The tumor volume was approximately 90-100 mm. 3Once the target was reached, xenografts were randomized and divided into groups. Animals were treated with either a vehicle (0.05% HPMC (hydroxypropyl methylcellulose) and 2% Tween-80 in ddH2O) or compound 5 (different doses every other day (qod.) - oral or intravenous administration and method of administration - 2 or 5 mg / kg). In another 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, oral) using JIMT-1 cells. Mice were sacrificed 20-30 days after the start of treatment, and tumors were collected and saved for subsequent analysis. To test the effect of compound 5 in an immunoqualified female Balb / c mouse model, a highly malignant mouse mammary cancer cell line was used. 1 × 10 6 Individual EMT-6 cells were prepared in PBS and injected into mouse MFPs. Similarly, tumors with a volume of 90-100 mm² were treated. 3 Upon reaching a certain stage, mice were randomized into two groups and administered either the vehicle or compound 5 at 25 mg / kg orally daily. Survival was measured at 1500 mm. 3 The calculation was performed using a predefined tumor volume cutoff.

[0200] Furthermore, compound 5 was tested immediately after the induction of lung metastases. Highly malignant and metastatic 4T1-Luc2 (luciferase-labeled) cells were 1 × 10⁶ cells in PBS. 6 The cells were prepared as a mouse and administered intravenously to Balb / c female mice. Metastasis development was monitored using an in vivo imaging system (IVIS), and bioluminescence was periodically quantified. When lung metastases developed, mice were randomized into two groups and administered either the vehicle or 50 mg / kg of compound 5 orally daily. Survival rates were calculated when mice died.

[0201] In addition to breast cancer models, the antitumor effect of compound 5 was tested in both immunodeficient nude mice (HCT-116) and syngeneic immunocompetent Balb / c mice (CT-26) as animal models of colon cancer. 4×10 6 HCT-116 and 1×10 6The cells were prepared in either Matrigel:PBS or PBS, respectively, and subcutaneously injected into the flank region of mice. Similarly, mice with tumor volumes of 90–100 mm² were treated. 3 Once the mice reached a certain stage, they were randomized into two groups and orally administered either the vehicle or compound 5 at 25-50 mg / kg daily for 20-25 days. The mice's body weight was measured periodically.

[0202] For toxicity analysis, nude female mice were administered either 100 mg / kg of compound 5 for 7 days, or a single oral dose of either a vehicle or 500 mg / kg of compound 5. The mice's body weight was measured periodically, and organs were collected to assess potential toxicity.

[0203] Bioinformatics analysis TACC3 differential plots between different tumor and normal tissues were created using patients from The Cancer Genome Atlas (TCGA) (Akbani et al., 2014), and the data were downloaded from http: / / firebrowse.org / . For TACC3 survival analysis and prognostic significance, various independent, publicly available cancer datasets were used. One is the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset (Curtis, Shah et al., 2012). TACC3 expression levels from the METABRIC Discovery and Validation set were used for overall survival in breast cancer patients. Patients in the 25th and 75th quartiles of TACC3 levels were used and 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 containing information on overall survival in 876 gastric cancer patients (Szasz et al., 2016). Disease-free survival data for prostate cancer patients, comprising 122 patients, were obtained from The Cancer Genome Atlas (TCGA) database using https: / / www.cancer.gov / tcga, with patients separated based on the 25th and 75th percentiles. Finally, recurrence-free survival data for lung cancer patients were obtained from the GSE31210 dataset, and similarly, the 25th and 75th percentiles of patients were used for this analysis (Okayama, Kohno et al. 2012). Gene set enrichment analysis (GSEA) of mitotic and DNA repair-related gene sets, available on the Broad Institute website (http: / / software.broadinstitute.org / gsea / index.jsp), was performed using breast cancer, and patients were divided into two groups (high vs. low) based on TACC3 expression levels in the METABRIC Validation dataset (n=995).For the analysis of TACC3 dependence in NCI-60 cell lines, we used dependency data combined with RNAi screening by Broad Institute, Novartis and Marcotte et al. (13), available at https: / / depmap.org / portal / . Multivariate Cox regression analysis was performed using the METABRIC dataset in SPSS software. TACC3 level, 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 expressed as mean ± standard deviation from three independent experiments unless otherwise specified. Statistical significance of two-group comparisons was determined by two-sided Student's t-tests. One-way ANOVA was used to compare doubling time curves of different cell lines. Pairwise significance of tumor volume between treatment groups for EMT6 xenografts was determined using multiple t-tests. P and corrected p(q) values ​​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] Built-in by reference All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of any conflict, including the definitions herein, this application shall prevail.

[0207] Equal portions While specific embodiments of the subject matter disclosure have been discussed, the above specification is illustrative and not limiting. Many variations of this disclosure will become apparent to those skilled in the art upon consideration of this specification and the following claims. The full scope of this disclosure should be determined by referring to the claims, together with the full scope of equivalents, and the specification, together with such variations. The present invention includes, for example, the following embodiments. [Section 1] Compound of formula (I), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X1 is N or CR6, X2 is N or CR3, R1 is an aryl or heteroaryl, R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. A compound in which R5 is a heterocyclyl, alkyl, or amino compound. [Section 2] The aforementioned compound, [ka] Not the compounds listed in item 1. [Section 3] The compound according to item 1 or 2, wherein R1 is an aryl (e.g., phenyl). [Section 4] The compound according to item 1 or 2, wherein R1 is a heteroaryl (e.g., benzofuran or pyrimidinyl). [Section 5] The compound according to item 1 or 2, wherein R1 is a heterocyclyl (e.g., benzodioxole or dihydrobenzofuran). [Section 6] A compound according to any one of claims 1 to 5, wherein R1 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amide, cyano, nitro, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl, or sulfonamide. [Section 7] The compounds described in any one of claims 1 to 5, wherein R1 is substituted with an alkyl (e.g., methyl, ethyl, isopropyl, fluoroethyl, or trifluoromethyl), an alkyloxy (e.g., methoxy, trifluoromethyloxy, difluoromethyloxy, ethoxy, or propyloxy), an alkylthio (e.g., methylthio), an aralkyloxy (e.g., benzyloxy), a hydroxyl, a halo (e.g., fluoro or chloro), or an amino (e.g., dimethylaminoalkyl). [Section 8] A compound according to any one of items 1 to 7, wherein R1 is substituted with a halo (e.g., fluoro). [Section 9] A compound according to any one of items 1 to 8, wherein R1 is substituted with an alkyloxy (e.g., methoxy). [Section 10] A compound according to any one of items 1 to 9, wherein R1 is substituted with an alkyl group (e.g., methyl, ethyl, or trifluoromethyl). [Section 11] A compound according to any one of items 1 to 7, wherein R1 is substituted with one halo (e.g., F). [Section 12] The compound according to item 11, wherein the halo (e.g., F) is para with respect to isoxazole. [Section 13] The compound according to item 11, wherein the halo (e.g., F) is ortho with respect to isoxazole. [Section 14] The compound according to item 11, wherein the halo (e.g., F) is meta with respect to isoxazole. [Section 15] A compound according to any one of items 1 to 7, wherein R1 is substituted with two halos (e.g., F). [Section 16] The compound according to item 15, wherein one halo (e.g., F) is meta with respect to isoxazole and one halo (e.g., F) is ortho with respect to the isoxazole. [Section 17] A compound according to any one of items 1 to 7, wherein R1 is substituted with an alkoxy (e.g., methoxy). [Section 18] The compound according to item 17, wherein the alkoxy (e.g., methoxy) is para with respect to isoxazole. [Section 19] The compound according to item 17, wherein the alkoxy (e.g., methoxy) is ortho with respect to isoxazole. [Section 20] The compound according to item 17, wherein the alkoxy (e.g., methoxy) is meta relative to isoxazole. [Section 21] A compound according to any one of items 1 to 7, wherein R1 is substituted with one alkoxy (e.g., methoxy) and one halo (e.g., F). [Section 22] The compound according to item 21, wherein the alkoxy (e.g., methoxy) is para relative to the isoxazole, and F is meta relative to the isoxazole. [Section 23] The compound according to item 21, wherein the alkoxy (e.g., methoxy) is para relative to the isoxazole, and F is ortho relative to the isoxazole. [Section 24] The compound according to item 21, wherein the halo (e.g., F) is para relative to the isoxazole and the alkoxy (e.g., methoxy) is meta relative to the isoxazole. [Section 25] A compound according to any one of items 1 to 7, wherein R1 is substituted with an alkoxy (e.g., methoxy) and two halos (e.g., F). [Section 26] The compound according to item 25, wherein the alkoxy (e.g., methoxy) is para relative to the isoxazole, and both halos (e.g., F) are meta relative to the isoxazole. [Section 27] The compound according to claim 25, wherein the alkoxy (e.g., methoxy) is para relative to the isoxazole, one halo (e.g., F) is meta relative to the isoxazole, and one halo (e.g., F) is ortho relative to the isoxazole. [Section 28] A compound according to any one of items 1 to 27, wherein R2 is alkyl (e.g., methyl or ethyl). [Section 29] A compound according to any one of claims 1 to 28, wherein R2 is substituted with an amino (e.g., dimethylamino or diethylamino) or a nitrile. [Section 30] A compound according to any one of items 1 to 27, wherein R2 is H. [Section 31] A compound according to any one of items 1 to 30, wherein X1 is N. [Section 32] A compound according to any one of items 1 to 30, wherein X1 is CR6. [Section 33] The compound described in item 32, wherein R6 is H. [Section 34] A compound according to any one of items 1 to 33, wherein X2 is N. [Section 35] A compound according to any one of items 1 to 33, wherein X2 is CR3. [Section 36] The compound according to item 35, wherein R3 is H or a halo (e.g., fluoro or chloro). [Section 37] A compound according to any one of items 1 to 36, wherein R4 is alkyl (e.g., methyl). [Section 38] A compound according to any one of items 1 to 37, wherein R5 is a heterocycline (e.g., azetidinyl, morpholino, pyrrolidinyl, piperazinyl, piperidinyl, oxaazabicyclooctanyl, oxaazabicycloheptnyl, thiomorpholino, thiomorpholino dioxide, hexahydroflopyrrolyl, or azabicyclohexanyl). [Section 39] A compound according to any one of claims 1 to 38, wherein R5 is a 6-membered heterocycline and the cycle skeleton contains one nitrogen. [Section 40] A compound according to any one of claims 1 to 38, wherein R5 is a 6-membered heterocycline and the cycle skeleton contains one nitrogen and one oxygen. [Section 41] A compound according to any one of claims 1 to 38, wherein R5 is a 7-membered heterocycline and the cycle skeleton contains one nitrogen. [Section 42] A compound according to any one of claims 1 to 38, wherein R5 is a 7-membered heterocycline and the cycle skeleton contains one nitrogen and one oxygen. [Section 43] A compound according to any one of claims 1 to 38, wherein R5 is an 8-membered heterocycline and the cycle skeleton contains one nitrogen. [Section 44] A compound according to any one of claims 1 to 38, wherein R5 is an 8-membered heterocycline and the cycle skeleton contains one nitrogen and one oxygen. [Section 45] The compound according to any one of claims 1 to 38, wherein R5 is a nitrogen-containing heterocycline, and the nitrogen is directly bonded to an aryl or heteroaryl ring having an R4 substituent. [Clause 46] A compound according to any one of claims 1 to 45, wherein R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amide, cyano, nitro, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or sulfonamide. [Section 47] A compound according to any one of claims 1 to 46, 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] A compound according to any one of items 1 to 47, wherein R5 is substituted with a halo (e.g., fluoro). [Section 49] A compound according to any one of items 1 to 48, wherein R5 is substituted with two alkyl moieties (for example, two methyl moieties). [Section 50] A compound according to any one of items 1 to 38, wherein R5 is 2,6-dimethylmopholine, 4-methylpiperidine, or 4-(trifluoromethyl)piperidine. [Section 51] A compound according to any one of items 1 to 37, wherein R5 is an amino acid. [Section 52] The compound according to item 51, wherein R5 is substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amide, cyano, nitro, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, sulfonamide, cycloalkyl, or heterocyclyl. [Section 53] The compounds according to item 51 or 52, wherein R5 is substituted with an 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 aforementioned compound has a structure represented by formula II, [ka] During the ceremony, R1 is a ring selected from the group consisting of unsubstituted phenyl or o-, m-, p-CH3, C2H5, CH(CH3)2, OCH3, OC2H5, OC3H7, SCH3, CF2CH3, CF3, OCF3, OCHF2, N(CH3)2, F, Cl, OH monosubstituted or disubstituted 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, morpholine-4-amine, piperidine, tetrahydro-2H-pyran-4-amine, piperidine-1-amine, 4-fluoropiperidine, 4,4-difluoropiperidine, 4-methylpiperidine, 4-(trifluoromethyl)piperidine, piperazine, N-methylpiperazine, pyrrolidine, 2-(4-piperidinyl)ethanol A luamine selected from the group consisting of ol, 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 either CH or N. The compounds described in item 1, and / or their pharmaceutically acceptable salts and / or solvates. [Section 55] The compound described in item 54, wherein R1 is phenyl. [Section 56] The compound described in item 54, wherein R1 is pyridyl. [Section 57] The compound described in item 54, wherein R1 is benzyloxy. [Section 58] The compound described in item 54, wherein R1 is piperonyl. [Section 59] A 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] A compound according to any one of items 54 to 58, wherein R1 is substituted with one F. [Section 61] The compound according to item 60, wherein F is para with respect to isoxazole. [Section 62] The compound according to item 60, wherein F is ortho with respect to isoxazole. [Section 63] The compound according to item 60, wherein F is meta with respect to isoxazole. [Section 64] A compound according to any one of items 54 to 58, wherein R1 is substituted with two F atoms. [Section 65] The compound according to claim 64, wherein the first F is meta relative to isoxazole and the second F is ortho relative to isoxazole. [Section 66] A compound according to any one of items 54 to 65, wherein R1 is substituted with OCH3. [Section 67] The compound according to item 66, wherein OCH3 is para with respect to isoxazole. [Section 68] The compound according to item 66, wherein OCH3 is ortho with respect to isoxazole. [Section 69] The compound according to item 66, wherein the OCH3 is meta with respect to isoxazole. [Section 70] A compound according to any one of items 54 to 58, wherein R1 is substituted with one OCH3 and one F. [Section 71] The compound according to item 70, wherein OCH3 is para relative to isoxazole and F is meta relative to isoxazole. [Section 72] The compound according to claim 70, wherein OCH3 is para with respect to isoxazole and F is ortho with respect to isoxazole. [Section 73] The compound according to item 70, wherein F is para relative to isoxazole and OCH3 is meta relative to isoxazole. [Section 74] A compound according to any one of items 53 to 58, wherein R1 is substituted with OCH3 and two Fs. [Section 75] The compound according to item 74, wherein OCH3 is para relative to isoxazole and both Fs are meta relative to isoxazole. [Section 76] The compound according to claim 74, wherein OCH3 is para relative to isoxazole, one F is meta relative to isoxazole, and one F is ortho relative to isoxazole. [Section 77] A compound according to any one of items 53 to 76, wherein R2 is H. [Section 78] A compound according to any one of items 53 to 76, wherein R2 is CH3. [Section 79] A compound according to any one of items 53 to 76, wherein R3 is H. [Section 80] A compound according to any one of items 53 to 76, wherein R3 is F. [Section 81] A compound according to any one of items 53 to 76, wherein R3 is Cl. [Section 82] A compound according to any one of items 53 to 81, wherein R5 is morpholine. [Section 83] A compound according to any one of items 53 to 81, wherein R5 is piperidine. [Section 84] A compound according to any one of items 53 to 81, wherein R5 is 4-fluoropiperidine. [Section 85] A compound according to any one of items 53 to 81, wherein R5 is 4,4-difluoropiperidine. [Section 86] A compound according to any one of the items 53 to 81, wherein R5 is 3-oxa-8-azabicyclo[3.2.1]octane. [Section 87] A compound according to any one of the items 53 to 81, wherein R5 is 8-oxa-3-azabicyclo[3.2.1]octane. [Section 88] A compound according to any one of items 53 to 87, wherein X1 is C. [Section 89] A compound according to any one of items 53 to 87, wherein X1 is N. [Section 90] The aforementioned compound, 3-(4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(3-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-Molfolinopyrimidine-4-yl)-3-phenylisoxazole-5-amine 3-(4-ethoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-morpholinopyrimidine-4-yl)-3-(4-propoxyphenyl)isoxazole-5-amine 3-(4-fluorophenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-chlorophenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-morpholinopyrimidine-4-yl)-3-(p-tolyl)isoxazole-5-amine 3-(4-ethylphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-isopropylphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-Molfolinopyrimidine-4-yl)-3-(4-(trifluoromethyl)phenyl)isoxazole-5-amine 3-(4-(1,1-difluoroethyl)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-(difluoromethoxy)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-Molfolinopyrimidine-4-yl)-3-(4-(trifluoromethoxy)phenyl)isoxazole-5-amine 3-(4-(methylthio)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(3-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(3,4-dimethoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(benzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(6-methoxypyridine-3-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 4-(5-((2-Morpholinopyrimidine-4-yl)amino)isoxazole-3-yl)phenol 3-(4-(benzyloxy)phenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-(4,4-difluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-thiomorpholinopyrimidine-4-yl)isoxazole-5-amine 4-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)thiomorpholine 1,1-dioxide 3-(4-methoxyphenyl)-N-(2-(piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-(pyrrolidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 2-(1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperidine-4-yl)ethane-1-ol 2-(4-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperazine-1-yl)ethane-1-ol 3-(4-methoxyphenyl)-N-(2-(4-methylpiperazine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-(piperazin-1-yl)pyrimidine-4-yl)isoxazole-5-amine Ethyl 1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperidine-4-carboxylate 1-(4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)piperidine-4-carboxylic acid N4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -Molfolinopyrimidine-2,4-diamine N 4 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(piperidine-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-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(5-fluoro-2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine N-(2-(4-fluoropiperidine-1-yl)-6-methylpyrimidine-4-yl)-3-(4-methoxyphenyl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-methyl-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-(dimethylamino)phenyl)-N-(2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-fluoropiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)pyrimidine-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(pyrrolidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine N 4 -(3-(3-fluoro-4-methoxyphenyl)isoxazole-5-yl)-N 2 -(tetrahydro-2H-pyran-4-yl)pyrimidine-2,4-diamine 3-(2,3-dihydrobenzofuran-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N 2 -Cyclopropyl-N 4 -(3-(4-methoxyphenyl)isoxazole-5-yl)pyrimidine-2,4-diamine N 2 -Isobutyl-N 4 -(3-(4 - Methoxyphenyl)isoxazole-5-yl)pyrimidine-2,4-diamine N 2 -(2-methoxyethyl)-N 4 -(3-(4-methoxyphenyl)isoxazole-5-yl)pyrimidine-2,4-diamine N 1 ,N 1 -diethyl-N 2 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 -(2-Morpholinopyrimidine-4-yl)ethane-1,2-diamine N 1 -(3-(4-methoxyphenyl)isoxazol-5-yl)-N 2 ,N 2 -dimethyl-N1-(2-morpholinopyrimidine-4-yl)ethane-1,2-diamine 2-((3-(4-methoxyphenyl)isoxazole-5-yl)(2-morpholinopyrimidine-4-yl)amino)acetonitrile 3-(5-methoxypyridine-2-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-morpholinopyridine-4-yl)isoxazole-5-amine 3-(4-methoxyphenyl)-N-(4-morpholino-1,3,5-triazine-2-yl)isoxazole-5-amine N 2 -(2,2-difluoroethyl)-N 4 -(3-(4-methoxyphenyl)isoxazole-5-yl)pyrimidine-2,4-diamine 2-((4-((3-(4-methoxyphenyl)isoxazole-5-yl)amino)pyrimidine-2-yl)amino)ethane-1-ol 3-(2-fluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2,3-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(3,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine N-(2-Molfolinopyrimidine-4-yl)-3-(3,4,5-trimethoxyphenyl)isoxazole-5-amine 3-(benzofuran-5-yl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine 3-(2,5-difluoro-4-methoxyphenyl)-N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)isoxazole-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)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(3-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(3-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-(trifluoromethyl)piperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-methylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-((2R,6S)-2,6-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetramethylmorpholino)pyrimidine-4-yl)isoxazole-5-amine N-(2-(3,3-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2,2-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(3,5-dimethylmorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-methylmorpholino)pyrimidine-4-yl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methylmorpholino)pyrimidine-4-yl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2-(trifluoromethyl)morpholino)pyrimidine-4-yl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(tetrahydro-1H-fluoro[3,4-c]pyrrole-5(3H)-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-(3-azabicyclo[3.1.0]hexane-3-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(7-azaspiro[3.5]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(4,4-dimethylpiperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-6-azaspiro[3,3]heptan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(1-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(6-azaspiro[2.5]octan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-8-azabicyclo[3.2.1]octan-8-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-(2,2-difluoromorpholino)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-5-azabicyclo[2.2.2]octan-5-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-(trifluoromethyl)morpholino)pyrimidine-4-yl)isoxazole-5-amine N-(2-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-5-azabicyclo[4.1.0]heptan-5-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(4-oxa-7-azaspiro[2.5]octan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(7-oxa-4-azaspiro[2.5]octan-4-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-8-azaspiro[4.5]decane-8-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-6-azaspiro[3,4]octan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-oxa-7-azaspiro[4,4]nonan-7-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(2,2,6,6-tetrafluoromorpholino)pyrimidine-4-yl)isoxazole-5-amine N-(2-(6-azabicyclo[3.1.1]heptan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(3-methyl-6-azabicyclo[3.1.1]heptan-6-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-(3,5-dimethylpiperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-fluoro-4-methoxyphenyl)-N-(2-(4-isopropylpiperidine-1-yl)pyrimidine-4-yl)isoxazole-5-amine N-(2-(4-(difluoromethyl)piperidine-1-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(6-azaspiro[3.5]nonan-6-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine N-(2-(2-azaspiro[3.5]nonan-2-yl)pyrimidine-4-yl)-3-(2-fluoro-4-methoxyphenyl)isoxazole-5-amine 3-(2-chloro-4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine Compounds as described in item 1 or 54, selected from the group consisting of and / or pharmaceutically acceptable salts and / or solvates thereof. [Section 91] The aforementioned compound, [ka] [ka] [ka] A compound selected from the group consisting of items 1 or 54. [Section 92] The aforementioned compound, [ka] [ka] [ka] [ka] The compound described in item 1, selected from a pharmaceutically acceptable salt thereof. [Section 93] A pharmaceutical composition comprising a compound described in 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 a compound or a pharmaceutically acceptable salt thereof described in any one of paragraphs 1 to 92. [Section 95] The method according to paragraph 94, wherein the TACC3-mediated disease or disorder is cancer. [Section 96] The method according to paragraph 94 or 95, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia, kidney cancer, or prostate cancer. [Section 97] The method according to any one of paragraphs 94 to 96, wherein the cancer is selected from the NCI-60 panel. [Section 98] The method according to any one of claims 94 to 97, wherein the compound is administered orally to the subject. [Section 99] A compound according to any one of items 1 to 92, for use in the treatment of TACC3-mediated diseases or disorders. [Section 100] The compound for use according to item 99, wherein the TACC3-mediated disease or disorder is cancer. [Section 101] The compound for use as described in item 99 or 100, wherein the cancer is breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia, kidney cancer, or prostate cancer. [Section 102] The compound for use according to paragraph 99 or 100, wherein the cancer is a cancer selected from the NCI-60 panel. [Section 103] The compound for use according to any one of claims 99 to 102, wherein the compound is for oral administration. [Section 104] A compound described in any one of items 1 to 92, for use in the treatment of cancer. [Section 105] An anticancer agent as described in item 104, which targets cancers that express TACC3. [Section 106] The compound according to item 104 or 105, wherein the anticancer agent is a TACC3 protein inhibitor. [Section 107] The anticancer agent according to item 106, wherein the targeted cancer expressing TACC3 is a breast cancer, colon cancer, melanoma cancer, lung cancer, central nervous system cancer, ovarian cancer, leukemia, renal cancer, and prostate cancer cell line listed in the NCI-60 panel. [Section 108] The anticancer agent described in any one of paragraphs 104 to 107, wherein the anticancer agent is for oral administration. [Section 109] The compound according to claim 104, wherein the compound is 3-(4-methoxyphenyl)-N-(2-morpholinopyrimidine-4-yl)isoxazole-5-amine (compound 5). [Clause 110] The compound for use according to item 109, characterized in that the compound has a structure that induces mitotic arrest, apoptosis, and DNA damage. [Section 111] The compounds described in item 110 function by activating SAC, which induces prolonged mitosis, apoptotic cell death, and severe spindle defects that further lead to DNA damage. [Section 112] The compounds described in item 111 inhibit the growth of cancer cells possessing FGFR3-TACC3 oncogenic fusions. [Section 113] A pharmaceutical composition comprising a compound described in sections 1 to 92 of the therapeutically effective amounts and / or a pharmaceutically acceptable salt and / or solvate thereof, and at least one pharmaceutically acceptable carrier. [Section 114] A composition comprising the compound described in item 113, wherein the composition further comprises a pharmaceutically acceptable chemotherapeutic agent or targeted therapy agent. [Section 115] A therapeutic combination of chemotherapy agents, targeted therapies, or drugs for an individual in need of cancer treatment, wherein an anticancer agent described in any one of paragraphs 1 to 92 is part of the therapeutic combination. [Section 116] A compound described in any one of items 1 to 92, for use as a pharmaceutical agent. [Section 117] A compound according to any one of items 1 to 92, for use in inhibiting and / or preventing tumor growth and metastasis. [Section 118] The compound according to item 117, wherein the cancer is a cancer that responds to inhibition of TACC3. [Section 119] The compound described in 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 any one of the compounds described in any one of the sections 1 to 90, comprising the reaction of an amine derivative described herein with an intermediate compound from Table 1, but not limited to the above. [Section 121] The aforementioned method is represented by the steps of scheme I, [ka] or comprising a pharmaceutically acceptable salt thereof, in the formula, X1 is N or CR6, X2 is N or CR3, R1 is an aryl or heteroaryl, R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. R5 is a heterocycline, alkyl, or amino compound. R 51 But it's a halo, R 52 However, it is a heterocycline or alkyl group. X 10 However, there is a base, X 11 However, it is a precious metal catalyst, X 12 The method according to item 120, wherein the ligand is a phosphine ligand. [Section 122] A compound described in any one of items 1 to 92 represented by Scheme I, [ka] or a method for preparing a pharmaceutically acceptable salt thereof, wherein the formula is: X1 is N or CR6, X2 is N or CR3, R1 is an aryl or heteroaryl, R2 is either 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, azide, alkylthio, alkenyl, alkynyl, cycloalkyl, or sulfonamide. R5 is a heterocycline, alkyl, or amino compound. R 51But it's a halo, R 52 However, it is a heterocycline or alkyl group. X 10 However, it is a base, X 11 However, it is a precious metal catalyst, X 12 However, the method involves using a phosphine ligand. [Section 123] The method according to item 121 or 122, wherein the base is a carbonate, oxide, tertiary amine, secondary amine, or hydride. [Section 124] The method according to item 123, wherein the oxide is an alkoxide (e.g., tert-butoxide). [Section 125] The method according to claim 123, wherein the tertiary amine is a tertiary alkylamine (e.g., diisopropylethylamine). [Section 126] The method according to item 123, wherein the hydride is a metal hydride (e.g., sodium hydride). [Section 127] 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 claims 121 to 127, wherein the noble metal catalyst is a palladium catalyst (e.g., palladium-II acetate). [Section 129] The method according to any one of claims 121 to 128, wherein the phosphine catalyst is an arylphosphine (e.g., triphenylphosphine). [Section 130] The method according to any one of claims 121 to 128, wherein the phosphine catalyst is xanthophos. [Section 131] The method according to any one of claims 121 to 130, wherein the method further comprises a solvent. [Section 132] The method according to claim 131, wherein the solvent is tertiary butanol, dimethylacetamide, or dioxane. [Section 133] The method according to any one of claims 121 to 132, further comprising heating. [Section 134] The method according to any one of items 121 to 133, wherein the method is carried out under an inert atmosphere.

Claims

1. Compound of formula (I), 【Chemistry 1】 or a pharmaceutically acceptable salt thereof (In the formula, X １ However, N or CR ６ And, X ２ However, N or CR ３ And, R １ However, they are aryl, heteroaryl, or heterocyclyl. R ２ However, it 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, amide, cyano, nitro, azide, alkylthio, cycloalkyl, or sulfonamide, R ５ However, it is an amino acid.

2. R １ The compound according to claim 1, wherein the compound is an aryl compound.

3. R １ The compound according to claim 1, wherein it is a heteroaryl compound.

4. R １ The compound according to claim 1, wherein it is a heterocyclyl.

5. R １ The compound according to any one of claims 1 to 4, wherein the compound is substituted with hydrogen, deuterium, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amide, cyano, nitro, azide, alkylthio, cycloalkyl, heterocyclyl, alkylsulfonyl, or sulfonamide.

6. R １ The compound according to any one of claims 1 to 4, wherein the compound is substituted with alkyl, alkoxy, alkylthio, aralkyloxy, hydroxyl, halo, or amino.

7. The compound according to any one of claims 1 to 5, wherein R1 is substituted with deuteroalkyl, methyl, ethyl, butyl, isopropyl, difluoromethyl, trifluoromethyl, difluoroethyl, fluoro, chloro, deuteroalkoxy, methoxy, ethoxy, difluoromethoxy, trifluoromethoxy, dimethylamino, methylthio, or azetidinyl.

8. X １ The compound according to any one of claims 1 to 7, wherein N is present.

9. X １ However, CR ６ The compound according to any one of claims 1 to 7.

10. R ６ The compound according to claim 9, wherein H is present.

11. X ２ The compound according to any one of claims 1 to 10, wherein N is present.

12. X ２ However, CR ３ The compound according to any one of claims 1 to 10.

13. R ３ The compound according to claim 12, wherein the compound is H or a halo.

14. R ４ The compound according to any one of claims 1 to 13, wherein the compound is alkyl.

15. R ５ The compound according to any one of claims 1 to 14, wherein the compound is substituted with alkyl, alkenyl, alkynyl, hydroxyalkyl, hydroxyl, acyl, acetyl, ester, cycloalkyl, or heterocyclyl.

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition according to claim 16 for use in the treatment of cancer.

18. The pharmaceutical composition according to claim 17, wherein the cancer is breast cancer, colon cancer, melanoma, lung cancer, central nervous system cancer, ovarian cancer, leukemia, kidney cancer, or prostate cancer.

19. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15 in the manufacture of a pharmaceutical product for treating cancer.

20. The use according to claim 19, wherein the cancer is breast cancer, colon cancer, melanoma, lung cancer, central nervous system cancer, ovarian cancer, leukemia, kidney cancer, or prostate cancer.

Images