Use of compound hit211363271 in the preparation of a medicament for the treatment of colorectal cancer

Abstract

The application belongs to the field of anti-tumor, and particularly relates to application of a compound HIT211363271 in preparation of a drug for treating colorectal cancer. The application finds that the compound HIT211363271 can obviously inhibit proliferation, migration and glycolysis related phenotype of colorectal cancer cells, and promote apoptosis of the colorectal cancer cells, and can also inhibit growth of a colorectal cancer organoid, and can be used for preparation of a drug for treating colorectal cancer, thereby providing a new candidate scheme for treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor therapy, specifically relating to the application of compound HIT211363271 in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors of the digestive system in clinical practice, with a high incidence and mortality rate. Although surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy are widely used in clinical practice, the overall efficacy of existing treatment regimens for patients with advanced, metastatic, and recurrent colorectal cancer remains limited. Therefore, the development of new therapeutic drugs and new uses for these drugs is of great significance.

[0003] Tumor metabolic reprogramming is a key biological characteristic of colorectal cancer development and progression. Colorectal cancer cells are typically accompanied by significant abnormalities in glucose metabolism. Enhanced glycolysis not only provides energy for rapid tumor cell proliferation but also promotes tumor progression by providing metabolic intermediates, regulating redox homeostasis, and altering the tumor microenvironment. Therefore, interventions targeting abnormal tumor glucose metabolism and related processes are an important direction in colorectal cancer treatment research. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention has discovered that compound HIT211363271 exhibits anti-colorectal cancer activity in cell phenotypes, glycolysis-related phenotypes, and organoid models, providing a new technical solution for the treatment of colorectal cancer. Specifically, this includes the following: In a first aspect, the present invention provides the use of compound HIT211363271 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug; the structural formula of compound HIT211363271 is shown in formula (Ⅰ) below:

[0005] Equation (Ⅰ).

[0006] Preferably, the tumor is colorectal cancer.

[0007] Preferably, the compound HIT211363271 inhibits the proliferation of colorectal cancer cells.

[0008] Preferably, the compound HIT211363271 inhibits the migration of colorectal cancer cells.

[0009] Preferably, the compound HIT211363271 promotes apoptosis in colorectal cancer cells.

[0010] Preferably, the compound HIT211363271 inhibits the glycolysis-related phenotype of colorectal cancer cells.

[0011] Preferably, the compound HIT211363271 inhibits the growth of colorectal cancer organoids.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for antitumor purposes, wherein the active ingredient of the pharmaceutical composition comprises compound HIT211363271 or a pharmaceutically acceptable salt thereof.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0014] Preferably, the pharmaceutical composition can be formulated into any one of the following dosage forms: tablets, granules, capsules, oral liquids, injections, or suspensions.

[0015] Thirdly, the present invention provides the use of the pharmaceutical composition described in the second aspect above in the preparation of antitumor drugs.

[0016] Preferably, the tumor is colorectal cancer.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention provides a technical solution for the use of compound HIT211363271 in the treatment of colorectal cancer, providing a new technical path for the development of new uses of commercial compounds.

[0018] (2) This invention shows that compound HIT211363271 can inhibit the proliferation, migration and glycolysis-related phenotypes of colorectal cancer cells and promote cell apoptosis, and has good in vitro antitumor activity.

[0019] (3) The present invention further verified the inhibitory effect of HIT211363271 on organoid growth in colorectal cancer organoid models, which improved the credibility and application value of the technical solution.

[0020] (4) This invention provides an experimental basis and technical reference for the development of drugs for the treatment of colorectal cancer. Attached Figure Description

[0021] Figure 1 Figure 1 shows the source and quality verification results of HIT211363271. A is the ^1H NMR spectrum of HIT211363271; B is the LCMS chromatogram of HIT211363271 at 220 nm; C is the LCMS chromatogram of HIT211363271 at 254 nm; D is the mass spectrometry detection of HIT211363271 in positive ion mode; and E is the mass spectrometry detection of HIT211363271 in negative ion mode.

[0022] Figure 2Figure 1 shows the experimental results of the effect of HIT211363271 on the viability of colorectal cancer cells and normal intestinal epithelial cells; Figure 2 shows the effect of HIT211363271 on the viability of HCT116 and Caco-2 cells; Figure 3 shows the effect of HIT211363271 on the viability of normal intestinal epithelial cells (HIEC).

[0023] Figure 3 Figure 1 shows the experimental results of the effect of HIT211363271 on the proliferation-related phenotypes of colorectal cancer cells. Among them, A is the CCK-8 time curve of HCT116 and Caco-2 cells after treatment with solvent control or HIT211363271 for 0–4 days; B is the colony formation experiment results of the corresponding groups; C is the EdU detection results of the corresponding groups.

[0024] Figure 4 The figures show the experimental results of the effects of HIT211363271 on the migration and apoptosis of colorectal cancer cells; where A is the scratch assay results of HCT116 and Caco-2 cells after treatment with solvent control or HIT211363271; and B is the Annexin V / PI flow cytometry apoptosis detection results of the corresponding groups.

[0025] Figure 5 Figure 1 shows the experimental results of the effect of HIT211363271 on glycolysis-related phenotypes in colorectal cancer cells. In this figure, A shows the glucose consumption of HCT116 and Caco-2 cells after treatment with solvent control or HIT211363271; B shows the lactate production of the corresponding groups; and C shows the ATP content of the corresponding groups.

[0026] Figure 6 Figure 1 shows the experimental results of the effect of HIT211363271 on the growth of colorectal cancer organoids; Figure 2 shows the observation results of colorectal cancer organoids in the solvent control group, HIT211363271 group and 5-Fu positive control group. Detailed Implementation

[0027] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used are conventional methods in the art, and the reagents, materials and instruments used are commercially available.

[0028] The human colorectal cancer cell lines HCT116 and Caco-2, as well as the normal intestinal epithelial cells HIEC used in the following examples, were all purchased from ATCC, identified by STR before culture, and tested negative for mycoplasma. The HCT116 and Caco-2 cells were mainly used for subsequent drug screening and functional experiments; the HIEC cells served as normal intestinal epithelial control cells and were used for cell viability reference experiments.

[0029] The colorectal cancer organoids used were derived from patient tissue isolates and cultured, and stably passaged. The related research has received ethical approval (approval number 2026-054). During culture, the organoid suspension was mixed with pre-cooled matrix gel at a volume ratio of 3:7 and inoculated into preheated 24-well culture plates. The plates were incubated at 37 °C for 30 min to allow the matrix gel to solidify. Then, complete colorectal cancer organoid culture medium was added, and the plates were cultured at 37 °C with 5% CO2. The culture medium was changed every 2 days. After the organoids reached stable growth, subsequent drug treatments were performed according to the experimental design.

[0030] The HIT211363271 stock solution was prepared using DMSO with a concentration of 50 mM. It can be further diluted to prepare working solutions as needed for subsequent in vitro and organoid experiments. 5-Fu can be used as a positive control for organoid evaluation.

[0031] Unless otherwise specified, the reagents and consumables used in the embodiments of this invention can be conventional commercially available products in the art. These reagents and consumables mainly include cell culture-related reagents, cell function detection-related reagents, metabolic detection-related reagents, and organoid culture-related reagents, etc.

[0032] Cell culture-related reagents may include penicillin-streptomycin antibiotics, RPMI 1640 medium, fetal bovine serum, serum-free cell cryopreservation solution, PBS phosphate buffer, and 0.25% trypsin digestion solution; cell function detection-related reagents may include CCK-8 assay kits, crystal violet staining solution, EdU cell proliferation assay kits, and Annexin V / PI apoptosis assay kits; metabolic detection-related reagents may include glucose assay kits, lactate assay kits, and ATP assay kits; organoid culture-related reagents may include matrix gel, colorectal cancer organoid culture medium, organoid digestion solution, organoid recovery solution, organoid cryopreservation solution, and anti-adhesion washing solution.

[0033] Unless otherwise specified, the experimental instruments and equipment used in the embodiments of this invention can be conventional commercial equipment in the field, including but not limited to: clean benches, cell culture incubators, inverted microscopes, fluorescence microscopes, flow cytometers, low-temperature high-speed centrifuges, low-speed centrifuges, microplate readers, multi-functional microplate readers, and other conventional experimental equipment.

[0034] Each in vitro experiment of this invention is preferably performed at least three times independently; data from each group are expressed as mean ± standard deviation. Comparisons between two groups can be performed using a two-tailed Student's t-test, and comparisons among multiple groups can be performed using one-way ANOVA, etc. A p-value < 0.05 is considered statistically significant.

[0035] Example 1: Source and Quality Control of HIT211363271 HIT211363271 was purchased from Shenzhen Taoshu Biotechnology Co., Ltd., manufacturer number C464-1425. According to the basic information provided by the supplier, the molecular formula of the compound is C. 24 H 29 FN4O4S has a relative molecular mass of 488.58. To confirm the structural information, purity, and relative molecular mass of HIT211363271 used in the experiment, [the following was performed / analyzed]. 1 The purchased samples were analyzed using 1H NMR and LCMS.

[0036] HIT211363271 has a clear origin. 1 H NMR detection results are as follows Figure 1 As shown in Figure A, the proton NMR spectrum of this compound is consistent with the structure of the target compound, and no obvious abnormal impurity peaks were observed. The LCMS detection results are as follows: Figure 1 As shown in Figures B and C, this compound exhibits a high percentage of main peak area at both 220 nm and 254 nm. Mass spectrometry results are as follows. Figure 1 As shown in Figures D and E, the main peak at m / z 489.2 was detected in positive ion mode, and the main peak at m / z 487.1 was detected in negative ion mode.

[0037] The above results indicate that the HIT211363271 used in this invention has the correct structure, high purity, and a relative molecular mass that meets expectations. Its quality is controllable and can meet the needs of subsequent cell function experiments and organoid experiments.

[0038] Example 2: Effect of HIT211363271 on the viability of colorectal cancer cells To evaluate the effect of HIT211363271 on the viability of colorectal cancer cells, the CCK-8 assay was used to detect its effect on the viability of HCT116, Caco-2 and normal intestinal epithelial cells HIEC.

[0039] Cells were seeded at a rate of 5000 cells / well in 96-well plates, with 6 replicates per group and blank wells. After cell attachment, different concentrations of HIT211363271 were added for incubation. After 48 h, 10 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37 ℃ for 2 h. The absorbance at 450 nm was then measured, and the relative cell viability of each group was calculated. Cell viability was calculated using the following formula: Cell viability (%) = [(OD value of drug-treated group - OD value of blank well) / (OD value of solvent control group - OD value of blank well)] × 100%.

[0040] Based on dose-response curves, the IC50 of HIT211363271 against HCT116, Caco-2, and HIEC cells at 48 h was calculated. 50 The values ​​were 43.84±2.27 μM, 40.31±6.24 μM, and 257.98±24.38 μM, respectively.

[0041] The results are as follows Figure 2 As shown in Figure A, HIT211363271 significantly reduced the viability of HCT116 and Caco-2 cells; Figure 2 As shown in Figure B, HIT211363271 exhibited relatively weak inhibitory effects on normal intestinal epithelial cells (HIEC), suggesting that HIT211363271 has a good inhibitory effect on colorectal cancer cells and possesses a certain degree of selectivity. Based on the above cell viability test results, HIT211363271 was used at a treatment concentration of 40 μM in subsequent cell function experiments.

[0042] Example 3: Effect of HIT211363271 on the proliferation of colorectal cancer cells To further evaluate the effect of HIT211363271 on the proliferation ability of colorectal cancer cells, CCK-8 time curves, colony formation assays, and EdU assays were used. DMSO was used as the solvent control group.

[0043] CCK-8 time-curve assay: HCT116 and Caco-2 cells were seeded at 1000 cells / well in 96-well plates, with 6 replicates per group and blank wells. After cell attachment, DMSO was added to the Vehicle group as a solvent control; HIT211363271 group was treated with HIT211363271 to a final concentration of 40 μM. The final DMSO concentration was maintained consistent across groups by supplementing with DMSO, and the final culture volume for each well was 100 μL. Cell viability was assessed on days 0, 1, 2, 3, and 4 post-treatment. Each time point was analyzed using independent 96-well plates. 10 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37 ℃ for 2 h. The absorbance at 450 nm was then measured, and cell growth curves were plotted.

[0044] Colony formation assay: HCT116 and Caco-2 cells were digested and prepared into single-cell suspensions, and seeded at 1000 cells / well in 6-well plates. After cell adhesion, DMSO was added to the Vehicle group as a solvent control; HIT211363271 group was treated with HIT211363271 to a final concentration of 40 μM. The final DMSO concentration was maintained consistent across groups by adding more DMSO, and the final volume of each well was 2 mL. Cells were incubated at 37 ℃ in a 5% CO2 incubator for 10–14 days. The culture medium was changed every 3 days, adding fresh medium containing the above treatment. After visible colonies formed, the culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 20 min, washed, air-dried, and photographed. The colony count was determined using ImageJ software.

[0045] EdU experiment: HCT116 and Caco-2 cells were mixed at 3×10⁻⁶ 4 Cells were seeded per well in 24-well plates. After cell adhesion, DMSO was added to the Vehicle group as a solvent control; HIT211363271 group was treated with HIT211363271 to a final concentration of 40 μM. The final DMSO concentration was maintained consistent across groups by adding more DMSO, and the final volume of each well was 1 mL. After culturing for 48 h, EdU detection was performed according to the kit instructions. Specifically, EdU working solution was added and incubated for 2 h, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with permeabilization solution for 15 min, click reaction solution was added and incubated at room temperature in the dark for 40 min, Hoechst 33342 was added and incubated at room temperature in the dark for 10 min. Finally, the EdU positivity rate was observed and calculated under an inverted fluorescence microscope.

[0046] The results are as follows Figure 3 As shown, compared with the solvent control group, the viability of HCT116 and Caco-2 cells decreased after HIT211363271 treatment ( Figure 3 As shown in A), the ability to form clones is weakened ( Figure 3 As shown in Figure B), the EdU positivity rate decreased ( Figure 3 As shown in Figure C), HIT211363271 can inhibit the proliferation of colorectal cancer cells.

[0047] Example 4: Effects of HIT211363271 on the migration and apoptosis of colorectal cancer cells To evaluate the effects of HIT211363271 on the migration and apoptosis of colorectal cancer cells, scratch assay and Annexin V Alexa Fluor APC / PI double staining flow cytometry were used for detection.

[0048] Scratch assay: HCT116 and Caco-2 cells were subjected to a 1×10⁻⁶ scratch assay. 6 Cells were seeded per well in 6-well plates and cultured at 37 ℃ in a 5% CO2 incubator until confluence reached 90% or higher. After vertical scratching with a sterile pipette tip, cells were gently washed with PBS to remove detached cells. Culture medium containing 1% serum was then added for further culture. DMSO was added as a solvent control in the Vehicle group; HIT211363271 was added to the HIT211363271 group to a final concentration of 40 μM. The final DMSO concentration was maintained consistent across groups by supplementing with DMSO, and the final culture volume for each well was 2 mL. Images were acquired at 0 h, 24 h, and 48 h. The scratch area was measured using ImageJ software, and the scratch healing rate was calculated using the following formula: Scratch healing rate (%) = [(0 h scratch area - scratch area at each time point) / 0 h scratch area] × 100%.

[0049] Apoptosis experiment: HCT116 and Caco-2 cells were cultured at 6 × 10⁻⁶ cells per cell line. 5 Cells were seeded per well in 6-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h before drug treatment. The Vehicle group received DMSO as a solvent control, while the HIT211363271 group received HIT211363271 to a final concentration of 40 μM. The final DMSO concentration was maintained across all groups by supplementing with DMSO, and the final culture volume for each well was 2 mL. After 48 h of treatment, cells were collected for subsequent apoptosis detection. After washing with pre-cooled PBS, cells were resuspended in 1× Binding Buffer according to the kit instructions, and Annexin VAlexa Fluor APC and PI staining solution were added. Cells were incubated in the dark for 15 min. Immediately afterwards, flow cytometry was used for detection, collecting at least 10,000 effective cell events per group, and data analysis was performed using the accompanying NovoExpress software. The sum of early and late apoptosis rates was used as the total apoptosis rate to evaluate the effect of HIT211363271 on apoptosis in colorectal cancer cells.

[0050] The results are as follows Figure 4 As shown, compared with the solvent control group, the migration rate of HCT116 and Caco-2 cells was significantly reduced after treatment with HIT211363271. Figure 4 As shown in Figure A), the total apoptosis rate was significantly increased ( Figure 4 As shown in Figure B), HIT211363271 can inhibit the migration of colorectal cancer cells and promote their apoptosis.

[0051] Example 5: Effect of HIT211363271 on glycolysis-related phenotypes in colorectal cancer cells To evaluate the effect of HIT211363271 on glycolysis-related phenotypes in colorectal cancer cells, glucose consumption, lactate production, and ATP content were measured. HCT116 and Caco-2 cells were cultured at 6 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates and cultured at 37 ℃ in a 5% CO2 incubator. After cell adhesion, the cells were treated with DMSO. The Vehicle group received DMSO as a solvent control, while the HIT211363271 group received HIT211363271 to a final concentration of 40 μM. The final DMSO concentration was maintained across all groups by supplementing with DMSO, and the final culture volume for each well was 2 mL. After 48 h of treatment, the cell culture supernatant was collected for glucose and lactate assays, and cells were also collected and lysed for ATP assays.

[0052] After diluting the culture supernatant to the detection range of the kit as needed, the glucose and lactate contents in the culture supernatant were detected using glucose and lactate assay kits, respectively. Glucose consumption was calculated based on the difference between the initial glucose concentration in the blank medium and the remaining glucose concentration in the cell culture supernatant, while lactate production was calculated based on the difference between the lactate concentration in the cell culture supernatant and the initial lactate concentration in the blank medium. The total protein concentration in the cell lysate was determined using a BCA protein quantification kit. Intracellular ATP levels were detected using an ATP assay kit and normalized to the total protein content.

[0053] The results are as follows Figure 5 As shown, compared with the solvent control group, HIT211363271 treatment reduced glucose consumption in HCT116 and Caco-2 cells ( Figure 5 As shown in Figure A), lactic acid production is reduced ( Figure 5 As shown in Figure B), the ATP content decreased after normalization to total protein ( Figure 5 As shown in Figure C), HIT211363271 can inhibit the glycolysis-related phenotype of colorectal cancer cells.

[0054] Example 6: Effect of HIT211363271 on the growth of colorectal cancer organoids After colorectal cancer organoids were cultured to full expansion and stable growth as described above, they were treated with a drug-containing medium prepared from complete colorectal cancer organoid culture medium according to the experimental design. The experiment included three groups: a Vehicle group, a 5-Fu group, and a HIT211363271 group. The Vehicle group received DMSO as a control; the HIT211363271 group received HIT211363271 at a final concentration of 40 μM; and the 5-Fu positive control group received 5-Fu at a final concentration of 20 μM. To eliminate the influence of solvents, the final solvent concentrations were kept consistent across all groups. Morphological changes, growth status, and survival of the organoids were observed and recorded under a microscope at 0, 24, 48, 72, and 96 h of treatment.

[0055] The results are as follows Figure 6 As shown, compared with the solvent control group, the organoids treated with HIT211363271 exhibited restricted growth, irregular structure, and some even showed shrinkage, collapse, or disintegration; the 5-Fu positive control group also showed a significant inhibitory effect. These results indicate that HIT211363271 has an inhibitory effect on the growth of colorectal cancer organoids.

[0056] This invention, through cell function experiments and organoid experiments, demonstrates the application value of HIT211363271 in the preparation of drugs for treating colorectal cancer. The results show that HIT211363271 can inhibit the proliferation, migration, and glycolysis-related phenotypes of colorectal cancer cells, promote apoptosis, and inhibit the growth of colorectal cancer organoids. Therefore, the uses of HIT211363271 provided by this invention can offer a reference for the development of drugs for treating colorectal cancer and have promising industrial application prospects.

Claims

1. The use of compound HIT211363271 or a pharmaceutically acceptable salt thereof in the preparation of an anti-colorectal cancer drug; the structural formula of compound HIT211363271 is shown in formula (Ⅰ) below: Equation (Ⅰ).

2. The application as described in claim 1, characterized in that, The compound HIT211363271 inhibits the proliferation and migration of colorectal cancer cells and promotes apoptosis of colorectal cancer cells.

3. The application as described in claim 1, characterized in that, The compound HIT211363271 inhibits the glycolysis-related phenotype of colorectal cancer cells.

4. The application as described in claim 1, characterized in that, The compound HIT211363271 inhibits the growth of colorectal cancer organoids.

5. The use of the pharmaceutical composition in the preparation of an anti-colorectal cancer drug, characterized in that, The active ingredient of the pharmaceutical composition includes compound HIT211363271 or a pharmaceutically acceptable salt thereof; the structural formula of compound HIT211363271 is shown in formula (Ⅰ) below: Equation (Ⅰ).

6. The application as described in claim 5, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

7. The application as described in claim 6, characterized in that, The pharmaceutical composition is any one of the following dosage forms: tablets, granules, capsules, oral liquids, injections, or suspensions.

Images