Use of thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs

Abstract

The application discloses a kind of thieno [2, 3-c] pyrazole compounds in preparation antitumor drug purposes, belong to pharmaceutical chemistry technical field.The application provides a kind of small molecule skeleton thieno [2, 3-c] pyrazole compound I and II, compound I is 2-oxo benzo [d] [1, 3] oxathiole-6-yl 1-(3-fluorophenyl)-3-methyl-1H-thieno [2, 3-c] pyrazole-5-carboxylate.Compound II is 1-(4-chlorophenyl)-N-[4-((4-ethylpiperazine-1-yl)methyl)phenyl]-3-methyl-1H-thieno [2, 3-c] pyrazole-5-formyl.Both have strong antitumor activity.Compound II has strong inhibition to a variety of human tumor cells, such as bone sarcoma MG63, breast cancer HCC1806, liver cancer LM3, lung cancer A549, colorectal cancer HCT-8 and the like, provides new candidate compounds for broad-spectrum antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the use of thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs. Background Technology

[0002] Cancer is currently one of the leading causes of death worldwide, posing a serious threat to human health. Over the past three decades, significant progress has been made in the development of anti-tumor drugs, with treatment strategies gradually shifting from traditional chemotherapy to molecularly targeted therapy and immunotherapy, forming a new treatment landscape with multiple mechanisms. Statistics show that small molecule drugs account for approximately 78% of the 174 widely used anti-tumor drugs globally. Despite the significant achievements of small molecule anti-tumor drugs in clinical applications, they still face many challenges: firstly, most drugs have a narrow anti-tumor spectrum, making it difficult to cover multiple cancer types; secondly, their efficacy is limited for certain refractory or highly heterogeneous tumors; and thirdly, long-term use can easily induce drug resistance in tumor cells. Therefore, developing small molecule anti-tumor candidate compounds with novel chemical structures and broad-spectrum, high efficacy is extremely necessary.

[0003] The present invention aims to provide a thieno[2,3-c]pyrazole small molecule compound with broad-spectrum antitumor activity. Summary of the Invention

[0004] The purpose of this invention is to provide the use of thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs, wherein the structural formula of the thieno[2,3-c]pyrazole compounds is shown in Formula I or II:

[0005] .

[0006] The object of this invention is achieved by the use of thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs.

[0007] This invention provides two small-molecule thieno[2,3-c]pyrazole compounds, I and II, both exhibiting strong antitumor activity. Compound II, in particular, demonstrates strong inhibitory effects on various human tumor cells, including osteosarcoma MG63, breast cancer HCC1806, liver cancer LM3, lung cancer A549, and colorectal cancer HCT-8, providing a novel candidate compound for broad-spectrum antitumor drugs. Attached Figure Description

[0008] Figure 1 The structural formula of compound I of this invention is shown below;

[0009] Figure 2 The structural formula of compound II of this invention is shown below;

[0010] Figure 3 This is a curve showing the inhibition of the activity of compound I of the present invention on two types of human tumor cells;

[0011] Figure 4 This is a graph showing the inhibition of the activity of compound II of the present invention against five types of human tumor cells;

[0012] Figure 5 The curve showing the inhibition of human breast cancer cell viability by the chemotherapy drug carboplatin in Comparative Example 1 (IC50) 50 The numerical value represents the concentration at which carboplatin inhibits cancer cell growth at half the level.

[0013] Figure 6 The image shows the inhibition curves (IC50) of the chemotherapy drug carboplatin against four types of human tumor cells (human osteosarcoma MG63, human colorectal cancer HCT-8, human liver cancer LM3, and human lung cancer A549) in Comparative Example 1. 50 The numerical value represents the concentration at which carboplatin inhibits cancer cell growth at half the level. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0015] Compounds I and II of this invention were purchased from a drug-like compound library containing 5,000 compounds developed by Shanghai Taoshu Biotechnology Co., Ltd.

[0016] Compound I: 2-oxobenzo[d][1,3]oxothiacyclopentane-6-yl-1-(3-fluorophenyl)-3-methyl-1H-thieno[2,3-c]pyrazole-5-carboxylate, with the structural formula shown in Formula I. Library ID: G784-0951, Plate layout: PHD198945, CAS Number: 1115332-69-8, Molecular formula: C 20 H 11 FN2O4S2 has a relative molecular mass of 426.44.

[0017] Compound II: 1-(4-chlorophenyl)-N-[4-((4-ethylpiperazin-1-yl)methyl)phenyl]-3-methyl-1H-thieno[2,3-c]pyrazole-5-carboxyl; Library ID: G789-1022, Plate layout: PHD198945, CASNumber: 1357704-5-0, Molecular formula: C 26 H 28 ClN5OS has a relative molecular mass of 494.05.

[0018] .

[0019] The conventional cancer cells used in the embodiments of this invention are all derived from Kunming Medical University, including human breast cancer cells HCC1806, human liver cancer cells LM3, human lung cancer cells A549, human ileocecal colorectal adenocarcinoma cells HCT-8, and human osteosarcoma cells MG63.

[0020] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available. RPMI 1640 medium, DMEM medium, and fetal bovine serum were purchased from Biological Industries; phosphate-buffered saline (PBS) and 0.25% trypsin (containing EDTA) were purchased from Gibco.

[0021] The cell lines were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 90% humidity.

[0022] The present invention provides the use of the thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs.

[0023] The tumor is selected from breast cancer, liver cancer, colorectal cancer, osteosarcoma, or lung cancer.

[0024] The present invention also provides an antitumor drug, wherein the antitumor drug uses a thieno[2,3-c]pyrazole compound as shown in Formula I or II and / or a pharmaceutically acceptable salt thereof as the active ingredient.

[0025] The dosage form of the antitumor drug is tablets, capsules, pills, oral solutions, or injections.

[0026] Example 1: Detection of the inhibitory effects of compounds I and II on breast cancer cells

[0027] The CCK-8 assay was used to detect cell growth inhibition. The CCK-8 assay, short for Cell Counting Kit-8, is a commonly used method for detecting cell proliferation and cytotoxicity. The principle of the CCK-8 assay is based on the compound WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt). In the presence of the electron carrier 1-Methoxy PMS (menaquinone phosphate), WST-8 can be reduced by intracellular mitochondrial dehydrogenases to produce a highly water-soluble orange-yellow formazan product. The amount of formazan produced is directly proportional to the number of viable cells, and the color intensity reflects cellular metabolic activity. By measuring the absorbance at 450 nm using a microplate reader, the number of viable cells can be indirectly reflected, thereby assessing cell proliferation, cytotoxicity, or the cellular effect of drugs.

[0028] Experimental method: Compound I and Compound II were dissolved in dimethyl sulfoxide (DMSO), and then diluted with DMSO to prepare test solutions with concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM, respectively. All experiments were performed in triplicate.

[0029] Human breast cancer cells HCC1806 (4×10) 3 Cells were seeded in 96-well plates with 99 μL of culture medium per well and incubated at 37°C with 5% CO2 for 24 h. Then, 1 μL of the test solution was added to each well. For the blank control group, only 1 μL of DMSO was added to the cell culture, and the plates were incubated for 72 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was gently mixed to avoid air bubbles. The plates were incubated for another 1 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the average OD value was calculated. The results were compared with the blank control group (cells treated with only DMSO) to analyze changes in cell proliferation or toxicity. The percentage of absorbance in the experimental group relative to the blank control group represents the cell viability or proliferation level; the blank control group is assumed to be 100%.

[0030] Table 1. Inhibition results of compound I on human breast cancer cells HCC1806

[0031] Drug concentration (μM) Cell viability (%) 0 100±1.569 0.1 92.707±1.698 0.5 81.145±4.094 1 79.097±2.365 5 73.634±1.05 10 34.559±2.47

[0032] Table 2 shows the inhibitory effects of compound II on human breast cancer cells HCC1806.

[0033] Drug concentration (μM) Cell viability (%) 0 100±1.372 0.1 86.536±1.201 0.5 63.176±2.1 1 41.427±2.637 5 0.094±0.001 10 0.09±0.001

[0034] Experimental results: as shown in Tables 1 and 2. Figure 3 , Figure 4 It is known that compounds I and II are cytotoxic to human breast cancer cells HCC1806.

[0035] Example 2: Detection of the inhibitory effects of compounds I and II on osteosarcoma cells

[0036] Experimental method: Compound I and compound II were dissolved in DMSO, respectively. Compound I was diluted with DMSO to prepare test solutions with concentrations of 10 μM and 0.1 μM, respectively. Compound II was diluted with DMSO to prepare test solutions with concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM, respectively.

[0037] Human osteosarcoma cells MG63 (4×10) 3 Cells were seeded in 96-well plates with 99 μL of culture medium per well and incubated at 37°C with 5% CO2 for 24 h. Then, 1 μL of the test solution was added to each well. For the blank control group, only 1 μL of DMSO was added to the cell culture, and the plates were incubated for 72 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was gently mixed to avoid air bubbles. The plates were incubated for another 1 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the average OD value was calculated. The results were compared with the blank control group (cells treated with only DMSO) to analyze changes in cell proliferation or cytotoxicity. The percentage of absorbance in the experimental group relative to the blank control group represents the cell viability or proliferation level; the blank control group is assumed to be 100%.

[0038] Table 3. Inhibition of compound I on human osteosarcoma cells MG63

[0039] Drug concentration (μM) Cell viability (%) 0 100±1.989 0.1 48.231±1.343 10 44.486±2.277

[0040] Table 4. Inhibition results of compound II on human osteosarcoma cells MG63

[0041] Drug concentration (μM) Cell viability (%) 0 100±11.619 0.1 57.039±1.825 0.5 55.966±5 1 52.575±8.353 5 0.108±0.004 10 0.078±0.006

[0042] Experimental results: From Tables 3 and 4 and Figure 3 , Figure 4 It is known that compounds I and II are cytotoxic to the human osteosarcoma cell line MG63.

[0043] Example 3: Detection of the inhibitory effect of compound II on lung cancer cells

[0044] Experimental method: Compound II was dissolved in DMSO and diluted with DMSO to prepare test solutions with concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM, respectively.

[0045] Human lung cancer cells A549 (4×10) 3 Cells were seeded in 96-well plates with 99 μL of culture medium per well and incubated at 37°C with 5% CO2 for 24 h. Then, 1 μL of the test solution was added to each well. For the blank control group, only 1 μL of DMSO was added to the cell culture, and the plates were incubated for 72 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was gently mixed to avoid air bubbles. The plates were incubated for another 1 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the average OD value was calculated. The results were compared with the blank control group (cells treated with only DMSO) to analyze changes in cell proliferation or cytotoxicity. The percentage of absorbance in the experimental group relative to the blank control group represents the cell viability or proliferation level; the blank control group is assumed to be 100%.

[0046] Table 5. Inhibition results of compound II on human lung cancer cells A549

[0047] Drug concentration (μM) Cell viability (%) 0 100±1.994 0.1 83.31±1.791 0.5 71.191±5.753 1 23.823±4.309 5 1.334±0.3 10 0.117±0.002

[0048] Experimental results: as shown in Table 5 and Figure 4 As shown, compound II is cytotoxic to human lung cancer cells A549.

[0049] Example 4: Detection of the inhibitory effect of compound II on ileocecal colorectal adenocarcinoma cells.

[0050] Experimental method: Compound II was dissolved in DMSO and diluted with DMSO to prepare test solutions with concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM, respectively.

[0051] HCT-8 colorectal adenocarcinoma cells from the ileocecal region (4×10⁻⁸) 3 Cells were seeded in 96-well plates with 99 μL of culture medium per well and incubated at 37°C with 5% CO2 for 24 h. Then, 1 μL of the test solution was added to each well. For the blank control group, only 1 μL of DMSO was added to the cell culture, and the plates were incubated for 72 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was gently mixed to avoid air bubbles. The plates were incubated for another 1 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the average OD value was calculated. The results were compared with the blank control group (cells treated with only DMSO) to analyze changes in cell proliferation or toxicity. The percentage of absorbance in the experimental group relative to the blank control group represents the cell viability or proliferation level; the blank control group is assumed to be 100%.

[0052] Table 6. Inhibition of compound II on human ileocecal colorectal adenocarcinoma cells HCT-8

[0053] Drug concentration (μM) Cell viability (%) 0 100±1.152 0.1 87.06±1.146 0.5 72.66±5.211 1 56.835±1.38 5 21.166±4.43 10 13.18±0.049

[0054] Experimental results: From Table 6 and Figure 4 It is known that compound II has cancer cell toxicity against the human ileocecal colorectal adenocarcinoma cell line HCT-8.

[0055] Example 5: Detection of the inhibitory effect of compound II on liver cancer cells.

[0056] Experimental method: Compound II was dissolved in DMSO and diluted with DMSO to prepare test solutions with concentrations of 10 μM, 5 μM, 1 μM, 0.5 μM and 0.1 μM, respectively.

[0057] Human liver cancer cells LM3 (4×10) 3 Cells were seeded in 96-well plates with 99 μL of culture medium per well and incubated at 37°C with 5% CO2 for 24 h. Then, 1 μL of the test solution was added to each well. For the blank control group, only 1 μL of DMSO was added to the cell culture, and the plates were incubated for 72 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was gently mixed to avoid air bubbles. The plates were incubated for another 1 h, and the absorbance of each well was measured at 450 nm using a microplate reader. The average OD value of each well was calculated, and the results were compared with the blank control group (cells treated with only DMSO) to analyze changes in cell proliferation or toxicity. The percentage of the absorbance value of the experimental group relative to the absorbance value of the blank control group represents the cell viability or proliferation level; the blank control group is assumed to be 100%.

[0058] Table 7. Inhibition results of compound II on human liver cancer cells LM3.

[0059] Drug concentration (μM) Cell viability (%) 0 100±1.994 0.1 83.31±1.791 0.5 71.191±5.753 1 23.823±4.309 5 1.334±0.3 10 0.117±0.002

[0060] Experimental results: According to Table 7 and Figure 4 The results showed that compound II had cancer cell toxicity against the human liver cancer cell line LM3.

[0061] In summary, as shown in Tables 1-4 and Figure 3 , Figure 4As shown, compounds I and II exhibit significant dose-dependent inhibitory activity against human breast cancer cells and human osteosarcoma cells; compound II also showed significant dose-dependent inhibitory activity against human liver cancer cells, human lung cancer cells, and human colorectal cancer cells. Cell viability gradually decreased with increasing drug concentration, indicating that both compounds I and II have a clear in vitro anti-proliferative effect.

[0062] Comparative Example 1

[0063] The inhibitory activity of carboplatin against human breast cancer cells HCC1806, human osteosarcoma cells MG63, human colorectal cancer cells HCT-8, human liver cancer cells LM3, and human lung cancer cells A549 was detected using the same methods as in Examples 1-5. The difference was that carboplatin was prepared with sterile PBS as the stock solution, and a series of concentration gradients from 0.1 to 100 μM were set up. 1 μL of carboplatin was added to each well, and an equal volume of PBS was added to the blank control group. The remaining operations were the same as in Examples 1-5.

[0064] Result: As Figure 5-6 As shown, carboplatin exhibited some inhibitory effect on the proliferation of all five types of tumor cells, but the overall inhibitory activity was weak.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of thieno[2,3-c]pyrazole compounds in the preparation of antitumor drugs, characterized in that, The structural formulas of the thieno[2,3-c]pyrazole compounds are shown in Formula I or II: AND, II; The tumor is breast cancer, liver cancer, colorectal cancer, osteosarcoma, or lung cancer.

2. The use of the thieno[2,3-c]pyrazole compound according to claim 1 in the preparation of antitumor drugs, characterized in that, The antitumor drug uses thieno[2,3-c]pyrazole compounds of formula I or II and / or their pharmaceutically acceptable salts as active ingredients.

3. The use of the thieno[2,3-c]pyrazole compound according to claim 1 in the preparation of antitumor drugs, characterized in that, The dosage form of the antitumor drug is tablets, capsules, pills, oral solutions, or injections.

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