Use of a small molecule compound in the preparation of a medicament for treating pancreatic cancer

Abstract

The application discloses anti-pancreatic cancer activity of a small molecule compound (5-ethoxy-2-((2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazine) methyl) phenol) derived from a small molecule library, so as to be used as an active ingredient for preparing a clinical treatment drug for resisting pancreatic cancer. Research finds that the small molecule compound can significantly inhibit human pancreatic cancer cell proliferation, and the IC 50 Value of CFPAC-1 cells is 0.99+ / -0.26 µM; can induce human pancreatic cancer cell apoptosis, and the apoptosis rate presents concentration dependence; can significantly reduce the volume of a mouse pancreatic cancer tumor after administration (20 mg / kg), has certain in-vivo anti-tumor activity, and has high anti-cancer activity, low toxicity and certain safety as an anti-pancreatic cancer treatment drug.

Description

Technical Field

[0001] This invention relates to the pancreatic cancer activity of a small molecule compound 7 and its application in the preparation of drugs for treating pancreatic cancer, belonging to the field of natural product chemistry. Background Technology

[0002] Pancreatic cancer (PC) is one of the deadliest malignant digestive tract tumors in the world, seriously threatening human life and health. In recent years, the mortality rate of pancreatic cancer has been rising year by year, with its incidence rate comparable to its mortality rate. By 2030, pancreatic cancer will become the second leading cause of cancer-related death after lung cancer. Pancreatic cancer has an insidious onset, is difficult to diagnose early, has an extremely poor prognosis, and is prone to recurrence and metastasis. Its 5-year survival rate is approximately 9%, and more than 80% of patients are diagnosed at a locally advanced stage or even with metastasis, earning it the title of "King of Cancers."

[0003] Currently, treatment options for pancreatic cancer mainly include radiotherapy, chemotherapy, targeted therapy, surgical resection, and immunotherapy. However, solid tumors are mostly heterogeneous, characterized by high connective tissue proliferation and an immunosuppressive tumor microenvironment. Therefore, existing treatments often suffer from drug resistance and drawbacks. Furthermore, approximately 80% of patients are already at an advanced stage at initial diagnosis, making surgical resection impossible. Therefore, the development of drugs for pancreatic cancer is urgently needed.

[0004] Small molecule compounds refer to compounds with a molecular weight of less than 1000. They are typically composed of a series of complex molecules consisting of oxygen, nitrogen, carbon, and hydrogen elements. In recent years, small molecule compounds have played a role in drug research, agriculture, food additives, materials, environmental protection, energy, medicine, and other fields. They possess excellent selectivity and specific biological activities, meeting many scientific and industrial needs.

[0005] The small molecule compound 5-ethoxy-2-((2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazone)methyl)phenol (compound 7) is derived from a small molecule library and its structure is shown below:

[0006]

[0007] Currently, there are no reports on the biological activity of small molecule compound 7. Summary of the Invention

[0008] The purpose of this invention is to study the anti-pancreatic cancer activity of small molecule compound 7, with the aim of using it as an active ingredient in the preparation of a clinical therapeutic drug for pancreatic cancer.

[0009] The following experiments will determine the bioactivity of small molecule compound 7 and its inhibitory effect on pancreatic cancer.

[0010] Effects of small molecule compound (compound 7) on the growth of pancreatic cancer cells

[0011] Cells in the logarithmic growth phase were taken at a concentration of 3 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates, with experimental, blank, and control groups established (the drug-treated group was the experimental group, the group without cells but with the same proportion of solvent was the blank group, and the group with the same proportion of solvent-treated cells was the control group). The AMG510 (a KRAS-targeting inhibitor for pancreatic cancer)-treated cell group served as the positive control group. The next day, after the cells adhered, the medium was prepared and changed according to the treatment of each group, and the cells were incubated at 37°C in a 5% CO2 incubator (Thermo Fisher Scientific, USA) for 72 h. The liquid in each well was discarded, and the cells were washed 1-2 times with PBS (Solepro Science & Technology Co., Ltd., China), the supernatant was discarded, and 100 μL of serum-free medium (Gibco, USA) containing 10%, 5 mg / mL MTT solution (MedChemexpress, USA) was added. The cells were then incubated at 37°C in the dark for 4 h. Discard any excess liquid in the plate. Add DMSO (Solepro Technology Co., Ltd., China) to each well to dissolve the blue-purple formazan crystals at the bottom of the plate. Finally, measure the absorbance of each well at 490 nm using a microplate reader (TECAN, Switzerland). Cell viability and inhibition rate are calculated using the following formula: half-inhibitory concentration (IC50) of the drug's anti-cell proliferation activity. 50 The results were calculated using Graph Pad Prism 8 software. Cell viability (%) = (OD490 of treatment group - OD490 of blank group) / (OD490 of control group - OD490 of blank group) × 100%, cell inhibition rate (%) = (OD490 of control group - OD490 of treatment group) / (OD490 of control group - OD490 of blank group) × 100%.

[0012]

[0013] like Figure 1 And Table 1 (IC50 of compound 7 against SW1990, CFPAC-1 and PANC-1 cells at 72 h) 50 As shown in the summary table, compound 7 inhibited the proliferation of three pancreatic cancer cell lines to varying degrees in a concentration-dependent manner. The results indicated that compound 7 had an IC50 concentration of [missing value] in SW1990, CFPAC-1, and PANC-1 cells. 50 The values ​​were 15.71±2.56μM, 0.99±0.26μM and 5.25±1.25μM, respectively, revealing that compound 7 has strong anti-proliferation inhibitory activity against CFPAC-1 cells, followed by PANC-1 cells, and has the worst inhibitory effect on SW1990 cells.

[0014] Effect of compound 7 on apoptosis of pancreatic cancer cells

[0015] Cells in the logarithmic growth phase were taken at a concentration of 1 × 10⁻⁶. 6 Cells were seeded per well in 6-well plates at concentrations of 0.5 µM, 1 µM, 2 µM, and 4 µM of compound 7, and cultured for 48 h. Cells were then digested with 0.25% trypsin and collected (digestion time should not be too long, otherwise false positives may occur). The supernatant was discarded, and cells were washed twice with pre-chilled PBS. Cells were centrifuged at 1000 r / min for 5 min and collected again. Cells were resuspended by adding 200 μL–500 μL of buffer according to an apoptosis detection kit (BD Biosciences, USA). 5 μL of Annexin V-FITC was added, and the mixture was gently pipetted to mix. Cells were incubated at room temperature in the dark for 15–30 min. Apoptosis was detected within 1 h using a flow cytometer (Beckman Biosciences, USA).

[0016] like Figure 2 As shown, with increasing drug concentration, the apoptosis rates of each group were 5.19%, 6.91%, 14.91%, 16.07%, and 25.27%, respectively, indicating that compound 7 can induce apoptosis in CFPAC-1 cells in a concentration-dependent manner.

[0017] Effect of compound 7 on the growth of pancreatic cancer model mice

[0018] Thirty-two pancreatic cancer model mice were randomly divided into four groups (n=8): control group, compound 7 treatment group, GEM treatment group, and combined treatment group. The tumor volume reached 100 mm². 3 Compound 7 (20 mg / kg) was administered by gavage once daily, and GEM (50 mg / kg) was administered intraperitoneally once weekly. The experiment lasted for 21 consecutive days, during which tumor volume and mouse weight were measured daily. Tumor volume was calculated using the formula V = 1 / 2 × A × B. 2 (A represents the long axis of the tumor, and B represents the short axis of the tumor) Calculation. After 21 days of continuous administration, mice were sacrificed, tumors were removed, weighed, and the average weight of tumors in each group was calculated. Tumor samples from each group were placed together and photographed for observation. Some samples were fixed in 4% paraformaldehyde solution, and others were frozen in liquid nitrogen. During the experiment, the therapeutic effect of compound 7 on the mouse tumor model was detected by measuring tumor volume and mouse weight daily.

[0019] like Figure 3As shown, there were no abnormal changes in the body weight of mice before and after drug administration, and all groups showed varying degrees of increase. Compared with the control group, the tumor volume of mice in all experimental groups decreased. The combined drug group showed the greatest inhibition of tumor volume, with a tumor inhibition rate of 60.63%, followed by the GEM treatment group and the compound 7 treatment group, with tumor inhibition rates of 46.90% and 53.16%, respectively. In addition, compared with the control group, HE staining of tumor tissue showed that the infiltration of tumor cells gradually decreased after treatment with compound 7.

[0020] Evaluation of the pharmacokinetics of compound 7 in rats

[0021] Compound 7 was administered orally (PO) at a dose of 20 mg / kg and intravenously (IV) at a dose of 2 mg / kg. It was dissolved in water and administered by gavage. Blood samples of 0.3 ml were collected from the retro-orbital venous plexus before administration (0 h) and at 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. The blood was rapidly added to 0.5 ml centrifuge tubes containing heparin and centrifuged at 8000 r / min for 5 min. The plasma was collected and stored at -20 °C and analyzed by LC-MS / MS (Shimadzu, Japan). Table 2 shows the pharmacokinetic evaluation of compound 7 in rats.

[0022]

[0023] As shown in Table 2, the plasma clearance rate of compound 7 in rats after oral administration was 94.10 L / h / kg, and the plasma volume distribution of compound 7 reached steady state (Vss = 490.38 L / kg). Following oral and intravenous administration, the terminal elimination half-lives of compound 7 in rats were 10.66 h and 6.33 h, respectively; the times to peak concentration were 7.60 h and 0.14 h, respectively; the peak concentrations were 38.74 μg / L and 205.34 μg / L, respectively; and the absolute bioavailability was 10.60%.

[0024] Evaluation of the acute toxicity of compound 7 in mice

[0025] Twenty Kunming mice were divided into a compound 7 treatment group and a blank control group, with 10 mice in each group (half male and half female). The dosage of compound 7 was 2000 mg / kg, and the mice were administered by gavage for 7 consecutive days. During the period, the behavior, weight and number of deaths of the mice were observed.

[0026] like Figure 4 and Figure 5 As shown, the body weight of mice in both the control and experimental groups increased to some extent after 7 days of treatment. HE staining confirmed that compound 7 had no significant toxicity to the heart, liver, spleen, lungs, and kidneys of mice. Table 3 shows the evaluation of mortality after acute toxicity treatment of compound 7 in mice.

[0027]

[0028] As shown in Table 3, after 7 days of continuous gavage administration of compound 7, no mice in any group died.

[0029] In summary, small molecule compound 7 has the following characteristics:

[0030] 1. Compound 7 can significantly inhibit the proliferation of human pancreatic cancer cells and has an effect on the IC50 of CFPAC-1 cells. 50 The value is 0.99 ± 0.26 µM;

[0031] 2. Compound 7 can induce apoptosis in human pancreatic cancer cells, and the apoptosis rate is concentration-dependent.

[0032] 3. Compound 7 (20 mg / kg) significantly reduced the volume of pancreatic cancer tumors in mice after administration, and has certain in vivo anti-tumor activity, and can be used as an anti-pancreatic cancer treatment drug;

[0033] 4. Compound 7 has no toxic side effects on normal rats when used as a drug and has a certain degree of safety. It can be used as an active ingredient against pancreatic cancer, either alone or in combination with existing chemotherapy drugs to treat pancreatic cancer. Attached Figure Description

[0034] Figure 1 The graph shows the cell viability assay results of the inhibitory effect of compound 7 on the proliferation of SW1990, CFPAC-1 and PANC-1 cells.

[0035] Figure 2 The figure shows the apoptosis-inducing effect of compound 7 on CFPAC-1 cells.

[0036] Figure 3 The figure shows the tumor inhibition results of compound 7 in a mouse model of pancreatic cancer.

[0037] Figure 4 This is a statistical graph showing the changes in body weight in mice after acute toxicity treatment with compound 7.

[0038] Figure 5 This is a statistical chart showing the pathological conditions of the heart, liver, spleen, lungs, and kidneys in mice after acute toxicity treatment with compound 7. Detailed Implementation

[0039] The active ingredient, compound 7, is prepared into an oral or injectable formulation using pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparation processes.

[0040] Compound 7 is combined with other drugs with anti-pancreatic cancer activity as active ingredients and is formulated into oral or injectable preparations using pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparation processes.

[0041] Oral preparations include powders, granules, capsules, soft capsules, powders, pills, tablets, oral liquids, etc.

Claims

1. The application of a small molecule compound in the preparation of a drug for treating pancreatic cancer, characterized in that: The small molecule compound is 5-ethoxy-2-((2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazone)methyl)phenol, with the following structural formula: 。 2. The use of the small molecule compound as described in claim 1 in the preparation of a drug for treating pancreatic cancer, characterized in that: The small molecule compound is used as the active ingredient and is prepared into an oral or injectable formulation using pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparation processes.

3. The use of the small molecule compound as described in claim 1 in the preparation of a drug for treating pancreatic cancer, characterized in that: The small molecule compound is combined with other drugs with anti-pancreatic cancer activity as active ingredients and prepared into oral or injectable formulations using pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparation processes.

4. The use of a small molecule compound as described in claim 2 or 3 in the preparation of a drug for treating pancreatic cancer, characterized in that: Oral preparations include powders, granules, capsules, powders, pills, tablets, and oral liquids.

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