Use of chiral tetrahydropyrazolo[1,5-a]pyrimidine derivatives for the preparation of antitumor drugs for the treatment of HER2-positive colorectal cancer

Abstract

The application discloses application of a chiral tetrahydropyrazolo[1,5-a]pyrimidine derivative in preparation of an antitumor drug for treating HER2 positive colorectal cancer. Through in vitro and in vivo experiments, it is confirmed that the compound has a significant inhibitory effect on proliferation and migration of HER2 positive colorectal cancer cells, and shows antitumor activity. The application provides a new idea for developing a novel tyrosine kinase inhibitor for treating HER2 positive colorectal cancer in clinic.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drugs, specifically involving a novel chiral tetrahydropyrazolo[1,5-a]pyrimidine derivative and its in vitro and in vivo anti-HER2-positive colorectal cancer cell activity. Background Technology

[0002] Colorectal cancer is the third most common malignant tumor worldwide, and its mortality rate ranks second among all cancers [Bray Fet al.]. CA Cancer J Clin [Li N et al. Cancer Lett. 2021;522:255-268.]. China is a high-burden country for colorectal cancer, accounting for about one-third of the global total of new cases [Li N et al. Cancer Lett. 2021;522:255-268.]. Among them, human epidermal growth factor receptor 2 (HER2) positive colorectal cancer is a special subtype of this disease, characterized by HER2 protein overexpression, HER2 gene amplification and / or point mutation. HER2 oncogenic activation is closely related to tumor metastasis, poor prognosis and resistance to anti-epidermal growth factor receptor (EGFR) therapy [Shan L et al.]. J Cancer Res Clin Oncol .2018;144(11):2275-2281; Ingold Heppner B et al. Br J Cancer . 2014;111(10):1977-1984; Yonesaka K et al. Sci Transl Med [2011;3(99):99ra86.]. Currently, the selection of targeted therapies for HER2-positive colorectal cancer is limited, and existing drugs have problems such as insufficient efficacy or easy drug resistance, which leads to serious challenges in clinical treatment.

[0003] Tyrosine kinase inhibitors (TKIs) are typically small organic compounds with a nitrogen-containing heterocyclic skeleton. Their structural features generally include a rigid aromatic heterocyclic core, a hinge region hydrogen-bonded group, and a hydrophobic substituted side chain that regulates target selectivity. They can competitively bind to the ATP-binding pocket of the HER2 protein, thereby inhibiting its tyrosine kinase activity. Studies have shown that, as HER2-targeted therapeutic drugs, these inhibitors can effectively inhibit downstream signaling pathways and have demonstrated significant clinical efficacy in the treatment of HER2-positive breast cancer and gastric cancer. Their clinical application is gradually expanding to the treatment of HER2-positive colorectal cancer [Fu X et al. Cancer Sci. 2023;114(3):1067-1074.]. Currently, there is a lack of TKI drugs targeting HER2-positive colorectal cancer, and in practical applications, they also face problems such as heterogeneity of efficacy and easy development of drug resistance [Li HS et al. Front Oncol. 2020;10:548867.]. There is an urgent need to develop novel TKI drugs targeting HER2-positive colorectal cancer. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a novel chiral tetrahydropyrazolo[1,5-a]pyrimidine derivative (i.e., compound 1) as shown in Formula I in the preparation of a drug for treating HER2-positive colorectal cancer. In vivo and in vitro experiments have shown that this compound exhibits strong anticancer activity against two types of HER2-positive colorectal cancer cells.

[0005] Formula I Furthermore, in the above technical solution, the colorectal cancer cells are LS174T and / or HCT116 cells.

[0006] Furthermore, in the above-mentioned technical solution, the drug comprises a chiral tetrahydropyrazolo[1,5-a]pyrimidine derivative as shown in Formula I and one or more pharmaceutically acceptable excipients.

[0007] Compared with the prior art, the present invention has the following beneficial effects: Compound 1 disclosed in this invention is easy to synthesize, operates under mild reaction conditions, requires low energy, has low raw material costs, is environmentally friendly, and yields high quantities. Experiments have demonstrated that this compound significantly inhibits the proliferation and migration of HER2-positive colorectal cancer cells (including LS174T and HCT116 cells), downregulates the expression level of HER2 protein, and reduces its phosphorylation modification. Simultaneously, it effectively inhibits the growth of LS174T and HCT116 cell xenografts in nude mice. These results indicate that this compound holds promise for providing important lead structures and strategic support for the development of novel HER2-positive colorectal cancer tyrosine kinase inhibitors. Attached Figure Description

[0008] Figure 1 To detect the expression level of HER2 protein in normal human colonic epithelial cells NCM460 and human colorectal cancer cells LoVo, HCT116, and LS174T using Western blot experiments; Figure 2 Line graphs showing cell viability after 24 hours of treatment with different concentrations of compound 1, as determined by the CCK-8 assay, for LS174T, HCT116, and NCM460 cells. Figure 3 To investigate the effect of 20 μM compound 1 on the proliferation of LS174T and HCT116 cells in a plate colony assay; Figure 4 To investigate the effect of 20 μM compound 1 on the migration ability of LS174T and HCT116 cells using a scratch assay; Figure 5 To detect the expression level of HER2 protein in LS174T and HCT116 cells after treatment with 30 μM compound 1 for 24 h using Western blot experiments; Figure 6 To detect the expression levels of p-HER2 and HER2 proteins in LS174T and HCT116 cells after treatment with 30 μM compound 1 for 0, 2, 3, and 4 h using Western blot experiments; Figure 7 To inoculate LS174T cells subcutaneously into nude mice, when the tumor was palpable, compound 1 (0, 25, 50 mg / kg / day) was administered orally by gavage for two weeks, with tumor volume measured every two days; Figure 8 To inoculate HCT116 cells subcutaneously into nude mice, when tumors were palpable, compound 1 (0, 25, 50 mg / kg / day) was administered orally by gavage for two weeks, with tumor volume measured every two days; Figure 9Line graphs showing cell viability of LS174T and HCT116 cells after 24 h of treatment with comparative drug compounds, as determined by the CCK-8 assay. Figure 10 Line graphs showing cell viability after 24 h and 48 h of treatment with different concentrations of (R)-3, as determined by the CCK-8 assay; Figure 11 Line graphs showing cell viability after 24 h and 48 h of treatment with different concentrations of (S)-3, as determined by the CCK-8 assay; Figure 12 The cell viability of normal human colonic epithelial NCM460 cells treated with different concentrations of (R)-3 and (S)-3 for 24 h was detected by CCK-8 assay, and the IC50 value and selectivity index (SI) were calculated based on the detection results. Figure 13 Line graphs showing cell viability after 24 h of treatment with different concentrations of compound 8, as determined by the CCK-8 assay, for LS174T, HCT116, and NCM460 cells. Detailed Implementation

[0009] The present invention will now be further illustrated by the following specific embodiments to facilitate understanding of the technical solution and beneficial effects of the present invention. These embodiments are merely examples and are not intended to limit the scope of the present invention.

[0010] The preparation method of compound 1 is as follows: it is prepared by asymmetric hydrogenation of pyrazolopyrimidine compound through a catalytic system of [Ir(COD)Cl]2 / (S)-SegPos. The specific operation steps are as follows: weighed [Ir(COD)Cl]2 and (S)-SegPos are added to an ampoule, then tetrahydrofuran is added in a glove box and stirred for ten minutes, then trichloroisocyanuric acid is added and stirred for ten minutes. Finally, the stirred catalyst is introduced into a reaction flask containing raw materials and copper trifluoromethanesulfonate, and tetrahydrofuran is added as solvent. The reaction flask is then placed in a reaction vessel, tightened, removed from the glove box, and hydrogen is introduced. The reaction is stirred at 50 degrees Celsius for 48 hours. The reaction is stopped, and the target product compound 1 is obtained by column chromatography.

[0011] Example 1: Detection of HER2 protein expression levels in human colorectal cancer cell lines method: The cells used included human colorectal cancer cells HCT116, LS174T, and LoVo, and normal human colonic epithelial cells NCM460. HCT116 and NCM460 cells were cultured in RPMI 1640 medium, while LS174T and LoVo cells were cultured in DMEM medium. All media were supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotic solution. All cells were cultured in a 37°C, 5% CO2 incubator.

[0012] Prepare protein samples. Prepare protein lysis buffer by adding 10 µL of phosphatase inhibitor A, 10 µL of phosphatase inhibitor B, 10 µL of protease inhibitor, and 10 µL of PMSF sequentially to 1 mL of pre-chilled Lysis Buffer (Seven). Mix well and store on ice. Wash the cell pellet twice with pre-chilled PBS. (The last part, "1×10," appears to be a typo and should be removed.) 7 Add 1 mL of lysis buffer per cell, gently pipette to mix, and incubate on ice for 30 min on a shaker. After lysis, centrifuge at 12000 rpm for 10 min at 4 °C, collect the supernatant and transfer to a new EP tube. Determine the protein concentration using a BCA protein assay kit (Seven), and calculate the required loading volume based on a protein loading rate of 40 μg per well. Add 4× protein loading buffer (Solarbio) to the obtained protein sample at a 3:1 ratio, mix well, boil at 100 °C for 10 min, cool, and store at -20 °C for later use.

[0013] Western blot assay for proteins. A 10% separating gel and a 5% stacking gel were prepared, and samples were added in the predetermined order and volume. Electrophoresis was performed in an ice bath with an initial voltage of 80 V. After the samples entered the separating gel, the voltage was adjusted to 120 V until the bromophenol blue indicator reached the bottom of the gel. Subsequently, a wet transfer system was used for membrane transfer. The gel, NC membrane, and filter paper were neatly assembled and immersed in the transfer buffer. Under ice-water bath conditions, a constant current of 180 mA was set, and transfer was performed for 2 hours. After transfer, the NC membrane was placed in a TBST solution containing 5% skim milk powder and blocked at room temperature for 2 hours. After blocking, the NC membrane was immersed in specific primary antibody diluted with TBST solution containing 5% skim milk and incubated overnight at 4°C. The primary antibody for the target protein was Anti-HER2 (Proteintech, 18299-1-AP, 1:3000), and the primary antibody for the internal control protein was Anti-β-actin (Proteintech, 66009-1-Ig, 1:2000). After incubation with the primary antibody, the membrane was washed three times with TBST solution for 10 min each time. Subsequently, the membrane was incubated with the corresponding horseradish peroxidase-labeled goat anti-rabbit IgG or goat anti-mouse IgG (Solarbio, 1:2000) at room temperature for 2 h. After incubation with the secondary antibody, the membrane was washed six times with TBST solution for 5 min each time. The reaction bands were detected using an ECL kit (Seven), and images were acquired using a chemiluminescence imaging system. The gray values ​​of the bands were quantitatively analyzed using ImageJ software. The relative expression level of the target protein was calculated using the gray value of the internal control protein β-actin as a reference. Results are expressed as mean ± standard deviation, and each experiment was independently repeated three times.

[0014] result: Compared with normal human colonic epithelial cells NCM460, the expression level of HER2 protein in human colorectal cancer cell lines HCT116 and LS174T showed an upregulated trend, while no significant difference was shown in LoVo. Among them, the HER2 expression level in LS174T was significantly higher than that in other cell lines. Figure 1 Therefore, LS174T and HCT116 cell lines were selected as HER2-positive colorectal cancer cells for the determination of the anticancer effect of the compound.

[0015] Example 2: Detection of the viability of NCM460, LS174T and HCT116 cells by compound 1 method: Cells in the logarithmic growth phase were seeded at a density of approximately 4000 cells per well in the middle wells of a 96-well plate. After complete cell adhesion, the medium was replaced with culture medium containing 0, 2.5, 5, 10, 20, 30, and 40 μM of Compound 1 for further culture. Each plate contained three types of wells: blank wells contained only normal complete culture medium, without cells or the compound; control wells used complete culture medium containing 0.5% DMSO; and experimental wells contained complete culture medium with different concentrations of Compound 1. After 24 hours of culture, 10 μL of CCK8 reagent (APEXBIO, K1018) was added to each well, and the plates were incubated for 1–4 hours. The OD value of each well at 450 nm was then measured using a microplate reader. Cell viability was calculated as follows: Cell viability = [(Experimental well OD value - Blank well OD value) / (Control well OD value - Blank well OD value)] × 100%. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 9.5.0 software. 50 The selectivity index (SI) of a compound is calculated as SI = (IC) / (IC) 50 in NCM460 cells) / (IC 50 (in LS174Tor HCT116 cells). Results are expressed as mean ± standard deviation, and each experiment was independently repeated 3 times.

[0016] result: The CCK8 experiment results showed that compound 1 inhibited the growth of three cell lines in a dose-dependent manner. Figure 2 Compound 1's IC50 value after 24 hours of treatment with NCM460, LS174T, and HCT116 cells was determined. 50 The values ​​were 38.62±0.31, 24.17±2.51, and 29.74±4.94 μM, respectively. The IC50 values ​​for NCM460 cells were... 50 With reference values, the SI values ​​of compound 1 for LS174T and HCT116 cells were 1.60 and 1.30, respectively.

[0017] Example 3: Detection of the effect of compound 1 on the proliferation ability of LS174T and HCT116 cells method: Cells in the logarithmic growth phase were seeded evenly in 6-well plates at a density of approximately 500 cells per well. After cell adhesion, the cells were cultured for 48 hours in control medium containing 0.5% DMSO and complete medium containing 20 μM compound 1, followed by replacement with normal medium and continued culture for 1–2 weeks. Culture was stopped when clear clones were visible to the naked eye, and the cells were stained with crystal violet and photographed. The number of clones formed in each group was counted and statistically analyzed using ImageJ software. The relative colony formation rate was calculated as (number of clones in the experimental group / number of clones in the control group) × 100%. Results are expressed as mean ± standard deviation. Each experiment was independently repeated three times.

[0018] result: The results of the plate colony formation assay showed that, compared with the 0 μM control group, 20 μM compound 1 could inhibit the proliferation of LS174T and HCT116 cells. Figure 3 ).

[0019] Example 4: Detection of the effect of compound 1 on the migration ability of LS174T and HCT116 cells method: Using a black marker and ruler, draw three equidistant horizontal lines on the back of a 12-well plate. Seed cells in the logarithmic growth phase evenly into the wells at the desired density, then incubate at 37°C in a 5% CO2 incubator. When cell confluence reaches 90% or higher, remove the culture medium and streak the cells vertically along the bottom of the plate using a sterile 200μL pipette tip. Wash away any suspended cells with sterile PBS. For the control group, add 1mL of serum-free medium containing 0.5% DMSO to each well; for the experimental group, add serum-free medium containing 20μM compound 1 to each well. Observe and photograph the same field of view under an inverted microscope at 0h and 24h after streaking. Measure the streaking area using ImageJ software and perform statistical analysis. Cell migration rate = [(streaking area at 0h - streaking area at 24h) / streaking area at 0h] × 100%. Results are expressed as mean ± standard deviation. Each experiment was independently repeated three times.

[0020] result: Scratch assay results showed that, compared with the 0 μM control group, 20 μM compound 1 could inhibit the migration ability of LS174T and HCT116 cells. Figure 4 ).

[0021] Example 5: Detection of the effect of compound 1 on HER2 protein expression levels in LS174T and HCT116 cells method: Cells in the logarithmic growth phase were seeded evenly in 12-well plates at the desired density and then incubated at 37°C in a 5% CO2 incubator. When the cells reached approximately 80% confluence, the culture medium was removed. For the control group, 1 mL of serum-free medium containing 0.5% DMSO was added to each well, while for the experimental group, 30 μM of compound 1 was added to each well. Incubation was continued for 24 h. After culture was stopped, protein samples were prepared and Western blot experiments were performed using the same method as in Example 1. Results are expressed as mean ± standard deviation, and each experiment was independently repeated three times.

[0022] result: Compared with the DMSO control group, treatment of LS174T and HCT116 cells with 30 μM compound 1 for 24 h reduced HER2 protein expression levels. Figure 5 ).

[0023] Example 6: Detection of the effect of compound 1 on the phosphorylation level of HER2 protein in LS174T and HCT116 cells method: Cells in the logarithmic growth phase were seeded evenly in 12-well plates at the desired density and then cultured in an incubator at 37°C with 5% CO2. When the cells reached approximately 80% confluence, the medium was replaced with serum-free medium. Subsequently, cells were treated in reverse order, with the medium replaced with serum-free medium containing 30 μM compound 1 at time points of 0h, 2h, 3h, and 4h to simulate cell responses at different treatment durations. After culture was stopped, protein sample preparation and Western blot experiments were performed using the same methods as in Example 1. The primary antibodies against the target proteins were Anti-HER2 (Proteintech, 18299-1-AP, 1:3000) and Anti-p-HER2 (Abclonal, AP0527, 1:1000). Results are expressed as mean ± standard deviation, and each experiment was independently repeated three times.

[0024] result: 30 μM compound 1 resulted in a time-dependent decrease in phosphorylation levels at the Y1221 / 1222 site of the HER2 protein in LS174T and HCT116 cells. Figure 6 ).

[0025] Example 7: Compound 1 inhibits the growth of LS174T xenografts in nude mice. method: LS174T cells in logarithmic growth phase (1×10⁻⁶) 7Compound 1 (cells in 0.2 mL PBS per mouse) was injected subcutaneously into the back of BALB / c nude mice. One week later, mice carrying homologous and similarly sized xenografts were randomly divided into three groups and treated orally (po) once daily (QD) for two weeks with 25 mg / kg, 50 mg / kg of compound 1, and the control solvent, respectively. Compound 1 was dissolved in distilled water containing 2% DMSO, 30% PEG300, and 5% Tween-80. During treatment, mouse weight was recorded every two days, and tumor size was measured with calipers. Tumor volume (V) was calculated using the formula V = (L × W²) / 2, where L is the longest diameter and W is the diameter perpendicular to tumor L. After the experiment, mice were anesthetized and euthanized by intraperitoneal injection of 15 μl / g 2.5% tribromoethanol, resulting in cervical dislocation and death.

[0026] result: Compared with the control group, compound 1 at 25 mg / kg and 50 mg / kg inhibited the growth of LS174T xenografts, with the 50 mg / kg compound 1 showing a more significant inhibitory effect on LS174T xenograft growth compared with the 25 mg / kg group. Figure 7 ).

[0027] Example 8: Compound 1 inhibits the growth of HCT116 xenografts in nude mice. method: HCT116 cells in logarithmic growth phase (5 × 10⁻⁶) 6 Compound 1 (cells in 0.2 mL PBS per mouse) was injected subcutaneously into the back of BALB / c nude mice. One week later, mice carrying homologous and similarly sized xenografts were randomly divided into three groups and treated orally (po) once daily (QD) for two weeks with 25 mg / kg, 50 mg / kg of compound 1, and the control solvent, respectively. Compound 1 was dissolved in distilled water containing 2% DMSO, 30% PEG300, and 5% Tween-80. During treatment, mouse weight was recorded every two days, and tumor size was measured with calipers. Tumor volume (V) was calculated using the formula V = (L × W²) / 2, where L is the longest diameter and W is the diameter perpendicular to tumor L. After the experiment, mice were anesthetized and euthanized by intraperitoneal injection of 15 μl / g 2.5% tribromoethanol, resulting in cervical dislocation and death.

[0028] result: Compared with the control group, compound 1 at 25 mg / kg and 50 mg / kg inhibited the growth of HCT116 xenografts, with compound 1 at 50 mg / kg showing a more significant inhibitory effect on HCT116 xenograft growth compared with the 25 mg / kg group. Figure 8 ).

[0029] Comparative Example Anticancer activity of a chiral tetrahydropyrazolopyrimidine compound library in HER2-positive colorectal cancer: In vitro screening experiments were conducted using LS174T and HCT116 cell lines, with lapatinib used as a positive control. The difference from Example 1 is that the drugs used are different; the drugs used in this comparative example are compounds listed in section A. Compound 1 in section A is not the same drug compound as compound 1 in Example 1. See [link to example]. Figure 9 Among them, compounds ( R )-3、( S Compounds )-3 and 8 showed relatively high sensitivity in both colon cancer cell lines, while other compounds exhibited relatively weaker activity. Different concentrations of ( ) were used... R )-3 or ( S Treatment of LS174T and HCT116 cells with )-3 for 24 and 48 hours, CCK8 assay results showed that both compounds inhibited cell proliferation in a concentration- and time-dependent manner. Figure 10 , Figure 11 To evaluate the selectivity of the compound for colon cancer cells, different concentrations of ( ) were used. R )-3 or ( S Human normal colonic epithelial NCM460 cells were treated with 0.3 for 24 hours, and cell viability was detected by CCK8 assay. Then IC50 was calculated. 50 Values ​​and Selectivity Index (SI) Figure 12 After 24 hours of treatment, ( R )-3 and ( S )-3 IC50 on NCM460 cells 50 The values ​​were 110.80 ± 12.30 μM and 104.00 ± 7.44 μM, respectively. (Based on the NCM460 IC...) 50 The value is used as the numerator. R The SI values ​​of )-3 for LS174T cells and HCT116 cells were 2.69 and 1.62, respectively, while ( S The SI values ​​for )-3 were 1.56 and 1.42, respectively. This indicates that ( R )-3 showed better selectivity for colon cancer cells, and compared to HCT116 cells, ( RCompound 8 showed stronger selectivity for LS174T cells compared to other compounds. Compound 8 exhibited stronger inhibitory effects on the growth of LS174T and HCT116 cells compared to other compounds. After treating NCM460, LS174T, and HCT116 cells with different concentrations of compound 8 for 24 hours, CCK8 assay results showed that the compound inhibited the proliferation of the three cell lines in a concentration-dependent manner. Figure 13 ). 24-hour IC50 of compound 8 50 The values ​​were 29.54 ± 2.86 μM (NCM460), 34.63 ± 3.93 μM (LS174T), and 26.11 ± 2.33 μM (HCT116). The IC50 values ​​for NCM460 cells were... 50 For reference, the SI values ​​for compound 8 were 0.85 and 1.13, respectively.

Claims

1. The application of chiral tetrahydropyrazolo[1,5-a]pyrimidine derivatives as shown in Formula I in the preparation of drugs for treating HER2-positive colorectal cancer. Formula I.

2. The application according to claim 1, characterized in that: The colorectal cancer cells were LS174T and / or HCT116 cells.

3. The application according to claim 1, characterized in that: The drug comprises a chiral tetrahydropyrazolo[1,5-a]pyrimidine derivative as shown in Formula I and one or more pharmaceutically acceptable excipients.

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