Novel HER2 inhibitor as well as preparation method and application thereof

Abstract

The invention provides a novel HER2 inhibitor as well as a preparation method and application thereof. The novel HER2 inhibitor is a small molecule compound, and the structural formula of the novel HER2 inhibitor is as shown in formula 1. NCI-N87 cells are selected as an HER2 positive gastric cancer cell model, and the targeting effect and anti-tumor activity of the compound shown in the formula 1 on an HER2 signal channel are verified. Experimental results show that the compound shown in the formula 1 has a targeting effect on HER2, can inhibit HER2 positive tumors, and is expected to become a new generation of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a novel HER2 inhibitor and a preparation method and application thereof. BACKGROUND

[0002] Human Epidermal Growth Factor Receptor 2 (HER2) is related to the occurrence and development of gastric cancer, breast cancer and many other cancers, and is one of the important targets of cancer targeted therapy.

[0003] The existing anti-HER-2 drugs have the following mechanisms of action: targeting HER-2 receptor: HER-2 (Human Epidermal Growth Factor Receptor 2) is a transmembrane protein that promotes cell proliferation, and overexpression can lead to malignant progression of tumors; blocking the signal pathway: by binding to the HER-2 receptor or inhibiting its activity, interfering with downstream pro-cancer signal transduction (such as PI3K / AKT, MAPK pathway); activating immune killing: some drugs enhance the immune system's clearance of tumor cells through antibody-dependent cellular cytotoxicity (ADCC).

[0004] The main drug types and representative drugs include monoclonal antibodies (such as trastuzumab, pertuzumab), small molecule tyrosine kinase inhibitors (such as small molecule tyrosine kinase inhibitors), antibody drug conjugates and bispecific antibodies (such as magituximab, which targets HER-2 and CD3, and activates T cells to kill tumors). Although monoclonal antibodies have the advantages of high specificity and strong efficacy, they can precisely target the HER2 receptor. However, their large molecular weight also leads to low oral bioavailability, and they need to be administered by injection. In addition, the production cost of monoclonal antibodies is high, and they can easily cause immune reactions and allergic reactions during treatment. The currently approved HER2 small molecule inhibitor drugs still face challenges such as drug resistance, insufficient targeting specificity and side effects in clinical applications, so the development of new HER2 small molecule inhibitors has become a research focus. SUMMARY

[0005] One of the purposes of the present application is to provide a compound.

[0006] The compound provided by the present application has the structural formula shown in Formula 1:

[0007] The compound shown in Formula 1 above is prepared by the method shown in the flowchart, which includes the following steps: Figure 1 1) Compound 1 and compound 2 are reacted in an organic solvent under basic conditions to obtain compound 3; ​

[0008] 2) Compound 3 is subjected to a reduction reaction in the presence of a reducing agent to obtain compound 4;

[0009] 3) Compound 4 is reacted with bromoacetonitrile (BrCH2CN) to obtain compound 6;

[0010] 4) Compound 6 is reacted with compound 7 in the presence of a palladium catalyst and a cesium salt to obtain the target compound, .

[0011] In the above method step 1), the base is potassium carbonate; The molar ratio of compound 1 to compound 2 can be 1:1-1:2; The temperature of the reaction can be 60-100°C, and the time can be 12-20 h, and specifically, the reaction can be carried out at 80°C for 16 h; In the above method step 2), the reducing agent can specifically be sodium borohydride, The molar ratio of compound 3 to sodium borohydride can be 1:2-3, and specifically, the molar ratio can be 1:2; The temperature of the reduction reaction can be room temperature, and the time can be 2-5 h, and specifically, the time can be 3 h; In the above method step 3), the molar ratio of compound 4 to bromoacetonitrile can be 1:4-6, and specifically, the molar ratio can be 1:5; The reaction is carried out under basic conditions, and the base can specifically be NaH; The temperature of the reaction can be room temperature, and the time can be 10-16 h, and specifically, the time can be 12 h; In the above method step 4), the palladium catalyst can specifically be Pd(dppf)Cl2; The cesium salt can specifically be cesium carbonate; The molar ratio of compound 6 to compound 7, the palladium catalyst, and the cesium salt can be 1:2-3:0.1-0.2:2-3, respectively; The reaction is carried out in an inert atmosphere; The temperature of the reaction can be 80-120°C, and specifically, the temperature can be 100°C, and the time can be 8-16 h, and specifically, the time can be 12 h.

[0012] Another object of the present application is to provide the use of the compound shown in formula 1 in the preparation of a HER2-positive tumor diagnosis and treatment reagent.

[0013] The use can specifically be the use of the compound shown in formula 1 in the preparation of a drug for preventing and / or treating a HER2-positive tumor.

[0014] The tumor is cancer, and specifically can be breast cancer, lung cancer, gastric cancer or intestinal cancer.

[0015] In the application, the drug has at least one of the following effects: 1) inhibiting the proliferation of tumor cells; 2) inhibiting the survival and clonal formation of tumor cells; 3) arresting the cell cycle of tumor cells.

[0016] The application also provides a drug for preventing and / or treating HER2-positive tumors, which contains the compound shown in formula 1.

[0017] The tumor is cancer, and specifically can be breast cancer, lung cancer, gastric cancer or intestinal cancer.

[0018] The application selects NCI-N87 cells as a HER2-positive gastric cancer cell model to verify the targeting effect of the compound shown in formula 1 on the HER2 signaling pathway and the anti-tumor activity. The experimental results show that the compound shown in formula 1 has a targeting effect on HER2, can inhibit HER2-positive tumors, and is expected to become a new generation of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a preparation flow chart of the compound shown in formula 1 in the application.

[0020] Figure 2 It is the nuclear magnetic hydrogen spectrum of the compound shown in formula 1 prepared in Example 1 of the application.

[0021] Figure 3 It is the molecular weight mass spectrum of the compound shown in formula 1 prepared in Example 1 of the application.

[0022] Figure 4 It is the HPLC purity result of the compound shown in formula 1 prepared in Example 1 of the application.

[0023] Figure 5 It is the effect of different concentrations of the compound shown in formula 1 on the activity of NCI-N87 cells determined by CCK-8 experiment in Example 2 of the application.

[0024] Figure 6 It is the relationship between the activity of HER2 cells and the concentration of the compound shown in formula 1 in Example 2 of the application.

[0025] Figure 7 It is the flow cytometry analysis of the apoptosis of NCI-N87 cells induced by the compound shown in formula 1 in Example 2 of the application.

[0026] Figure 8Statistics of the apoptosis rate of NCI-N87 cells induced by the compound represented by formula 1 in Example 2 of the present application, n = 3, compared with the control group, p <0.05,** p <0.01,*** p <0.001,**** p <0.0001.

[0027] Figure 9 Flow cytometry analysis of the cell cycle arrest of NCI-N87 cells induced by the compound represented by formula 1 in Example 2 of the present application.

[0028] Figure 10 Statistics of the proportion of the cell cycle arrest of NCI-N87 cells induced by the compound represented by formula 1 in Example 2 of the present application, n = 3, compared with the control group, p <0.05,** p <0.01,*** p <0.001,**** p <0.0001.

[0029] Figure 11 Observation results of the inhibition of NCI-N87 cell colony formation by the compound represented by formula 1 in Example 2 of the present application.

[0030] Figure 12 Statistics of the inhibition rate of NCI-N87 cell colony formation by the compound represented by formula 1 in Example 2 of the present application, n = 4, compared with the control group, p <0.05,** p <0.01,*** p <0.001,**** p <0.0001.

[0031] Figure 13 Subcutaneous tumor volume growth status after in vivo administration of the compound represented by formula 1 in Example 2 of the present application at different doses, n = 5.

[0032] Figure 14 Comparison of the subcutaneous tumor volume at the treatment endpoint after in vivo administration of the compound represented by formula 1 in Example 2 of the present application at different doses, n = 5.

[0033] Figure 15 Effect of the compound represented by formula 1 in Example 2 of the present application at different doses on the survival rate of tumor-bearing nude mice, n = 5.

[0034] Figure 16Effect of different doses of the compound of Formula 1 on the body weight of tumor-bearing nude mice in Example 2 of the present application, n =5. DETAILED DESCRIPTION

[0035] The present application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0036] The experimental methods in the following examples are all conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0037] Example 1, Preparation of the compound of Formula 1 Step 1: Synthesis of compound 3 Compound 1 (5.0 g, 41 mmol), compound 2 (20.5 g, 81.9 mmol) and K2CO3 (17 g, 123 mmol) were dissolved in 50 mL of DMF, stirred at 80 °C for 16 h, after the reaction was complete, concentrated under reduced pressure, and purified by flash chromatography to obtain compound 3 as a gray solid (7 g, 58.7%).

[0038] Step 2: Synthesis of compound 4 Compound 3 (7.0 g, 24 mmol) was dissolved in 50 mL of MeOH, NaBH4 (1.8 g, 48 mmol) was added, stirred at room temperature for 3 h, concentrated under reduced pressure, and purified by flash chromatography to obtain compound 4 as a gray solid (7 g, 99%).

[0039] Step 3: Synthesis of compound 6 Compound 4 (5.0 g, 17 mmol) and NaH (3.4 g, 85 mmol) were dissolved in 80 mL of THF, stirred at 0 °C for 30 min, then compound 5 (10.23 g, 85.28 mmol) was added, stirred at room temperature for 12 h, quenched with saturated NH4Cl (50 mL), extracted with EtOAc (3 x 50 mL), the organic layers were combined, washed with 30 mL of brine, dried over MgSO4, the solvent was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound 6 (1.5 g, 26%) as a gray solid.

[0040] Step 4, Synthesis of the target compound A mixture of compound 6 (1.5 g, 4.5 mmol), compound 7 (1.8 g, 9 mmol), Pd(dppf)Cl2(300 mg, 0.45 mmol), Cs2CO3(2.94 g, 9 mmol) and dioxane / H2O (20 mL / 5 mL) was added to a three-necked flask, degassed, filled with nitrogen, and the reaction mixture was stirred at 100 °C for 12 h, the solvent was evaporated under reduced pressure, and the residue was further purified by column chromatography to obtain the target compound (68 mg, 5%) as a white solid.

[0041] Figure 2 The nuclear magnetic resonance spectrum of the obtained compound is shown in

[0042] The structural formula of the target compound is shown in Figure 2 The nuclear magnetic resonance spectrum of the obtained compound is shown in 23 H 21 NO 2。 The molecular weight detection result of the target compound is shown in Figure 3 The main mass signal is concentrated at m / z = 366.2, which is C 23 H 21 NNaO2 single charged positive ion [M + Na] + The HPLC detection result of the target compound is shown in Figure 4 The purity is greater than 99%.

[0043] Example 2, biological activity research The biological effects of the compound on the proliferation, apoptosis and cycle regulation of positive HER2 high gastric cancer cells were systematically evaluated. According to the HER2 expression profile analysis of gastric cancer cell lines in The Human Protein Atlas database (https: / / www.proteinatlas.org / ), the NCI-N87 cell line (human gastric adenocarcinoma cells) showed significant HER2 protein overexpression characteristics. Therefore, NCI-N87 cells were selected as a HER2-positive gastric cancer cell model to verify the targeting effect of the compound on the HER2 signaling pathway and the anti-tumor activity.

[0044] The experimental design adopted a multi-level functional verification strategy: first, the inhibitory effect of the compound on the long-term proliferation ability of tumor cells was evaluated by colony formation experiment; second, the change of cell apoptosis rate was detected by Annexin V-FITC / PI double staining method; finally, the blocking effect of the compound on tumor cell cycle was elucidated by combining with PI single staining cell cycle analysis, and finally the in vivo therapeutic effect of the compound on transplanted tumor was clarified. Through the above systematic research, it is aimed to reveal the molecular mechanism of the candidate compound in inhibiting the growth of HER2-positive gastric cancer from the levels of cell function and animal, and to lay an experimental foundation for the further application of the compound.

[0045] Table 1, Experimental reagents and consumables

[0046] I. Experimental method (1) Cell colony formation experiment 1) Select NCI-N87 cells in logarithmic growth phase, and use 0.25% trypsin containing EDTA to process the cells. Cell suspension is inoculated into Φ35 mm culture dishes at a density of 6x10 5 cells / dish. When the cell density is close to 70% of the culture dish, drug treatment (100 μM, 300 μM and 500 μM of the compound shown in formula 1; the compound shown in formula 1 is dissolved with DMSO, and the stock solution concentration is 10 mg / mL) is performed. After the cells are continuously cultured for 48 h, the cells are collected and a single cell suspension is prepared. The cell density is determined by using an automatic cell counting system. The same volume of DMSO as the 500 μM group is added to the cells as a control group.

[0047] 2) After gentle shaking to ensure uniform distribution of the cells, they are transferred to a constant temperature incubator for adherent culture and overnight incubation.

[0048] 3) The cell state and colony growth are observed every 48 h, and the total culture period is controlled to be 18 d.

[0049] 4) After termination of the culture, the supernatant is discarded, the cells are washed with PBS twice, 1 mL of cell fixing solution is added, and the cells are fixed at room temperature for 30 min. After the liquid is discarded, the cells are naturally dried for 15 min.

[0050] 5) 1 mL of 0.1% crystal violet solution is added to each dish, which is immersed at room temperature for 20 min. Then, tap water is used to gently wash until the background is transparent. The dishes are naturally air-dried at room temperature, and the number of cell colonies formed in each group is counted: colony formation rate = colony number / inoculated cell number x inoculated cell number.

[0051] (2) Flow cytometry detection of cell apoptosis 1) Well-grown cells are selected, the culture medium is discarded, and the cells are washed with PBS twice. After trypsin digestion, termination of digestion, centrifugation, and resuspension, the cells are inoculated into a 6-well plate. When the cell density is close to 70% of the culture dish, drug treatment (100 μM, 300 μM and 500 μM of the compound shown in formula 1) is performed. The groups are the same as in the colony experiment.

[0052] 2) The 6-well plate is placed in a cell culture incubator for 48 h, and the culture medium in each well is collected. The wells are slowly washed twice with 4°C pre-cooled PBS. After the cells are digested with 500 μL of 0.25% trypsin without EDTA, the digestion is terminated with the collected original culture medium to maintain the integrity of the cell membrane. Then, the cell suspension is transferred to a centrifuge tube.

[0053] 3) Collect cells by centrifugation at 1200 rpm for 3 min, and wash twice with PBS.

[0054] 4) Add 200 μL of 1x binding buffer working solution, resuspend the cells, and transfer them to a 1.5 mL labeled EP tube.

[0055] 5) Set up 10 μL of Annexin V-FITC single staining tube and 5 μL of PI single staining tube for each sample in the experimental group, and add 10 μL of Annexin V-FITC and 5 μL of PI reagent to each sample in the control group and experimental group.

[0056] 6) Incubate at room temperature for 15 min in the dark, mix gently to form a single cell suspension, and detect by flow cytometry and analyze the results.

[0057] (3) Cell cycle detection 1) Single cell suspension preparation a. Select well-grown cells, discard the culture medium, wash twice with PBS, and perform trypsin digestion, stop digestion, centrifugation, resuspension, and then inoculate the cells into a Φ35 mm culture dish. When the cell density is close to 70% of the culture dish, perform drug treatment (100 μM, 300 μM, and 500 μM of the compound represented by Formula 1); group the same clone experiment, and collect the cells after 48 h.

[0058] b. Collect the supernatant into a 15 mL centrifuge tube.

[0059] c. Wash once with 1x PBS, and collect the cells again into a 15 mL centrifuge tube.

[0060] d. Add 1 mL of trypsin without EDTA, and stand for 4 min. Observe the cell digestion under a microscope. When the cell morphology becomes round, add trypsin to the 15 mL centrifuge tube; e. Gently tap the bottom of the dish to help the cells fall off the surface and form a single cell suspension; f. Add 1x PBS to collect the cells into a 15 mL centrifuge tube; g. Centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0061] 2) Cycle staining a. Add 500 μL of 1x PBS solution to make 1x10 6 Cell suspension in a 15 mL centrifuge tube.

[0062] b. Slowly add ice-cold anhydrous ethanol to 2 mL, the final concentration reaches 75%, and store at 4°C overnight.

[0063] c. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant.

[0064] d. Add 1 mL of 1xPBS, suspend the cells, and centrifuge again for 1 time.

[0065] e. Discard the supernatant, add 300 μL of PI / RNase staining solution, mix well, and stain at room temperature for 15 min in the dark. Filter the single-cell suspension using a 400-mesh screen.

[0066] f. Machine detection.

[0067] (4) Tumor-bearing mouse model construction and drug treatment 1) Tumor-bearing mouse model construction Select four-week-old, adaptively fed for 7 days, female immunodeficient BALB / c nude mice, and use a syringe to inoculate NCI-N87 cell suspension under sterile conditions into the right axillary subcutaneously. The cell suspension is adjusted to 5 million cells / 100 μL, and the same volume is inoculated in each nude mouse. Then, in the IVC system, about 14 d, when the tumor volume reaches 100 mm³, randomly group for subsequent drug treatment.

[0068] 2) Drug treatment The experiment sets a control group (Control) (diluted with PBS DMSO with the same volume as the 5 mg / kg group) and different dose treatment groups (0.25 mg / kg, 2.5 mg / kg and 5 mg / kg). The drug is administered through the tail vein, once every 3 days, for 15 days.

[0069] 3) Tumor volume monitoring Use a vernier caliper to measure the long diameter (L) and short diameter (W) of the tumor every 3 days, and calculate the tumor volume according to the following formula:

[0070] Where V is the tumor volume (mm³), L is the maximum diameter of the tumor, and W is the minimum diameter of the tumor.

[0071] 4) Data recording and analysis During the experiment, record the changes in body weight and tumor volume of the nude mice, and when the tumor volume reaches 1500 mm³, it is considered as the treatment endpoint, and the tumor tissue is isolated and photographed. After the experiment, compare the tumor inhibition effect of each treatment group with the control group by statistical method, and draw the tumor growth curve.

[0072] II. Analysis of experimental results Figure 5 The inhibition of cell proliferation by different concentrations of the drug at 24 h and 48 h is shown. Cell viability is negatively correlated with drug concentration, with a dose-dependent effect. At the same concentration, the cell viability of the 48 h group is significantly lower than that of the 24 h group, indicating that the inhibition of cell proliferation by the drug is enhanced as the drug action time is prolonged. This time-dependent effect is usually related to the accumulation of the drug or the cell cycle arrest effect. In the low concentration range (0-100 μM), the cell viability decreases rapidly, indicating that the drug can significantly inhibit cell proliferation at a lower dose. In the high concentration range (>100 μM), the cell viability tends to be stable, and the cells have basically stopped proliferating or tend to be in a state of death.

[0073] Figure 6 The effect of different concentrations of the drug on cell proliferation for 24 h is shown, and the IC 50 The value is 321 μM. The fitted curve shows a classic Sigmoid-shaped dose-effect curve, indicating that the effect of the drug conforms to the Hill equation model. From the curve shape, at low concentrations (logarithmic value <4), the cell viability remains around 100%, indicating that the drug does not significantly affect cell proliferation at this time. With the increase of drug concentration, the cell viability decreases sharply between Log(Conc.) and Log(Conc.), and finally tends to zero, indicating that this interval is the main range of drug action. The experimental data shows a small error line (standard deviation), indicating good experimental repeatability and reliable data. In addition, under the condition of high concentration of the drug (Log(Conc.)>6), the cell viability decreases to a very low level, indicating that the drug has strong toxicity to cells at high concentrations.

[0074] The flow cytometry results are shown in Figure 7 , and the apoptosis statistics are shown in Figure 8 Compared with the control group, the apoptosis rate significantly increases with the increase of drug concentration (100 μM, p <0.001; 300 μM and 500 μM, p <0.0001).

[0075] Cell cycle analysis is shown in Figure 9 , Figure 10 Compared with the control group, 100 μM ( p <0.0001) and 300 μM ( p <0.01) significantly increase the G0 / G1 phase arrest of cells, and 500 μM significantly increases the G2 / M arrest of cells ( p<0.001), indicating that the cell cycle arrest effect caused by different concentrations of the compound shown in formula 1 is different, and low concentration can cause G0 / G1 phase arrest, and G2 / M phase arrest gradually increases with the increase of the treatment concentration.

[0076] Cell clone formation as shown in Figure 11 , the number of cell clone plaques gradually decreases with the increase of drug concentration, and the clone plaques of the 500 μM group almost completely disappear. The clone formation rate as shown in Figure 12 , the clone formation rates of the 100 μM, 300 μM and 500 μM groups are reduced to about 35%, 30% and 10% respectively, indicating that the drug has a significant inhibitory effect on cell survival and clone formation. Therefore, the drug can significantly inhibit the survival and clone formation of NCI-N87 cells in a dose-dependent manner.

[0077] As shown in Figure 13 , 14 , compared with the control group, the growth of transplanted tumors in the 0.25 mg / kg, 2.5 mg / kg and 5 mg / kg groups was inhibited, indicating that the compound shown in formula 1 can inhibit the growth of transplanted tumors in a dose-dependent manner in tumor-bearing mice, and the compound shown in formula 1 has a certain therapeutic potential for HER2-positive gastric cancer.

[0078] As shown in Figure 15 , the survival time of tumor-bearing mice in the 0.25 mg / kg, 2.5 mg / kg and 5 mg / kg groups reached 39 days, 42 days and 57 days respectively, while the survival rate of tumor-bearing mice in the control group was only 27 days, indicating that the compound shown in formula 1 can improve the survival rate of tumor-bearing mice.

[0079] As shown in Figure 16 , compared with the initial body weight, the body weight of tumor-bearing mice in the 0.25 mg / kg, 2.5 mg / kg and 5 mg / kg groups did not decrease, indicating that the compound shown in formula 1 has little toxicity to tumor-bearing mice.

[0080] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. The compound shown in Formula 1, 。 2. A method for preparing the compound shown in Formula 1 of claim 1, comprising the following steps: 1) reacting compound 1 and compound 2 under alkaline conditions in an organic solvent to obtain compound 3; 2) Compound 3 undergoes a reduction reaction in the presence of a reducing agent to give compound 4; 3) Compound 4 reacts with bromoacetonitrile (BrCH2CN) to give compound 6; 4) Compound 6 and compound 7 react in the presence of a palladium catalyst and a cesium salt to give the target compound. 。 3. The use of the compound of Formula 1 in claim 1 in the preparation of HER2-positive tumor diagnostic reagents.

4. The application according to claim 3, characterized in that, The application refers to the use of the compound shown in Formula 1 in the preparation of a drug for the prevention and / or treatment of HER2-positive tumors.

5. The application according to claim 4, characterized in that, The tumor is cancer, specifically breast cancer, lung cancer, stomach cancer, or colorectal cancer.

6. The application according to claim 4, characterized in that, In the aforementioned application, the drug has at least one of the following effects: 1) Inhibits the proliferation of tumor cells; 2) Inhibits tumor cell survival and clone formation; 3) Blocking of the tumor cell cycle.

7. A medicament for the prevention and / or treatment of HER2-positive tumors, said medicament comprising the compound shown in Formula 1 of claim 1.

8. The application according to claim 7, characterized in that, The tumor is cancer, specifically breast cancer, lung cancer, stomach cancer, or colorectal cancer.

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