2-(substituted) phenyl-2H-indazole7-carboxamide compound as well as preparation method and application thereof
By synthesizing 2-(substituted)phenyl-2H-indazole 7-carboxamide compounds, the problems of drug resistance and toxic side effects of existing anti-breast cancer compounds have been solved, achieving a highly efficient inhibitory effect on malignant tumor cells and good drug-like properties, making it suitable for large-scale production.
Patent Information
- Application Number
- CN202511659983.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-02-13
AI Technical Summary
Existing anti-breast cancer compounds suffer from problems such as easy development of drug resistance, significant toxic side effects, limited response rate, and recurrence and metastasis. Their synthesis methods are cumbersome, yields are low, and their pharmacokinetic properties are poor, which limits their potential for clinical application.
2-(substituted)phenyl-2H-indazole 7-carboxamide compounds were developed. Novel compounds were synthesized through ammonolysis, nucleophilic substitution and condensation reactions. The solvent and catalyst conditions were optimized to prepare compounds with excellent inhibitory effects on the proliferation of malignant tumor cells.
It achieves a highly effective inhibitory effect on malignant tumor cells, especially showing significant anti-proliferative properties in human cervical cancer cells HeLa. It has good drug-like properties and stability, making it suitable for large-scale production.
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Figure CN121517359A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a kind of 2- (substituted) phenyl-2H-indazole 7-carboxamide compound and its preparation method and use. BACKGROUND
[0002] Breast cancer is one of the most common and lethal malignant tumors in women worldwide, with a complex pathogenesis. The main treatment methods include surgical resection, radiotherapy, chemotherapy, endocrine therapy and targeted therapy, etc. Although existing chemotherapeutic drugs (such as paclitaxel, doxorubicin, etc.) and targeted drugs (such as trastuzumab) have achieved certain efficacy in clinical application, they still face many challenges, including severe side effects, frequent drug resistance, limited patient response rate and easy recurrence and metastasis.
[0003] In recent years, drug development based on small molecule inhibitors has become an important direction in the field of anti-breast cancer, especially the design and optimization of compounds targeting key signaling pathways (such as PI3K / AKT / mTOR, CDK4 / 6, etc.). In addition, new strategies such as antibody drug conjugates (ADC) are gradually applied to breast cancer treatment, aiming to improve drug selectivity and reduce systemic toxicity. However, the existing synthetic methods still have defects such as complicated steps, low yield, insufficient stability or poor pharmacokinetic properties, which limit their clinical application potential.
[0004] Therefore, it is urgent to develop anti-breast cancer compounds with novel structure, significant activity, lower toxicity and good drugability, as well as efficient, environmentally friendly and suitable for large-scale production synthesis process, to provide new solutions for further improving the safety and effectiveness of breast cancer treatment. SUMMARY
[0005] In order to solve the technical problem that existing anti-tumor compounds are prone to drug resistance in application, the present application provides a kind of 2- (substituted) phenyl-2H-indazole 7-carboxamide compound and its preparation method and use.
[0006] In order to solve the above technical problems, first of all, the present application adopts the following technical solutions: The present application provides a kind of 2- (substituted) phenyl-2H-indazole 7-carboxamide compound, characterized in that the compound and its pharmaceutically acceptable salt have the structure shown in formula I and formula II:
[0007] Secondly, the present application also provides the use of a pharmaceutical composition containing the above-mentioned compound for treating or inhibiting the proliferation of malignant tumor cells.
[0008] Thirdly, the present application provides a preparation method of the above-mentioned compound, comprising the following steps: S1: Ammonolysis reaction of compound 1 methyl 2H-indazole-7-carboxylate with ammonium hydroxide solution to generate compound 2 2H-indazole-7-carboxamide S2: Nucleophilic substitution reaction of compound 2 2H-indazole-7-carboxamide with compound 3 4-fluorobenzaldehyde under basic condition to generate compound 5 2-(4-aminophenyl)-2H-indole-7-carboxamide S3: Condensation reaction of compound 5 2-(4-aminophenyl)-2H-indole-7-carboxamide with 4-4-methylphenyl-3-aminothiourea, 4-(4-fluorophenyl)-3-aminothiourea under acidic condition to generate compound I and II The chemical reaction formula of the 2-(substituted)phenyl-2H-indazole-7-carboxamide compound provided by the present application is as follows:
[0009] The solvent for the ammonolysis reaction in step S1 is a polar protic solvent, selected from methanol, ethanol, isopropanol, etc., with methanol being preferred; the reaction temperature is 0-25°C, with 25°C being preferred.
[0010] The reaction solvent for the nucleophilic substitution reaction in step S2 is selected from aprotic solvents such as DMF, DMAC, DMSO, etc., with DMF and DMAC being preferred; the base is selected from cesium carbonate, sodium carbonate, potassium carbonate, sodium tert-butoxide, potassium tert-butoxide, sodium amide, potassium amide, potassium hydroxide, sodium hydride, etc., with potassium carbonate being preferred; the reaction temperature for the nucleophilic substitution is 100-150°C, with 145°C being preferred.
[0011] The reaction solvent for the condensation reaction in step S3 is a polar protic solvent, selected from methanol, ethanol, isopropanol, etc., with methanol being preferred; the acid catalyst used is selected from formic acid, acetic acid, hydrochloric acid, etc., with acetic acid being preferred. The condensation reaction temperature is 50-80°C, with 80°C being preferred.
[0012] The present application provides a 2-(substituted)phenyl-2H-indazole-7-carboxamide compound, a preparation method and use thereof. The 2-(substituted)phenyl-2H-indazole-7-carboxamide compound or its medically acceptable pharmaceutical salt, i.e., the compound of formula I and formula II, has a definite and excellent performance effect in the application of anti-malignant cell proliferation.
[0013] The benzopyrazole derivatives with phenyl ring connected to the structure provided by the present application are a new series of anti-tumor proliferation compounds. In the structural features, the benzopyrazole ring is the basic core structure, and after introducing specific substituent groups into the above basic core structure, it has excellent therapeutic effect on treating and / or preventing various malignant tumor proliferations of animals (including humans). The compounds of formula I and formula II have outstanding inhibitory effect in the anti-proliferation experiment of human cervical cancer cells (Hela cells), and the compound can effectively resist the proliferation of Hela cells. The conclusion proves that the compounds of formula I and formula II have the possibility of being developed as new cancer treatment drugs. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 NMR H spectrum of formula I Figure 2 NMR C spectrum of formula I Figure 3 Mass spectrum of formula I Figure 4 NMR H spectrum of formula II Figure 5 NMR C spectrum of formula II Figure 6 Mass spectrum of formula II DETAILED DESCRIPTION
[0015] The present application discloses 2-(substituted)phenyl-2H-indazole 7-carboxamide compounds, preparation methods and uses thereof. Those skilled in the art can refer to the content herein, and appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.
[0016] In order for those skilled in the art to better understand the present application, the present application will be further described in detail below in combination with specific embodiments.
[0017] The raw material compounds described in the present application can be purchased through commercial channels, and the compounds shown in formula I and formula II can be synthesized according to the following synthetic route by using conventional compound synthesis methods. Example 1
[0018] The reaction bottle was added with compound 1 (10 mmol, 1.0 eq) dissolved in methanol, and NH3.H2O solution (50 mmol, 5.0 eq) was added. The mixture was stirred at room temperature (about 25°C) for 96 hours. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the solvent was removed from the reaction liquid by reduced pressure distillation. The obtained product was separated by column chromatography to obtain compound 2.
[0019] The reaction bottle was added with compound 2 (3.51 mmol, 1.0 eq), compound 3 (5.26 mmol, 1.5 eq), potassium carbonate (10.53 mmol, 3 eq) and DMF. The mixture was reacted at 145°C for about 5 hours. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the reaction liquid was extracted with ethyl acetate and saturated brine for three times. The organic phase was back-extracted, washed, dried, concentrated and separated by column chromatography to obtain intermediate 5 with a yield of about 80%.
[0020] To the mixture was added compound 5 (1.21 mmol, 1 eq), 4-4-methylphenyl-3- aminothiourea (1.57 mmol, 1.3 eq), 4-(4-fluorophenyl)-3-aminothiourea (1.57 mmol, 1.3 eq), and acetic acid was adjusted to pH 6. The mixture was reacted at 80°C for 5 hours. After the reaction was completed, the filtrate was rotary evaporated to obtain a crude product. The crude product was separated and purified by column chromatography (V (EA): V / (PE)=1:3) to obtain target products I and II with a yield of 81%. Example 2
[0021] The reaction bottle was added with compound 1 (10 mmol, 1.0 eq) dissolved in methanol, and triethylamine solution (50 mmol, 5.0 eq) was added. The mixture was stirred at room temperature (about 25°C) for 96 hours. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the solvent was removed from the reaction liquid by reduced pressure distillation. The obtained product was separated by column chromatography to obtain compound 2.
[0022] The reaction bottle was added with compound 2 (3.51 mmol, 1.0 eq), compound 3 (5.26 mmol, 1.5 eq), sodium carbonate (10.53 mmol, 3 eq) and DMF. The mixture was reacted at 130°C for about 6 hours. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the reaction liquid was extracted with ethyl acetate and saturated brine for three times. The organic phase was back-extracted, washed, dried, concentrated and separated by column chromatography to obtain intermediate 5 with a yield of about 70%.
[0023] To the mixture was added compound 5 (1.21 mmol, 1 eq), 4-4-methylphenyl-3- aminothiourea (1.57 mmol, 1.3 eq), 4-(4-fluorophenyl)-3-aminothiourea (1.57 mmol, 1.3 eq), formic acid was adjusted to pH 6, and the mixture was reacted at 65 °C for 6.5 h. After the reaction was completed, the filtrate was rotary evaporated to obtain a crude product. The crude product was separated and purified by column chromatography (V (EA): V / (PE) = 1:3) to obtain the target products I and II in a yield of 74%. Example 3
[0024] Compound 1 (10 mmol, 1.0 eq) was dissolved in ethanol, and triethylamine solution (50 mmol, 5.0 eq) was added. The mixture was stirred at about 50 °C for 24 h. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the solvent was removed from the reaction solution by reduced pressure distillation. The obtained product was separated by column chromatography to obtain compound 2.
[0025] Compound 2 (3.51 mmol, 1.0 eq), compound 3 (5.26 mmol, 1.5 eq), sodium tert-butoxide (10.53 mmol, 3 eq), and DMF were added to a reaction bottle, and the mixture was reacted at 100 °C for about 7 h. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the reaction solution was extracted with ethyl acetate and saturated brine three times, and the organic phase was back-extracted. The organic phase was washed, dried, concentrated, and separated by column chromatography to obtain intermediate 5 in a yield of about 68%.
[0026] To the mixture was added compound 5 (1.21 mmol, 1 eq), 4-4-methylphenyl-3- aminothiourea (1.57 mmol, 1.3 eq), 4-(4-fluorophenyl)-3-aminothiourea (1.57 mmol, 1.3 eq), formic acid was adjusted to pH 5, and the mixture was reacted at 45 °C for 8 h. After the reaction was completed, the filtrate was rotary evaporated to obtain a crude product. The crude product was separated and purified by column chromatography (V (EA): V / (PE) = 1:3) to obtain the target products I and II in a yield of 60%. Example 4
[0027] Compound 1 (10 mmol, 1.0 eq) was dissolved in ethanol, and NH3.H2O solution (50 mmol, 5.0 eq) was added. The mixture was stirred at about 25 °C for 120 h. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the solvent was removed from the reaction solution by reduced pressure distillation. The obtained product was separated by column chromatography to obtain compound 2.
[0028] The reaction bottle is added with compound 2 (3.51 mmol, 1.0 eq), compound 3 (5.26 mmol, 1.5 eq), potassium hydroxide (10.53 mmol, 3 eq) and DMF, and after being reacted at 120 DEG C for about 8 hours, the reaction progress is detected by thin layer chromatography, and after the reaction is completed, the reaction liquid is extracted with dichloromethane and saturated brine for three times, the organic phase is back-extracted, and after the organic phase is washed, dried and concentrated, the intermediate 5 is separated by column chromatography, and the yield is about 68%.
[0029] Compound 5 (1.21 mmol, 1 eq), 4-4-methylphenyl-3-aminothiourea (1.57 mmol, 1.3 eq), 4-(4-fluorobenzene)-3-aminothiourea (1.57 mmol, 1.3 eq) are added to the mixed solution, diluted with dilute hydrochloric acid to pH 6, and reacted at 60 DEG C for 8 h. After the reaction is completed, the filtrate is dried to obtain a crude product, and the crude product is separated and purified by column chromatography (V (EA): V / (PE)= 1:3) to obtain the target products I and II with a yield of 67%. The target compounds of structural formula I and formula II prepared in examples 1, 2, 3 and 4 are analyzed by nuclear magnetic resonance hydrogen spectrum and mass spectrum data, and the data analysis is shown in Table 1.
[0030] Table 1 Nuclear magnetic resonance hydrogen spectrum and mass spectrum data of compounds shown in formula I and formula II
[0031] Example 5 The compounds of formula I and formula II prepared in examples 1, 2, 3 and 4 and other control compounds are tested for cytotoxicity and toxicity quantification: 5.1 Preparation of positive control and compound solution The positive control niraparib 3.0 mg is weighed, dissolved with DMSO to prepare a 20 mg / mL stock solution, and stored at -20 DEG C for standby, and the target compound solution is also prepared in the same way, and before use, the test compound stock solution is diluted to the required concentration with 90% DMEM high glucose medium.
[0032] 5.2 MTT method for inhibiting cancer cell proliferation experiment Preparation of phosphate buffer solution (PBS): commercially available PBS powder is dissolved with ultrapure water, the pH value is adjusted to about 7.4, after high temperature sterilization, 0.22 μm microporous filter membrane is used for sterilization, and the obtained solution is stored at 4 DEG C for standby.
[0033] Preparation of MTT solution: 0.25 g of MTT powder was accurately weighed and dissolved in 50 mL of previously prepared and sterilized PBS solution, and magnetically stirred for 30 minutes under dark conditions until completely dissolved. The resulting solution had a concentration of 5 mg / mL. After filtration through a 0.22 μm microporous filter, it was aliquoted and stored at -20°C under dark conditions.
[0034] Logarithmic phase human cervical cancer cells Hela were trypsinized, resuspended, and adjusted to a cell concentration of 5 x 10 4 / mL, and inoculated into a 96-well plate at 100 μL / well. After 24 h of adherent growth, the culture medium was aspirated, and different concentrations of compounds (5, 10, and 20 μg / mL) were added. Three replicate wells were set up for each concentration, and PBS solvent controls of the corresponding concentrations, cell zero groups, and positive control groups were set up. The cells were incubated at 37°C, 5% CO2for 48 h, then 20 μL of 5 mg / mL MTT was added, and incubated at 37°C, 5% CO2for 4 h. The supernatant was aspirated, 150 μL of DMSO was added to each well, and shaken for 10 min. The OD value was detected at 490 nm on a microplate reader. The cell inhibition rate and half-maximal inhibitory concentration (IC 50 ) were calculated.
[0035] The data are shown in Table 2, which lists the evaluation results of the effects of compounds of formulae I and II and other control compounds on the proliferation activity of Hela cells.
[0036] Table 2 Evaluation results of the effects of compounds on the proliferation activity of human cervical cancer cells (Hela)
[0037] As can be seen from the data in Table 2, in the MTT method for inhibiting cancer cell proliferation, formulae I and II exhibited outstanding anti-proliferation properties of cervical cancer cells at a dose of 20 μg / mL. Compared with the positive control, formulae I and II could significantly inhibit the proliferation of cervical cancer cells Hela, and the inhibitory effect was clear and excellent. The above results show that for the parent nucleus structure of 2- (substituted) phenyl-2H-indazole 7-carboxamide compounds, when different groups are introduced onto the tail end aminothiourea N atom, only specific groups exhibit good anti-proliferation properties of cancer cells, and the structural type of the group has no obvious regularity on the anti-proliferation activity of the entire molecule, so it can be known that the type of the group has unpredictable effects on the anti-proliferation activity of the entire molecule.
[0038] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. A 2-(substituted)phenyl-2H-indazole 7-carboxamide compound, characterized in that, The compounds and their pharmaceutically usable salts have the structures shown in Formula I and Formula II:
2. A pharmaceutical composition, characterized in that, The active ingredient is the compound of claim 1, and it also contains one or more pharmaceutical carriers or one or more known pharmaceutical diluents.
3. The use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in an antitumor drug.
4. A method for preparing the compound according to claim 1, characterized in that, Includes the following steps: Step a): Compound 1 undergoes an ammonolysis reaction in an ammonia solution to generate compound 2. Step b): Compound 3 and compound 4 undergo a nucleophilic substitution reaction under basic conditions to generate compound 5. Step c): Compound 5 undergoes an amidation reaction with 4-4-tolyl-3-aminothiourea and 4-(4-fluorobenzene)-3-aminothiourea under acidic conditions to produce compounds shown in formulas I and II; 5. In the preparation method as described in claim 4, the ammonolysis reaction solvent in step a) is selected from methanol, ethanol, isopropanol, and trifluoroethanol; the base is selected from ammonia, N,N-diisopropylethylamine (DIPEA), triethylamine, or pyridine.
6. In the preparation method as described in claim 4, the temperature in step a) is selected from 0-25°C.
7. In the preparation method as described in claim 4, the reaction solvent for the nucleophilic substitution reaction in step b) is selected from aprotic solvents such as DMF, DMAC, and DMSO, and the base is selected from one of cesium carbonate, sodium carbonate, potassium carbonate, sodium tert-butoxide, potassium tert-butoxide, sodium amino, potassium amino, potassium hydroxide, and sodium hydride.
8. In the preparation method shown in claim 4, the temperature in step b) is selected from 100-150°C.
9. The acidic catalyst used in the condensation reaction described in step c) is selected from formic acid, acetic acid, and hydrochloric acid. The reaction solvent is a polar protic solvent, selected from methanol, ethanol, isopropanol, etc., and the reaction temperature is 50-80℃.