A 5-bromoindole-2-carboxylic acid methyl ester derivative and a method for preparing the same

Abstract

The application discloses a 5-bromoindole-2-carboxylic acid methyl ester derivative and a preparation method and application thereof in protein inhibitors, and relates to the technical field of inhibitor synthesis and preparation. The 5-bromoindole-2-carboxylic acid methyl ester derivative is prepared by the application, and compared with a comparative product, the synthetic route steps of the 5-bromoindole-2-carboxylic acid methyl ester derivative prepared by the application present a more simple trend, are easier to be modified and prepared, widen the substrate application range, and can satisfy the differentiated demand for novel molecular structure in the field of kinase inhibitors. The epidermal growth factor receptor (EGFR) inhibitor prepared from the 5-bromoindole-2-carboxylic acid methyl ester derivative of the application has higher inhibition efficiency in biological activity, more significant improvement in physiological solubility, can present more excellent and stable target binding results, and is helpful to improving the application effect of the compound in scenes such as anti-tumor drug development.

Description

Technical Field

[0001] This invention relates to the field of inhibitor synthesis and preparation technology, specifically to a methyl 5-bromoindole-2-carboxylic acid derivative and its preparation method. Background Technology

[0002] Epidermal growth factor receptor (EGFR), a transmembrane protein with tyrosine kinase activity, is closely related to the development and progression of various malignant tumors due to abnormal activation of its signaling pathway, making it a key drug target in the treatment of tumors such as non-small cell lung cancer. In the development of small molecule inhibitors targeting this target, indole compounds, with their unique heterocyclic structure and good biocompatibility, are often used as the core framework for constructing drug molecules. In particular, indole-2-carboxylic acid ester derivatives, with their hydrogen-bonded acceptor and donor sites, can interact with the ATP-binding pocket of kinases, showing potential research value in regulating cell proliferation and inducing apoptosis.

[0003] Among existing medicinal chemistry modification strategies, site-specific functionalization of the indole skeleton is a common method to optimize its pharmacological activity. For the specific structural unit methyl 5-bromoindole-2-carboxylate, exploring modifications at different sites is significant for enhancing EGFR inhibitory activity. For example, introducing hydrophobic groups with large spatial volumes or extending its conjugated system at specific sites on the indole ring can theoretically improve the fit between the molecule and the hydrophobic pocket of the target protein, while simultaneously regulating the molecule's electron cloud distribution and lipophilicity, thereby affecting the membrane permeability and metabolic stability of the drug molecule. However, current research on structural modifications of this type of skeleton, such as simultaneously introducing fused-ring aromatic groups on the nitrogen atom and constructing a large conjugated alkynyl system on the benzene ring side chain, is relatively limited.

[0004] Existing technologies for synthesizing highly functionalized indole derivatives often face challenges such as complex synthetic routes, demanding reaction conditions, or limited substrate compatibility. Furthermore, some reported compounds still have room for improvement in terms of selectivity or bioavailability against the EGFR target. Therefore, developing a more efficient synthetic route capable of constructing methyl 5-bromoindole-2-carboxylic acid derivatives with novel substitution modes and exploring their application potential in the field of EGFR inhibitors is a key technical issue that requires attention in the current research and development of anti-tumor drugs. Summary of the Invention

[0005] This invention aims to provide a methyl 5-bromoindole-2-carboxylic acid derivative with a novel molecular structure, good chemical stability, and excellent EGFR inhibitory activity, as well as its preparation method, and to apply it to the field of EGFR inhibitors to overcome the problems of insufficient inhibitor structural diversity and cumbersome preparation routes in the prior art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a methyl 5-bromoindole-2-carboxylic acid derivative, wherein the methyl 5-bromoindole-2-carboxylic acid derivative is a compound represented by Formula 1: Formula 1; R1 is selected from any one of hydrogen, hydroxyl, alkyl with 1-5 carbon atoms, alkoxy with 1-5 carbon atoms, acyl with 1-5 carbon atoms, and aryl with 6-10 carbon atoms; The derivatives can be prepared into pharmaceutically acceptable salts, solvates, and isotope derivatives.

[0007] Furthermore, the alkyl groups of carbon atoms 1-5 are selected from either methyl or ethyl. The alkoxy groups of carbon atoms 1-5 are selected from either methoxy or isopropoxy. The acyl groups of carbon atoms 1-5 are selected from formyl groups; The aryl groups at carbon atoms 6-10 are selected from phenyl groups.

[0008] Furthermore, the 5-bromoindole-2-carboxylic acid methyl ester derivative is any one or more of the compounds shown in the following structures: ; ; ; A method for preparing a methyl 5-bromoindole-2-carboxylic acid derivative includes the following steps: ; The first step involves synthesizing intermediate 1 from raw material 1 and raw material 2 via an N-acylation reaction. The second step involves synthesizing the 5-bromoindole-2-carboxylic acid methyl ester derivative shown in Formula 1 via a Nogashira coupling reaction between intermediate 1 and raw material 3.

[0009] Furthermore, R1 in raw material 1 is selected from any one of hydrogen, hydroxyl, methyl, ethyl, methoxy, isopropoxy, phenyl, and formyl.

[0010] Furthermore, the raw material 1 is selected from any one or more of the compounds shown in the following structures: , , , , , , , .

[0011] Application of a methyl 5-bromoindole-2-carboxylic acid derivative in protein inhibitors.

[0012] Furthermore, the protein inhibitor is an epidermal growth factor receptor (EGFR) inhibitor.

[0013] Application of a methyl 5-bromoindole-2-carboxylic acid derivative in the preparation of antitumor drugs.

[0014] Furthermore, the tumor is one or more of the following: non-small cell lung cancer, breast cancer, liver cancer, kidney cancer, gastrointestinal stromal tumor, and thyroid cancer.

[0015] Furthermore, the tumor is non-small cell lung cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The preparation route is simpler: Compared with the prior art, the synthesis method provided by this invention reduces the complexity of the operation steps by optimizing the reaction path and coupling conditions, and the reaction process is more controllable. While ensuring the high purity of the product, it effectively improves the overall yield and is more conducive to the large-scale preparation of this type of derivative.

[0017] 2. More Stable Target Binding: Thanks to the triphenylamine and specific large conjugated alkyne system introduced into the molecular structure, the derivatives of this invention exhibit a more effective tendency to interact with the ATP-binding pocket of the kinase compared to traditional modified indole compounds. This structural design enhances the hydrophobic interactions and electronic effects between molecules, overcoming the shortcomings of some existing drug molecules that have unstable binding or poor selectivity to target proteins, thus providing a superior structural basis for the efficacy of drug molecules.

[0018] 3. More balanced drug performance: In the activity test against epidermal growth factor receptor, the derivative of this invention showed a strong inhibitory trend at the nanomolar level, with its enzyme activity and cell inhibition rate data significantly superior to some existing positive control drugs. Furthermore, protein expression level tests showed that it has a stronger blocking ability on phosphorylation signaling pathways, indicating that this type of derivative has superior pharmacological activity in applications such as anti-tumor drug development. Attached Figure Description

[0019] Figure 1 This is a molecular docking diagram of compound 1 described in this invention with the epidermal growth factor receptor EGFR.

[0020] Figure 2 This is a molecular docking diagram of compound 5 described in this invention with the epidermal growth factor receptor EGFR.

[0021] Figure 3The images show Western blotting (WB) images of the EGFR proteins of the embodiments and comparative compounds described in this invention. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Synthesis of Compound 1: ; In the first step, under a nitrogen atmosphere, 4.82 g of diisopropylethylamine, 0.23 g of 4-dimethylaminopyridine, and 5.00 g of raw material 1 were added to 60 mL of 1,4-dioxane. 3.55 g of raw material 2 was dissolved in 20 mL of 1,4-dioxane. The solution was slowly added dropwise to the above solution while maintaining the temperature below -10°C. After the addition was complete, the mixture was stirred at 40°C for 12 h. After the reaction solution cooled to room temperature, 1,4-dioxane was removed by rotary evaporation under reduced pressure. The residue was dissolved in 100 mL of ethyl acetate, and quenched slowly with 50 mL of ice water. The organic phase was collected by separation and washed successively with 1 M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. After drying with anhydrous sodium sulfate, the mixture was filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography using a petroleum ether / ethyl acetate system as the eluent. The combined product fractions were monitored by TLC, evaporated to dryness, and then dried under vacuum to obtain 5.28 g of intermediate 1. In the second step, under nitrogen protection, 5.28 g of intermediate 1, 3.37 g of raw material 3, and 60 mL of a mixed solvent of dioxane and diisopropylamine (40 mL / 20 mL) were added sequentially to the dry reaction system. After stirring until homogeneous, 0.08 g of palladium acetate, 0.2 g of 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and 0.07 g of CuI were added sequentially. The mixture was stirred until homogeneous, heated to 85 °C, and refluxed for 6 h. After the reaction solution cooled to room temperature, it was first filtered through a diatomaceous earth-lined sand core funnel, and the filter cake was thoroughly washed with dichloromethane. The filtrates were combined and evaporated under reduced pressure. The residue was redissolved in dichloromethane and washed sequentially with saturated ammonium chloride solution, water, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane. The purified fractions were combined by TLC monitoring, evaporated to dryness and then dried under vacuum to give 5.35 g of compound 1.

[0024] Product structure identification: Mass spectrometry (MS) of intermediate 1 [M+H] +:422; Mass spectrometry (MS) of compound 1 [M+H] + :611; NMR of intermediate 1: ; NMR of compound 1: .

[0025] Examples 2 to 4: Compounds 2, 4, and 5 were synthesized sequentially in Examples 2 to 4, following the preparation method of Example 1, except that raw material 1 was replaced, and the rest remained the same as in Example 1. Specific structures of raw material 1, compounds 2, 4, and 5, and their mass spectrometry (MS [M+H]) are detailed below. + The data is shown in Table 1.

[0026] Table 1 ; Comparative compound 1: .

[0027] Comparative compound 2: Gefitinib.

[0028] Comparative compound 3: Icotinib.

[0029] 1. Assay for epidermal growth factor receptor (EGFR) inhibitory activity (ICF) 50 OD values ​​were measured using an enzyme-linked immunosorbent assay (ELISA). Poly(Glu,Tyr) substrate was coated onto 96-well microplates, which were then blocked before the reaction. Purified EGFR kinase and different concentrations of the analyte inhibitor were added to the wells. The concentration gradient of the analyte compounds was set at 10 μM, 3 μM, 1 μM, 300 nM, 100 nM, 30 nM, 10 nM, and 3 nM (DMSO was used as a solvent control), with three replicates (n=3) for each concentration. ATP was added to initiate the kinase reaction after incubation, and the plate was washed three times with PBST buffer after the reaction. HRP-labeled anti-phosphotyrosine antibody was then added for incubation, followed by washing and the addition of TMB chromogenic solution. Finally, sulfuric acid was added to terminate the reaction. OD values ​​were measured using a microplate reader. 450nm The absorbance value was used to calculate the half-maximal inhibitory concentration (IC50) of the compound on EGFR enzyme activity using a fitted curve. 50 The data (nM) are shown in Table 2.

[0030] 2. In vitro tumor cell inhibition rate test (IC) 50Cytotoxicity was determined using the CCK-8 assay. A549 cells (human non-small cell lung cancer cells) in logarithmic growth phase were seeded in 96-well plates. After cell attachment, the concentration gradient of the test compound was set at 50 μM, 15 μM, 5 μM, 0.5 μM, 0.15 μM, and 0.05 μM, with three parallel wells for each concentration. After co-culturing for 48 hours, the CCK-8 reagent was added, and OD values ​​were measured after incubation. 450nm Absorbance was used to calculate cell viability and determine IC50. 50 Values ​​(μM), data are shown in Table 2. 3. Protein expression level test: Western blotting was used. A549 cells treated with 1 μM of the test compound for 24 hours were collected, total cell protein was extracted and quantified by BCA. Equal amounts of protein samples were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibody against phosphorylated EGFR (p-EGFR, Tyr1068) and internal control GAPDH antibody, followed by incubation with secondary antibody. Chemiluminescence imaging was used to develop the images, analyze the band gray values, and calculate the relative inhibition rate of the compound on EGFR phosphorylation level. Data are shown in Table 2. Table 2 ; The compounds in Examples 1 to 4 of this invention all exhibited potent inhibitory activity at the nanomolar (nM) level against EGFR kinase and A549 non-small cell lung cancer cells, with IC50 values ​​of [missing value]. 50 The values ​​were lower than those of the unmodified core and the positive control drug. This indicates that the triphenylamine-alkynyl large conjugated side chain introduced into the molecule can effectively penetrate deep into the kinase active pocket, significantly enhancing the binding affinity between the molecule and the target protein by increasing hydrophobic interactions and π-π stacking effects, demonstrating the significant advantage of this specific structural modification strategy in enhancing antitumor activity. Western blot experiments confirmed that the compound of this invention induces tumor cell death by specifically blocking the EGFR (Tyr1068) phosphorylation signaling pathway, and the phosphorylation inhibition rate at specific concentrations showed a good positive correlation with cytotoxicity data, exhibiting a clear targeted therapeutic mechanism.

[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A methyl ester derivative of 5-bromoindole-2-carboxylic acid, characterized in that, The derivative is a compound represented by Formula 1: Formula 1; R1 is selected from any one of hydrogen, hydroxyl, alkyl with 1-5 carbon atoms, alkoxy with 1-5 carbon atoms, acyl with 1-5 carbon atoms, and aryl with 6-10 carbon atoms.

2. The methyl 5-bromoindole-2-carboxylic acid ester derivative according to claim 1, characterized in that, The alkyl groups of carbon atoms 1-5 are selected from methyl and ethyl; The alkoxy groups of carbon atoms 1-5 are selected from either methoxy or isopropoxy. The acyl groups of carbon atoms 1-5 are selected from formyl groups; The aryl groups at carbon atoms 6-10 are selected from phenyl groups.

3. The methyl 5-bromoindole-2-carboxylic acid derivative according to claim 1, characterized in that, The 5-bromoindole-2-carboxylic acid methyl ester derivative is any one or more of the compounds shown in the following structures: ； ； ； 。 4. A method for preparing a methyl 5-bromoindole-2-carboxylic acid derivative according to any one of claims 1 to 3, characterized in that, Includes the following steps: ； The first step involves synthesizing intermediate 1 from raw material 1 and raw material 2 via an N-acylation reaction. The second step involves synthesizing the 5-bromoindole-2-carboxylic acid methyl ester derivative shown in Formula 1 via a Nogashira coupling reaction between intermediate 1 and raw material 3.

5. The method for preparing a methyl 5-bromoindole-2-carboxylic acid derivative according to claim 4, characterized in that, R1 in raw material 1 is selected from any one of hydrogen, hydroxyl, methyl, ethyl, methoxy, isopropoxy, phenyl, and formyl.

6. The use of a methyl 5-bromoindole-2-carboxylic acid ester derivative according to any one of claims 1 to 3 in the preparation of a protein inhibitor, characterized in that, The protein inhibitor is an epidermal growth factor receptor (EGFR) inhibitor.

7. The use of a methyl 5-bromoindole-2-carboxylic acid ester derivative as described in any one of claims 1 to 3 in the preparation of an antitumor drug.

8. The use of a methyl 5-bromoindole-2-carboxylic acid ester derivative according to claim 7 in the preparation of antitumor drugs, characterized in that, The tumor is one or more of the following: non-small cell lung cancer, breast cancer, liver cancer, kidney cancer, gastrointestinal stromal tumor, and thyroid cancer.

9. The use of a methyl 5-bromoindole-2-carboxylic acid ester derivative according to claim 8 in the preparation of antitumor drugs, characterized in that, The tumor is non-small cell lung cancer.

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