CN106008306A - Substituted indole derivatives, and preparation method and application thereof - Google Patents

Abstract

The invention discloses substituted indole derivatives, and a preparation method and application thereof. The substituted indole compounds or pharmaceutically acceptable salts, esters or prodrugs have the structure disclosed as General Formula I. The invention also discloses a preparation method of the substituted indole compounds and application of the composition containing one or more of the compounds in preparing drugs for treating and preventing human hepatitis C.

Description

Substituted indole derivatives and their preparation methods and applications

technical field

The invention belongs to the technical field of medicine, and specifically relates to a class of substituted indole derivatives. The invention also relates to a preparation method of the derivatives and their use in preparing anti-hepatitis C drugs.

Background technique

Hepatitis C virus (HCV) RNA polymerase (Non-Structure 5B, NS5B) plays an irreplaceable role in the life cycle of HCV, a new type of high efficiency, low toxicity, anti-drug resistance and good pharmacokinetics Natural NS5B inhibitors are an important direction for the development of anti-HCV drugs. Several compounds among HCV NS5B Thumb Site I inhibitors are in the clinical research stage, but the poor pharmacokinetic properties of these inhibitors limit their clinical application. Therefore, the research and development of anti-hepatitis C drugs with new structures and new mechanisms is of great significance.

Heterocyclic compounds have a wide range of antiviral activities. They are generally used as the basic structural core of the pharmacophore to meet the space requirements of the special target of the drug, or as active substituents or components of the ring system to produce corresponding biological activity. The reason why drugs rely on heterocycles is that heterocycles are less likely to be metabolized and decomposed in the body than aliphatic or aromatic compounds, and have better biocompatibility. Indole is an important class of aromatic heterocycles, and its derivatives have a wide range of biological activities and clinical applications. Based on the good anti-HCV activity of indole analogues, the present invention designs and synthesizes a series of substituted indole derivatives. Such compounds and their applications are not seen in the prior art.

Contents of the invention

Aiming at the deficiencies in the prior art, a substituted indole derivative and its preparation method are provided. The present invention also provides the screening results of the anti-hepatitis C virus activity of the substituted indole derivative and its application in the preparation of anti-hepatitis C drugs. Applications.

Technical scheme of the present invention is as follows:

1. Substituted indole derivatives

A substituted indole derivative, or a pharmaceutically acceptable salt, ester or prodrug thereof, has a structure represented by general formula I:

in,

R ₁ and R ₂ are independently selected from substituted benzene rings, substituted naphthalene rings, various substituted six-membered heterocyclic rings, various substituted five-membered heterocyclic rings, various substituted six-membered and five-membered heterocyclic rings, various substituted six-membered and five-membered heterocyclic rings, various Substituted six-membered heterocyclic rings, various substituted five-membered and five-membered heterocyclic rings, various substituted benzo five-membered heterocyclic rings or various substituted benzo six-membered heterocyclic rings, various aliphatic rings, various A saturated fatty chain or various unsaturated fatty chains;

R ₃ is various carboxylic acids, carboxylic esters or amides.

Preferably according to the present invention, in general formula I, R ₁ and R ₂ are each independently selected from phenyl, p-methylphenyl, p-methoxyphenyl, 2-furyl, 3-thienyl, m-methyl Phenyl, m-fluorophenyl or p-trifluoromethylphenyl; R ₃ is carboxyl or methylcarboxylate.

According to the present invention, it is further preferred that the substituted indole compound is one of the following specific compounds:

The "pharmaceutically acceptable salt" mentioned in the present invention means that within the scope of reliable medical evaluation, the salt of the compound is suitable for contacting with human or lower animal tissues without undue toxicity, irritation and allergy reactions, etc., have a fairly reasonable ratio of benefit to risk, are usually water or oil soluble or dispersible, and are effective for their intended use. Included are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are acceptable for the intended use and chemically compatible with the compounds of formula I herein. For a list of suitable salts see S.M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

The "prodrugs" mentioned in the present invention refer to pharmaceutically acceptable derivatives, so that the biotransformation products obtained from these derivatives are active drugs as defined by the compounds of formula I.

2. Preparation method of substituted indole derivatives

The preparation method of the substituted indole derivative of the present invention, the step is one of the following routes:

Route 1: Using methyl indole-6-carboxylate 1 as the initial raw material, first undergo an affinity substitution reaction with chloroacetylmorpholine in N,N-dimethylformamide solution to generate intermediate compound 2; then the intermediate compound 2 reacts with N-bromosuccinimide in dichloromethane solution to generate intermediate compound 3; then intermediate compound 3 reacts with various substituted boronic acids in dioxane solution to generate target product a, followed by different Target product a reacts with sodium hydroxide solution in methanol solution to obtain target product b;

Synthetic route one is as follows:

Among them, R ₁ and R ₂ are as shown in the above general formula I;

Reagents and conditions: (i) chloroacetylmorpholine, sodium hydride, N,N-dimethylformamide, room temperature; (ii) dichloromethane, N-bromosuccinimide; (iii) substituted boronic acid, Tetrakis(triphenylphosphine)palladium, potassium phosphate, dioxane, 90°C; (iv) methanol, aqueous sodium hydroxide solution, reflux.

The substituted boronic acid is phenylboronic acid, p-methoxyphenylboronic acid, m-fluorophenylboronic acid, 3-furanboronic acid, p-tolueneboronic acid, m-methylphenylboronic acid, 2,4-dimethylphenylboronic acid, 2- Thiopheneboronic acid, 2,3-dimethylphenylboronic acid, or 2-fluoro-3-methoxyphenylboronic acid.

Route 2: Using intermediate compound 3 as raw material, react with phenylboronic acid in dioxane solution to generate intermediate compound 4, and then react with different substituted boronic acids to generate target product a; then compound a is oxidized with hydrogen in methanol solution Sodium aqueous solution reacts to generate target product b;

Synthetic route two is as follows:

Wherein, R ₁ is shown in the above general formula I;

Reagents and conditions: (i) phenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium phosphate, dioxane, 90°C; (ii) substituted boronic acid, tetrakis(triphenylphosphine)palladium, potassium phosphate, dioxane Six rings, 90 ° C; (iii) methanol, sodium hydroxide aqueous solution, reflux.

The substituted boronic acid is phenylboronic acid, p-methoxyphenylboronic acid or 3-furan boronic acid.

The room temperature described in the present invention is 25°C.

In the more detailed preparation method of the substituted indole derivatives of the present invention, the steps are one of the following routes:

Route 1:

(1) Weigh 5.26g raw material 6-indole carboxylate methyl ester into a 100mL flask, add 20mL N,N-dimethylformamide to dissolve, add 791.9mg sodium hydride, stir at room temperature for 30 minutes, then add 3.90mL Chloroacetylmorpholine, stirred and reacted at room temperature for 12 hours, after the reaction was completed by TLC, spin-dried the solvent, added 20mL water and 20mL dichloromethane, separated the organic phase, washed twice with 20mL water, dried over anhydrous sodium sulfate, and spin-dried the solvent to obtain white Solid, dried under vacuum to obtain intermediate compound 2;

(2) Weigh 4.53g of intermediate compound 2 in a 250mL flask, add 50mL of dichloromethane to dissolve, then add 5.61g of N-bromosuccinimide in batches, stir at room temperature for 12 hours, and detect the reaction by TLC After completion, add 30 mL of water to quench the reaction, wash the organic phase twice with 30 mL of water, dry over anhydrous sodium sulfate, and spin to dry the solvent to obtain a white solid by flash column chromatography, which is dried in vacuum to obtain intermediate 3;

(3) Weigh 92.0 mg of intermediate compound 3 and place it in a 25 mL microwave reactor, add 10 mL of distilled dioxane to dissolve, add 420 μmol of different substituted phenylboronic acids, 11.6 mg of tetrakis(triphenylphosphine) palladium and 89.2 mg Tripotassium phosphate; react in a microwave reactor at 150°C for 30 minutes, after the TLC detection is complete, filter the reaction solution with diatomaceous earth, spin the solvent, dissolve the solid in 10mL of dichloromethane, wash twice with 10mL of water, anhydrous sulfuric acid Sodium drying, spin-drying solvent, Flash column chromatography, ethanol recrystallization of the chromatographic product, to obtain the target product a01;

(4) Weigh 200.00 μmol of compound a01 into a 25 mL flask, add 15 mL of methanol to dissolve, heat up and reflux, slowly add 1M sodium hydroxide solution dropwise to the reaction solution, and monitor the pH value of the solution in real time. When the pH value of the solution is stabilized above 10, stop the drop. Add sodium hydroxide solution, and use a thin-layer plate to detect that the reaction is complete, adjust the pH value to 1 with 1M hydrochloric acid, then spin dry the solvent, add 10mL ethyl acetate to dissolve, wash twice with 10mL water, dry over anhydrous sodium sulfate, spin dry the solvent, The target product b01 was obtained by ethanol recrystallization.

Route two:

(1) Weigh 92.0 mg of intermediate 3 in a 50 mL microwave reactor, add 10 mL of redistilled dioxane to dissolve, add 24.4 mg of phenylboronic acid, 11.6 mg of tetrakis(triphenylphosphine) palladium and 46.7 mg of tripotassium phosphate; React in a microwave reactor at 150°C for 30 minutes to obtain intermediate 4;

(2) Add 220.00 μmol p-methoxyphenylboronic acid, 11.6 mg tetrakis(triphenylphosphine) palladium and 46.7 mg tripotassium phosphate to the reaction solution of the previous step reaction; react in a microwave reactor at 150° C. for 30 minutes, and detect by TLC The reaction is complete, the reaction solution is filtered with diatomaceous earth, the solvent is spin-dried, the solid is dissolved in 20 mL of ethyl acetate, washed twice with 20 mL of water, dried over anhydrous sodium sulfate, the solvent is spin-dried, Flash column chromatography, and the chromatographic product is recrystallized from ethanol. Obtain target product a02;

(3) Weigh 200.00μmol of compound a02 into a 25mL flask, add 15mL of methanol to dissolve, heat up and reflux, slowly add 1M sodium hydroxide solution dropwise to the reaction solution, and monitor the pH value of the solution in real time until the pH value is stable above 10 , stop adding the sodium hydroxide solution dropwise, and use a thin-layer plate to detect that the reaction is complete, adjust the pH value to 1 with 1M hydrochloric acid, and then spin dry the solvent, dissolve in 10mL ethyl acetate, wash twice with 10mL water, dry over anhydrous sodium sulfate, and spin dry Solvent, ethanol recrystallization to obtain the target product b02.

3. Application of substituted indole derivatives

The 23 specific compounds synthesized above were tested for affinity determination with NS5B protein in vitro by using a surface plasmon resonance instrument. As can be seen from Table 1, the substituted indole derivatives of the present invention are a series of novel non-nucleoside HCV inhibitors, and have different affinities with NS5B. Therefore, the present invention also provides:

Use of substituted indole derivatives as non-nucleoside HCV inhibitors. Specifically, it is used as an HCV inhibitor for the preparation of anti-hepatitis C drugs.

An anti-HCV pharmaceutical composition, comprising the substituted indole derivatives of the present invention and one or more pharmaceutically acceptable carriers or excipients.

The invention provides a substituted indole derivative with a brand new structure, a preparation method thereof, an anti-HCV activity screening result and its first application in the antiviral field. Experiments prove that the indole derivatives of the invention can be used as HCV inhibitors and have high application value. Specifically, it is used as an HCV inhibitor for the preparation of anti-hepatitis C drugs.

detailed description

The following examples help to understand the present invention, but the content of the present invention cannot be limited.

The synthetic route involved in the embodiment is as follows:

Synthetic route one:

Synthetic route two:

Embodiment 1: the preparation of intermediate 2

Weigh 6-methyl indolecarboxylate (5.26g, 30.00mmol) in a 100mL flask, add 20mL DMF to dissolve, add sodium hydride (791.9mg, 33.00mmol) and stir at room temperature for 30 minutes, then add chloroacetylmorpholine (3.90 mL, 30.00mmol), stirred and reacted at room temperature for 12 hours, TLC detected that the reaction was complete and spin-dried the solvent, added 20mL water and 20mL DCM for extraction, separated the organic phase, washed with water (2×20mL), dried over anhydrous sodium sulfate, and spin-dried the solvent to obtain The white solid was dried in a vacuum oven to obtain Intermediate 2, a light yellow solid, yield: 92%, melting point: 156-158°C.

Spectrum data of Intermediate 2: ¹ H NMR (400MHz, DMSO-d6, ppm) δ: 8.06(s, 1H, indole-H), 7.64-7.63(m, 2H, indole-H), 7.49(d, J= 3.20Hz, 1H, indole-H), 6.55(dd, J ₁ ＝0.40Hz, J ₂ ＝2.80Hz, 1H, indole-H), 5.22(s, 2H, CH ₂ ), 3.86(s, 3H, COOCH ₃ ),3.71-3.68(m,2H,CH ₂ ),3.61-3.59(m,4H,CH ₂ ,CH ₂ ),3.46-2.44(m,2H,CH ₂ ). ¹³ C NMR(100MHz,DMSO- d6, ppm) δ: 167.76 (COO), 166.61 (CON), 136.55 (indole-C), 134.33 (indole-C), 132.28 (indole-C), 122.54 (indole-C), 120.55 (indole-C) ,120.13(indole-C),112.62(indole-C),101.67(indole-C),66.53(morpholine-CH ₂ ),66.42(morpholine-CH ₂ ),52.25(CH ₃ ),47.55(CH ₂ ), 445.23 (morpholine-CH ₂ ), 42.32 (morpholine-CH ₂ ). ESI-MS: 303.5 [M+H] ⁺ , 320.4 [M+NH ₄ ] ⁺ , 325.4 [M+Na] ⁺ . _{C16H18N2O4 ( 302.13 )} .

Embodiment 2: the preparation of intermediate 3

Weigh Intermediate 2 (4.53g, 15.00mmol) in a 250mL flask, add 50mL of dichloromethane to dissolve, then add N-bromosuccinimide (5.61g, 30.50mmol) in batches, and stir at room temperature for 12 hours After TLC detection, 30mL of water was added to quench the reaction, the organic phase was washed with water (2×30mL), dried over anhydrous sodium sulfate, and the solvent was spin-dried to obtain a white solid by flash column chromatography, which was dried in a vacuum oven to obtain intermediate 3. White solid, yield: 94%, melting point: decomposed at 195°C.

Spectrum data of intermediate 3: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.23(s, 1H, indole-H), 7.80(dd, J ₁ =1.20Hz, J ₂ =12.00Hz, 1H, indole-H), 7.53 (d, J=8.00Hz, 1H, indole-H), 5.46 (s, 2H, CH ₂ ), 3.88 (s, 3H, COOCH ₃ ), 3.74-3.71 (m, 2H, CH ₂ ),3.66-3.64(m,2H,CH ₂ ),3.61-3.58(m,2H,CH ₂ ),3.46-3.43(m,2H,CH ₂ ).ESI-MS:303.5[M+H] ⁺ ,320.4[M+NH ₄ ] ⁺ ,325.4[M+Na] ⁺ .C ₁₆ H ₁₈ N ₂ O ₄ (302.13).ESI-MS: 461.3[M+H] ⁺ ,463.2[M+H] ⁺ , 476.2[M+NH ₄ ] ⁺ ,478.2[M+NH ₄ ] ⁺ ,476.2[M+NH ₄ ] ⁺ ,483.2[M+Na] ⁺ ,485.3[M+Na] ⁺ .C ₁₆ H ₁₆ Br ₂ N ₂ O ₄ (457.95).

Embodiment 3: the preparation of compound a01

Weigh Intermediate 3 (92.0 mg, 200.00 μmol) in a 25 mL microwave reactor, add 10 mL redistilled dioxane to dissolve, add phenylboronic acid (420.00 μmol), tetrakis(triphenylphosphine) palladium (11.6 mg, 10.00 μmol), tripotassium phosphate (89.2mg, 420.00μmol). React in a microwave reactor at 150°C for 30 minutes, TLC detects that the reaction is complete, filter the reaction solution with diatomaceous earth, spin to dry the solvent, dissolve the solid in 10mL of dichloromethane, wash with water (2×10mL), and dry over anhydrous sodium sulfate. The solvent was spin-dried, followed by Flash column chromatography, and the chromatographic product was recrystallized from ethanol to obtain the target product a01. White solid, yield: 64%, melting point: 252-254°C.

Spectrum data of compound a01: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.16(s, 1H, indole-H), 7.78-7.74(m, 2H, indole-H), 7.45(s, 3H, Ph-H),7.34-7.27(m,4H,Ph-H),7.26-7.20(s,3H,Ph-H),5.12(s,2H,CH ₂ ),3.89(s,3H,COOCH ₃ ) ,3.54-3.50(m,2H,CH ₂ ),3.48-3.45(m,2H,CH ₂ ),3.44-3.38(m,4H,CH ₂ ,CH ₂ ).ESI-MS: 455.5[M+H] ⁺ ,472.5[M+NH ₄ ] ⁺ ,477.4[M+Na] ⁺ .C ₂₈ H ₂₆ N ₂ O ₄ (454.19).

Embodiment 4: the preparation of compound a03

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 4-methoxyphenylboronic acid substituent. Yellow needle crystal, yield: 52.2%, melting point: 208-209°C.

Spectrum data of compound a03: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.09(s, 1H, indole-H), 7.74(d, J=8.00Hz, 1H, indole-H), 7.67(d ,J=8.00Hz,1H,indole-H),7.20(t,J=8.00Hz,4H,Ph-H),7.02(d,J=8.00Hz,2H,Ph-H),6.91(d,J =8.00Hz, 2H, Ph-H), 5.08(s, 2H, CH ₂ ), 3.88(s, 3H, COOCH ₃ ), 3.79(s, 3H, OCH ₃ ), 3.74(s, 3H, OCH ₃ ) ,3.56-3.50(m,4H,CH ₂ ,CH ₂ ),3.48-3.42(m,4H,CH ₂ ,CH ₂ ).ESI-MS: 515.6[M+H] ⁺ ,532.6[M+NH ₄ ] ⁺ ,537.5[M+Na] ⁺ .C ₃₀ H ₃₀ N ₂ O ₆ (514.21).

Embodiment 5: the preparation of compound a04

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 3-fluorophenylboronic acid substituent. Pale yellow solid, yield: 84.1%, melting point: 166-168°C.

Spectrum data of compound a04: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.20(s, 1H, indole-H), 7.85-7.75(m, 2H, indole-H), 7.65-7.60(m, 1H, Ph-H), 7.55-7.50 (m, 1H, Ph-H), 7.42-7.35 (m, 2H, Ph-H), 7.30-7.25 (m, 1H, Ph-H), 7.17-7.14 ( m,1H,Ph-H),7.10-7.07(m,1H,Ph-H),7.02-6.80(m,1H,Ph-H),5.23(s,1H,CH ₂ ),5.18(s,1H ,CH ₂ ),3.90(s,3H,COOCH ₃ ),3.58(s,1H,CH ₂ ),3.53-3.45(m,5H,CH ₂ ,CH ₂ ,CH ₂ ),3.44-3.40(m,2H ,CH ₂ ).ESI-MS: 508.5[M+NH ₄ ] ⁺ ,513.6[M+Na] ⁺ .C ₂₈ H ₂₄ F ₂ N ₂ O ₄ (490.17).

Embodiment 6: the preparation of compound a06

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 4-trifluoromethylphenylboronic acid substituent. White solid, yield: 92.4%, melting point: 204-206°C.

Spectrum data of compound a06: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.23 (s, 1H, indole-H), 7.87 (d, J=8.00Hz, 2H, Ph-H), 7.83-7.78 (m,2H,indole-H),7.71(d,J=8.00Hz,2H,Ph-H),7.55(d,J=8.00Hz,2H,Ph-H),7.46(d,J=8.00Hz ,2H,Ph-H),5.21(s,2H,CH ₂ ),3.90(s,3H,COOCH ₃ ),3.52-3.44(m,6H,CH ₂ ,CH ₂ ,CH ₂ ),3.42-3.39( m,2H,CH ₂ ).ESI-MS:591.4[M+H] ⁺ ,508.3[M+NH ₄ ] ⁺ ,513.3[M+Na] ⁺ .C ₃₀ H ₂₄ F ₆ N ₂ O ₄ (590.16) .

Embodiment 7: the preparation of compound a08

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by 2-thiopheneboronic acid substituent. Pale yellow solid, yield: 58.9%, melting point: 210-212°C.

Compound a08 spectral data: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.18 (s, 1H, indole-H), 7.91-7.64 (m, 3H, indole-H, indole-H, thiophene-H ),7.59-7.45(m,1H,thiophene-H),7.32-7.28(m,1H,thiophene-H),7.25-7.23(m,1H,thiophene-H),7.21-7.19(m,1H,thiophene -H),7.10-7.06(m,1H,thiophene-H),5.29(s,1H,CH ₂ ),5.18(s,1H,CH ₂ ),3.89(s,3H,COOCH ₃ ),3.60-3.53 (m,4H,CH ₂ ,CH ₂ ),3.49-3.48(m,2H,CH ₂ ),3.45-3.43(m,2H,CH ₂ ).ESI-MS: 467.11[M+H] ⁺ ,489.09[ _{M +} Na] ⁺ _{.C24H22N2O4S2 ( 466.10} ).

Embodiment 8: the preparation of compound a09

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 4-methylphenylboronic acid substituent. White solid, yield: 93.7%, melting point: 144-146°C.

Spectrum data of compound a09: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.11(s, 1H, indole-H), 7.75-7.73(m, 1H, indole-H), 7.70-7.68(m, 1H, indole-H),7.27-7.23(m,2H,Ph-H),7.18-7.15(m,2H,Ph-H),7.13-7.10(m,4H,Ph-H),5.08(s, 2H,CH ₂ ),3.88(s,3H,COOCH ₃ ),3.54-3.50(m,4H,CH ₂ ,CH ₂ ),3.46-3.42(m,4H,CH ₂ ,CH ₂ ),2.35(s, 3H, CH ₃ ), 2.28 (s, 3H, CH ₃ ). ESI-MS: 483.4 [M+H] ^{+ .} C ₃₀ H ₃₀ N ₂ O ₄ (482.22).

Embodiment 9: the preparation of compound a10

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 3-methylphenylboronic acid substituent. Yellow solid, yield: 76.9%, melting point: 170-172°C.

Spectrum data of compound a10: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.13(s, 1H, indole-H), 7.77-7.70(m, 2H, indole-H), 7.30-7.26(m, 2H, Ph-H), 7.18-7.15 (m, 2H, Ph-H), 7.12-7.10 (m, 1H, Ph-H), 7.05 (t, J=8.00Hz, 2H, Ph-H), 6.97 (d, J=8.00Hz, 1H, Ph-H), 5.08 (s, 2H, CH ₂ ), 3.88 (s, 3H, COOCH ₃ ), 3.58-3.56 (m, 1H, CH ₂ ), 3.54-3.52 (m,1H,CH ₂ ),3.48-3.46(m,2H,CH ₂ ),3.45-3.43(m,3H,CH ₂ ,CH ₂ ),2.30(s,3H,CH ₃ ),2.25(s, 3H, CH ₃ ). ESI-MS: 483.4[M+H] ⁺ , 500.4 [M+NH ₄ ] ⁺ , 505.5 [M+Na] ^{+ .} C ₃₀ H ₃₀ N ₂ O ₄ (482.22).

Embodiment 10: the preparation of compound a11

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 3-methylphenylboronic acid substituent. Pale yellow solid, yield: 87.2%, melting point: 176-178°C.

Spectrum data of compound a11: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.15(s, 1H, indole-H), 7.75-7.63(m, 2H, indole-H), 7.11(s, 1H, Ph-H),7.07(s,1H,Ph-H),7.03(s,1H,Ph-H),6.88(s,1H,Ph-H),5.13(s,1H,CH ₂ ),5.05( s,1H,CH ₂ ),3.88(s,3H,COOCH ₃ ),3.59-3.56(m,2H,CH ₂ ),3.55-3.53(m,2H,CH ₂ ),3.44-3.42(m,4H, CH ₂ ,CH ₂ ), 2.35(s,3H,CH ₃ ), 2.34(s,3H,CH ₃ ), 2.25(s,3H,CH ₃ ), 2.19(s,3H,CH ₃ ).ESI-MS :511.25[M+H] ⁺ ,533.24[M+Na] ⁺ .C ₃₂ H ₃₄ N ₂ O ₄ (510.25).

Embodiment 11: the preparation of compound a12

The operation is the same as in Example 3, except that the phenylboronic acid substituent is replaced by a 3-fluoro-4-methoxyphenylboronic acid substituent. Yellow solid, yield: 60.5%, melting point: 187-189°C.

Spectrum data of compound a12: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.14(s, 1H, indole-H), 7.77(d, J=8.00Hz, 1H, indole-H), 7.70(d ,J=8.00Hz,1H,indole-H),7.28(t,J=8.00Hz,1H,Ph-H),7.17(t,J=8.00Hz,2H,Ph-H),7.05(t,J =8.00Hz,3H,Ph-H),5.13(s,2H,CH ₂ ),3.89(s,6H,COOCH ₃ ,CH ₃ ),3.83(s,3H,CH ₃ ),3.53-3.52(m, 4H,CH ₂ ,CH ₂ ),3.49-3.48(m,2H,CH ₂ ),3.44-3.43(m,2H,CH ₂ ).ESI-MS: 551.19[M+H] ⁺ ,573.17[M+Na ] ⁺ .C ₃₀ H ₂₈ F ₂ N ₂ O ₆ (550.19).

Embodiment 12: the preparation of compound a13

The operation is the same as that in Example 3, except that the phenylboronic acid substituent is replaced by a 3,4-dimethylphenylboronic acid substituent. White solid, yield: 82.9%, melting point: 116-118°C.

Spectrum data of compound a13: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.10(s, 1H, indole-H), 7.72(s, 1H, indole-H), 7.68(s, 1H, indole-H) H),7.16(s,2H,Ph-H),7.07(s,1H,Ph-H),7.03(s,1H,Ph-H),6.96(s,1H,Ph-H),6.85(s ,1H,Ph-H),5.06(s,2H,CH ₂ ),3.89(s,3H,COOCH ₃ ),3.56-3.54(m,3H,CH ₂ ,CH ₂ ),3.49-3.45(m,5H ,CH ₂ ,CH ₂ ,CH ₂ ),2.27(s,3H,CH ₃ ),2.22(s,3H,CH ₃ ),2.19(s,6H,CH ₃ ,CH ₃ ).ESI-MS:511.6[ M+H] ⁺ , 528.5 [M+NH ₄ ] ⁺ , 533.3 [M+Na] ^{+ .} C ₃₂ H ₃₄ N ₂ O ₄ (510.25).

Embodiment 13: Preparation of compound b01

Weigh compound a01 (200.00 μmol) in a 25mL flask, add 15mL methanol to dissolve, heat up and reflux, slowly add 1M sodium hydroxide solution dropwise to the reaction solution, and monitor the pH value of the solution in real time until the pH value is stable above 10, Stop adding sodium hydroxide solution dropwise, and use TLC to check that the reaction is complete, adjust the pH value to 1 with 1M hydrochloric acid, then spin dry the solvent, add 10mL ethyl acetate to dissolve, wash with water (2×10mL), dry over anhydrous sodium sulfate, and spin dry Solvent, ethanol recrystallization to obtain the corresponding target product b01. White solid, yield: 87%, melting point: 200-202°C.

Spectrum data of compound b01: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.71(s, 1H, COOH), 8.15-8.12(m, 1H, indole-H), 7.77-7.74(m, 1H, indole-H),7.71-7.69(m,1H,indole-H),7.45-7.44(m,3H,Ph-H),7.32-7.27(m,4H,Ph-H),7.25-7.18(m, 3H,Ph-H),5.09(s,2H,CH ₂ ),3.52-3.51(m,2H,CH ₂ ),3.48-3.47(m,2H,CH ₂ ),3.44-3.43(m,4H,CH ₂ , CH ₂ ). ¹³ C NMR (100MHz, DMSO-d ₆ , ppm) δ: 168.60 (COOH), 166.35 (CO), 141.23 (indole-C), 137.12 (Ph-C), 134.57 (indole-C ),131.26(Ph-C),131.09(2×Ph-C),130.08(indole-C),129.78(2×Ph-C),129.32(Ph-C),129.04(2×Ph-C), 128.89(2×Ph-C),125.60(Ph-C),124.64(indole-C),121.63(indole-C),118.84(indole-C),115.18(indole-C),113.22(indole-C) ,66.66(morpholine-CH ₂ ),66.50(morpholine-CH ₂ ),53.37(CH ₂ ),45.52(morpholine-CH ₂ ),45.39(morpholine-CH ₂ ).ESI-MS:439.6[MH] ^- .C ₂₇ H ₂₄ N ₂ O ₄ (440.17).

Embodiment 14: the preparation of compound b04

The operation is the same as in Example 13, except that the substituent of compound a01 is replaced by compound a04. Pale yellow solid, yield: 92.2%, melting point: 178-180°C.

Spectrum data of compound b04: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.76 (s, 1H, COOH), 8.17 (s, 1H, indole-H), 7.88 (dd, J ₁ =1.20Hz, J ₂ =8.00Hz,1H,indole-H),7.83(d,J=8.00Hz,1H,indole-H),7.54-7.48(m,1H,Ph-H),7.40-7.39(m,1H, Ph-H),7.36-7.34(m,1H,Ph-H),7.32-7.30(m,1H,Ph-H),7.15-7.13(m,1H,Ph-H),7.12-7.10(m, 1H,Ph-H),7.08-7.06(m,1H,Ph-H),7.01-6.98(m,1H,Ph-H),5.16(s,2H,CH ₂ ),3.57-3.55(m,1H ,CH ₂ ),3.53-3.48(m,5H,CH ₂ ,CH ₂ ,CH ₂ ),3.43-3.42(m,2H,CH ₂ ). ¹³ C NMR(100MHz,DMSO-d ₆ ,ppm)δ: 168.58 (COOH), 166.38 (CO), 163.94 (Ph-CF), 163.57 (Ph-CF), 161.42 (Ph-CF), 161.09 (Ph-CF), 140.06, 138.33, 137.16, 136.80, 136.72, 133.25, 133.10,131.34,130.85,129.59,127.51,125.94,125.21,124.43,121.97,118.90,117.94,117.72,116.27,116.05,114.40,113.51,113.30,66.68(morpholine-CH ₂ ),66.58(morpholine-CH ₂ ), 45.61 (morpholine-CH ₂ ), 45.43 (morpholine-CH ₂ ), 42.63 (CH ₂ ).

Embodiment 15: the preparation of compound b06

The operation was the same as in Example 13, except that the substituent of compound a01 was replaced by compound a06, white solid, yield: 83.5%, melting point: 216-218°C.

Spectrum data of compound b06: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.91(s, 1H, COOH), 8.23(d, J=12.00Hz, 1H, indole-H), 7.88(q, J =4.00Hz, 2H, Ph-H), 7.83(t, J=8.00Hz, 1H, indole-H), 7.78(t, J=8.00Hz, 1H, indole-H), 7.71(d, J=8.00 Hz,2H,Ph-H),7.59(d,J=8.00Hz,1H,Ph-H),7.55(d,J=8.00Hz,1H,Ph-H),7.46(d,J=8.00Hz, 2H,Ph-H),5.19(s,1H,CH ₂ ),5.01(s,1H,CH ₂ ),3.51-3.47(m,3H,CH ₂ ,CH ₂ ),3.42-3.40(m,2H, CH ₂ ), 3.35-3.33 (m, 3H, CH ₂ , CH ₂ ). ¹³ C NMR (100MHz, DMSO-d ₆ , ppm) δ: 170.36 (COOH), 168.46, 168.38, 166.21 (CO), 140.31, 140.03,138.59,138.38,137.47,137.07,135.06,134.78,132.08,130.45,130.39,129.65,129.55,129.95,125.95,125.76,125.44,122.41,122.17,119.18,118.94,114.93,114.56,113.45,113.23,66.63( morpholine-CH ₂ ),66.53(morpholine-CH ₂ ),46.09(CH ₂ ),45.66(morpholine-CH ₂ ),45.41(morpholine-CH ₂ ).ESI-MS:575.4[MH] ^- .C ₂₉ H _{22 F2N2O4 ( 576.15} ).

Embodiment 16: Preparation of compound b09

The operation is the same as that of Example 13, except that the substituent of compound a01 is replaced by compound a09, white solid, yield: 87.1%, melting point: 268 ° C decomposition.

Spectrum data of compound b09: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.77(s, 1H, COOH), 8.08(d, J=4.00Hz, 1H, indole-H), 7.74(d, J =8.00Hz, 1H, indole-H), 7.67(d, J=8.00Hz, 1H, indole-H), 7.26(t, J=8.00Hz, 2H, Ph-H), 7.16(d, J=8.00 Hz, 2H, Ph-H), 7.12(s, 4H, Ph-H), 5.06(d, J=12.40Hz, 2H, CH ₂ ), 3.88(s, 3H, COOCH ₃ ), 3.54-3.50(m ,4H,CH ₂ ,CH ₂ ),3.46-3.44(m,4H,CH ₂ ,CH ₂ ),2.35(s,3H,CH ₃ ),2.28(s,3H,CH ₃ ). ¹³ C NMR (100MHz , DMSO-d ₆ , ppm) δ: 168.67 (COOH), 166.52 (CO), 141.06 (indole-C), 138.60 (Ph-C), 137.04 (Ph-C), 135.41 (indole-C), 131.69 ( Ph-C), 130.59(2×Ph-C), 130.91(2×Ph-C), 130.21(Ph-C), 129.66(2×Ph-C), 128.52(2×Ph-C), 128.29( indole-C), 124.50(indole-C), 121.49(indole-C), 118.75(indole-C), 114.95(indole-C), 113.04(indole-C), 66.67(morpholine-CH ₂ ), 66.53( morpholine-CH ₂ ), 45.51(morpholine-CH ₂ ), 45.39(morpholine-CH ₂ ), 42.58(CH ₂ ), 21.38(CH ₃ ), 21.16(CH ₃ ).ESI-MS: 467.5[MH] ^- . _{C29H28N2O4 ( 468.20 )} .

Embodiment 17: Preparation of compound b10

The operation is the same as in Example 13, except that the substituent of compound a01 is replaced by compound a10, a light yellow solid, yield: 90.2%, melting point: 246-248°C.

Spectral data of compound b10: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.65 (s, 1H, COOH), 8.10 (d, J=4.00Hz, 1H, indole-H), 7.76 (dd, J ₁ = 12.00Hz, J2 = _1.20Hz , 1H, indole-H), 7.70 (d, J = 8.00Hz, 1H, indole-H), 7.42-7.29 (m, 2H, Ph-H), 7.27-7.25 (m,2H,Ph-H),7.16-7.13(m,2H,Ph-H),7.11(s,1H,Ph-H),7.05(t,J=8.00Hz,2H,Ph-H), 6.97(d, J=8.00Hz, 1H, Ph-H), 5.16(d, J=40.00Hz, 2H, CH ₂ ), 3.54-3.52(m, 2H, CH ₂ ), 3.48-3.47(m, 2H , CH ₂ ), 3.44-3.43 (m, 4H, CH ₂ , CH ₂ ), 2.30 (s, 3H, CH ₃ ), 2.25 (s, 3H, CH ₃ ). ¹³ C NMR (100MHz, DMSO-d ₆ ,ppm)δ:168.68(COOH),166.54(CO),141.20(Ph-C),138.05(Ph-C),137.80(Ph-C),137.12(indole-C),134.54(indole-C), 131.25(Ph-C),130.37(Ph-C),130.14(Ph-C),129.88(Ph-C),128.87(Ph-C),128.70(indole-C),128.40(Ph-C),127.07 (Ph-C),126.96(Ph-C),124.52(Ph-C),121.53(indole-C),118.90(indole-C),115.09(indole-C),113.12(indole-C),102.31( indole-C),66.69(morpholine-CH ₂ ),66.54(morpholine-CH ₂ ),45.59(morpholine-CH ₂ ),45.39(morpholine-CH ₂ ),42.61(CH ₂ ),21.59(CH ₃ ),21.50 (CH ₃ ).ESI-MS: 467.5 [MH] ^- .C ₂₉ H ₂₈ N ₂ O ₄ (468.20).

Embodiment 18: Preparation of compound b11

The operation is the same as that of Example 13, except that the substituent of compound a01 is replaced by compound a11, white solid, yield: 85.9%, melting point: 244° C. decomposes.

Spectrum data of compound b11: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.67 (s, 1H, COOH), 8.12-8.01 (m, 1H, indole-H), 7.81 (dd, J ₁ =8.00 Hz,J ₂ =1.20Hz,1H,indole-H),7.69-7.65(m,1H,indole-H),7.62-7.52(m,1H,Ph-H),7.11-7.02(m,2H,Ph -H),6.88-6.84(m,3H,Ph-H),5.14-5.03(m,2H,CH ₂ ),3.58-3.53(m,4H,CH ₂ ,CH ₂ ),3.46-3.44(m, 4H,CH ₂ ,CH ₂ ),2.35(s,3H,CH ₃ ),2.33(s,3H,CH ₃ ),2.25(s,3H,CH ₃ ),2.19(s,3H, ^CH ₃ ). C NMR (100MHz, DMSO-d ₆ , ppm) δ: 168.70 (COOH), 166.64 (CO), 145.00 (Ph-C), 141.73 (indole-C), 137.79 (2×Ph-C), 137.49 (2 ×Ph-C),134.46(indole-C),131.16(Ph-C),130.24(Ph-C),128.64(2×Ph-C),128.16(indole-C),127.65(2×Ph-C ), 126.96(Ph-C), 124.40(indole-C), 121.41(indole-C), 118.97(indole-C), 115.01(indole-C), 113.08(indole-C), 66.70(morpholine-CH ₂ ),66.61(morpholine-CH ₂ ),45.67(morpholine-CH ₂ ),45.39(morpholine-CH ₂ ),42.61(CH ₂ ),21.48(CH ₃ ),21.45(CH ₃ ),21.40(CH ₃ ), 21.37 ( _CH3 ).

Embodiment 19: Preparation of compound b13

The operation is the same as that of Example 13, except that the substituent of compound a01 is replaced by compound a13, a light yellow solid, yield: 84.4%, melting point: 276° C. decomposes.

Spectrum data of compound b13: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.62(s, 1H, COOH), 8.06(s, 1H, indole-H), 7.73(d, J=8.00Hz, 1H , indole-H), 7.66(d, J=8.00Hz, 1H, indole-H), 7.18-7.15(m, 2H, Ph-H), 7.06(s, 1H, Ph-H), 7.03(d, J＝8.00Hz, 1H, Ph-H), 6.96(d, J＝8.00Hz, 1H, Ph-H), 6.86(d, J＝8.00Hz, 1H, Ph-H), 5.03(s, 2H, CH ₂ ),3.55-3.53(m,2H,CH ₂ ),3.49-3.47(m,2H,CH ₂ ),3.46-3.44(m,4H,CH ₂ ,CH ₂ ),2.26(s,3H,CH ₃ ), 2.21(s,3H,CH ₃ ), 2.19(s,3H,CH ₃ ), 2.18(s,3H,CH ₃ ). ¹³ C NMR(100MHz,DMSO-d ₆ ,ppm)δ:168.71( COOH), 166.63(CO), 141.05(Ph-C), 137.33(Ph-C), 137.01(indole-C), 136.68(indole-C), 136.41(Ph-C), 134.18(Ph-C), 132.13(Ph-C),131.56(Ph-C),130.87(Ph-C),130.35(Ph-C),130.05(indole-C),129.96(Ph-C),128.74(Ph-C),128.72 (Ph-C),127.32(Ph-C),124.30(indole-C),121.38(indole-C),118.84(indole-C),114.92(indole-C),113.01(indole-C),66.69( morpholine-CH ₂ ),66.55(morpholine-CH ₂ ),45.57(morpholine-CH ₂ ),45.39(morpholine-CH ₂ ),42.59(CH ₂ ),20.00(CH ₃ ),19.95(CH ₃ ),19.71( CH ₃ ), 19.49 (CH ₃ ). ESI-MS: 495.5 [MH] ^- .C ₃₁ H ₃₂ N ₂ O ₄ (496.24).

Example 20: Preparation of Intermediate 4

Weigh Intermediate 3 (92.0mg, 200.00μmol) in a 50mL microwave reactor, add 10mL redistilled dioxane to dissolve, add phenylboronic acid (24.4mg, 200.00μmol), tetrakis(triphenylphosphine) palladium (11.6mg , 10.00 μmol), tripotassium phosphate (46.7mg, 220.00mmol). React in a microwave reactor at 150° C. for 30 minutes, and proceed to the next reaction directly.

Intermediate 4 spectral data: ESI-MS: 457.4[M+H] ⁺ ,459.4[M+H] ⁺ ,474.3[M+NH ₄ ] ⁺ ,476.3[M+NH ₄ ] ⁺ ,481.3[M+Na] ⁺ ,483.3[M+Na] ⁺ .C ₂₂ H ₂₁ BrN ₂ O ₄ (456.07).

Embodiment 21: Preparation of compound a02

Added 4-methoxyphenylboronic acid substituent (220.00 μmol), tetrakis(triphenylphosphine) palladium (11.6 mg, 10.00 μmol) and tripotassium phosphate (46.7 mg, 220.00 mmol) to the reaction solution of the previous step reaction. React in a microwave reactor at 150°C for 30 minutes, TLC detects that the reaction is complete, filter the reaction solution with diatomaceous earth, spin to dry the solvent, dissolve the solid in 20 mL of ethyl acetate, wash with water (2×20 mL), and dry over anhydrous sodium sulfate. The solvent was spin-dried, followed by Flash column chromatography, and the chromatographic product was recrystallized from ethanol to obtain the corresponding target product a02. White needle crystals, yield: 31.8%, melting point: 208-210°C.

Spectrum data of compound a02: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.13(s, 1H, indole-H), 7.76(d, J=8.00Hz, 1H, indole-H), 7.69(d ,J=8.00Hz,1H,indole-H),7.44(s,3H,Ph-H),7.29(s,2H,Ph-H),7.17(d,J=8.00Hz,2H,Ph-H) ,6.89(d,J＝8.00Hz,2H,Ph-H),5.09(s,2H,CH ₂ ),3.88(s,3H,COOCH ₃ ),3.73(s,3H,OCH ₃ ),3.52-3.50 (m, 2H, CH ₂ ), 3.47-3.46 (m, 2H, CH ₂ ), 3.43-3.41 (m, 4H, CH ₂ , CH ₂ ).

Embodiment 22: Preparation of compound a05

The operation is the same as in Example 21 except that the 4-methoxyphenylboronic acid substituent is replaced by the 3-furan boronic acid substituent. White solid, yield: 33.2%, melting point: 170-172°C.

Spectrum data of compound a05: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 8.16(s, 1H, indole-H), 8.02(s, 1H, furan-H), 7.93(s, 1H, furan- H),7.80(s,1H,indole-H),7.75-7.73(m,1H,indole-H),7.67-7.65(m,1H,Ph-H),7.56-7.54(m,1H,Ph- H),7.39-7.37(m,2H,Ph-H),7.28(s,1H,Ph-H),6.71-6.36(m,1H,furan-H),5.32(s,1H,CH ₂ ), 5.24(s,1H,CH ₂ ),3.88(s,3H,COOCH ₃ ),3.64-3.61(m,2H,CH ₂ ),3.58-3.56(m,4H,CH ₂ ,CH ₂ ),3.46-3.44 (m,2H, _CH2 ).

Embodiment 23: Preparation of compound b02

Weigh compound a02 (200.00μmol) in a 25mL flask, add 15mL of methanol to dissolve, heat up and reflux, slowly add 1M sodium hydroxide solution dropwise to the reaction solution, and monitor the pH value of the solution in real time, and stop when it is stable above 10. Add sodium hydroxide solution dropwise, and use TLC to detect that the reaction is complete, adjust the pH value to 1 with 1M hydrochloric acid, and then spin dry the solvent, dissolve in 10mL ethyl acetate, wash with water (2×10mL), dry over anhydrous sodium sulfate, spin dry the solvent, Recrystallization from ethanol gave the corresponding target product b02. White solid, yield: 92.3%, melting point: decomposed at 232°C.

Spectrum data of compound b02: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.72 (s, 1H, COOH), 8.12 (d, J=8.00Hz, 1H, indole-H), 7.76 (dd, J ₁ = 8.00Hz, J2 = _8.00Hz , 1H, indole-H), 7.67 (d, J = 8.00Hz, 1H, indole-H), 7.45 (t, J = 4.00Hz, 3H, Ph-H), 7.30-7.27(m,2H,Ph-H),7.17(d,J=8.00Hz,2H,Ph-H),6.89(d,J=8.00Hz,2H,Ph-H),5.07(s,2H ,CH ₂ ),3.73(s,3H,CH ₃ ),3.52-3.50(m,2H,CH ₂ ),3.47-3.46(m,2H,CH ₂ ),3.43-3.42(m,4H,CH ₂ , CH ₂ ). ¹³ C NMR (100MHz, DMSO-d ₆ , ppm) δ: 168.68 (COOH), 166.48 (CO), 158.00 (Ph-CO), 140.79 (indole-C), 137.04 (indole-C), 131.35(Ph-C), 131.09(2×Ph-C), 130.89(2×Ph-C), 130.35(Ph-C), 129.23(indole-C), 129.01(2×Ph-C), 126.69( Ph-C), 124.49(indole-C), 121.45(indole-C), 118.85(indole-C), 114.91(indole-C), 114.43(2×Ph-C), 113.06(indole-C), 66.69 (morpholine-CH ₂ ), 66.50 (morpholine-CH ₂ ), 55.45 (CH ₃ ), 45.50 (morpholine-CH ₂ ), 45.38 (morpholine-CH ₂ ), 42.59 (CH ₂ ).

Embodiment 24: Preparation of compound b05

The operation is the same as that of Example 13, except that the substituent of compound a01 is replaced by compound a05, white solid, yield: 89.5%, melting point: 204°C decomposition.

Spectral data of compound b05: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.73(s, 1H, COOH), 8.15(d, J=8.00Hz, 1H, indole-H), 8.01(s, 1H , furan-H), 7.92 (t, J = 1.60Hz, 1H, furan-H), 7.81 (td, J ₁ = 8.00Hz, J ₂ = 1.60Hz, 1H, indole-H), 7.73 (dd, J ₁ = 8.00Hz, J2 = _1.60Hz , 1H, indole-H), 7.64 (d, J = 8.00Hz, 1H, Ph-H), 7.53 (d, J = 8.00Hz, 1H, Ph-H), 7.41-7.35 (m, 2H, Ph-H), 7.29 (tt, J ₁ =8.00Hz, J ₂ =1.60Hz, 1H, Ph-H), 6.71-6.36 (d, J = 180.00Hz, 1H, furan -H),5.29(s,1H,CH2),5.22(s,1H,CH2),3.62-3.60(m,2H,CH2),3.57-3.55(m,4H,CH2,CH2),3.46-3.44( m, 2H, CH2). ¹³ C NMR (100MHz, DMSO-d ₆ , ppm) δ: 168.66 (COOH), 166.18 (CO), 158.00 (Ph-CO), 144.67 (furan-C), 144.59 (furan- C),136.81(indole-C),134.55(indole-C),133.05(indole-C),130.11(Ph-C),129.87(2×Ph-C),128.89(2×Ph-C),126.72 (furan-C),122.57(Ph-C),121.50(indole-C),118.48(indole-C),115.51(indole-C),114.51(indole-C),112.47(indole-C),111.46( furan-C), 70.59 (morpholine-CH ₂ ), 69.96 (morpholine-CH ₂ ), 66.82 (CH ₂ ), 45.53 (morpholine-CH ₂ ), 45.51 (morpholine-CH ₂ ).

Embodiment 25: Preparation of compound a07

Weigh intermediate 4 (200.00μmol) into a 25mL flask, add 10mL anhydrous tetrahydrofuran to dissolve, and then add 10μmol [1,1'-bis(diphenylphosphino)ferrocene] dichloride under nitrogen protection environment Palladium, 340 μmol of tetramethylethylenediamine, 340 μmol of sodium borohydride, stirred at room temperature for 19 hours, then stopped the reaction, poured the reaction solution into 20 mL of cold water, added 10 mL of ethyl acetate to extract twice, combined the organic phases, and spin-dried the solvent. Compound a07 was obtained by flash column chromatography as a white solid, yield: 51.5%, melting point: 218-219°C.

Spectrum data of compound a07: ¹ H NMR (400MHz, CDCl ₃ , ppm) δ: 7.97(s, 1H, indole-H), 7.84(d, J=8.00Hz, 1H, indole-H), 7.65(d, J =8.00Hz, 1H, indole-H), 7.46-7.44(m, 5H, Ph-H), 6.63(s, 1H, indole-H), 4.88(s, 2H, CH ₂ ), 3.93(s, 3H ,COOCH ₃ ),3.69-3.68(m,2H,CH ₂ ),3.65-3.62(m,4H,CH ₂ ,CH ₂ ),3.42-3.40(m,2H,CH ₂ ).ESI-MS:379.5[ M+H] ⁺ , 396.5 [M+NH ₄ ] ⁺ , 401.5 [M+Na] ^{+ .} C ₂₂ H ₂₂ N ₂ O ₄ (378.16).

Embodiment 26: Preparation of compound b07

The operation is the same as in Example 13, except that the substituent of compound a01 is replaced by compound a07, yield: 89.4%, melting point: 266°C decomposition.

Spectrum data of compound b07: ¹ H NMR (400MHz, DMSO-d ₆ , ppm) δ: 12.62 (s, 1H, COOH), 8.03 (s, 1H, indole-H), 7.71 (dd, J ₁ =8.00Hz, J ₂ =4.00Hz,1H,indole-H),7.64(d,J=8.00Hz,1H,indole-H),7.52(d,J=4.00Hz,2H,Ph-H),7.49-7.47(m ,3H,Ph-H),6.67(s,1H,indole-H),5.17(s,2H,CH ₂ ),3.58-3.56(m,4H,CH ₂ ,CH ₂ ),3.54-3.53(m, 2H, CH ₂ ), 3.47-3.46 (m, 2H, CH ₂ ). ¹³ C NMR (100MHz, DMSO-d ₆ , ppm) δ: 168.75 (COOH), 166.64 (CO), 144.88 (indole-C), 138.20(indole-C), 132.10(Ph-C), 131.49(Ph-C), 129.31(2×Ph-C), 129.28(2×Ph-C), 129.08(indole-C), 124.02(indole- C), 121.20 (indole-C), 120.11 (indole-C), 113.02 (indole-C), 102.48 (indole-C), 66.73 (morpholine-CH ₂ ), 66.64 (morpholine-CH ₂ ), 45.83 (morpholine -CH ₂ ), 45.41 (morpholine-CH ₂ ), 42.58 (CH ₂ ). ESI-MS: 363.4 [MH] ^- .C ₂₁ H ₂₀ N ₂ O ₄ (364.14).

Example 27: Affinity determination experiment between target compound and NS5B

Experimental principle

Optical surface plasmon resonance (Surface Plasmon Resonance, SPR) is an optical physical phenomenon. When a beam of P-polarized light is incident on the end face of the prism within a certain angle range, surface plasmon waves will be generated at the interface between the prism and the metal film (Au or Ag). When the propagation constant of the incident light wave matches that of the surface plasmon wave, free electrons in the metal film are caused to resonate, that is, surface plasmon resonance. During the analysis, a layer of biomolecular recognition film is first fixed on the surface of the sensor chip, and then the sample to be tested flows over the chip surface. If there are molecules in the sample that can interact with the biomolecular recognition film on the chip surface, the surface of the gold film will be aroused. Changes in the refractive index eventually lead to changes in the SPR angle. By detecting the changes in the SPR angle, information such as the concentration, affinity, kinetic constant, and specificity of the analyte can be obtained.

Materials and Methods

Experimental materials and equipment

experimental method

CM5 chip surface activation and protein coupling

The surface of CM5 chip was activated with 0.2mol/L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.05mol/L N-succinimide (NHS) solution. Then the Anti-histidineantibody in the His Capture Kit kit was diluted to 50 μg/mL with the Immobilization buffer in the kit, and the samples were injected using the set flow rate (10 μL/min) and time (7min) mode, using 0.05 mmol/L NHS and 0.2 The mol/L EDC activates the carboxyl groups on the surface of the chip, and couples the Anti-histidine antibody protein to the Fc1 and Fc2 channels of the chip until the two channels reach the set coupling level (9000-15000RU). Finally, the surface of the chip was blocked with ethanolamine for 7 minutes, and then the chip was washed twice with 1.05X PBS (5% DMSO), and the mobile phase in the system was replaced with a system with DMSO to remove uncoupled proteins.

Solvent Calibration

Put the pre-configured DMSO gradient solution in the detector, add a sufficient amount of NS5B dilution solution and chip regeneration solution to the instrument at the same time, call the capture mode in the Knitic assay tool of the instrument, and inject the DMSO gradient solution at the same time as the reference Channel Fc1 and working channel Fc2 were subjected to binding reaction at 25°C and pH 7.4. The flow rate of NS5B diluent was set at 10 μL/min, the injection time was 2 min, and the stabilization time was 4 min. The flow rate of the DMSO gradient solution was set at 30 μL/min, the injection time was 2 min, and the stabilization time was 1 min. The flow rate of the regeneration solution was set at 30 μL/min, the injection time was 1 min, and the stabilization time was 1 min.

Affinity Kinetic Analysis of Small Molecule Inhibitors and NS5B

The pre-configured gradient concentration of small molecule samples to be tested was placed in the detector, and each sample was set at 12.5 μM as the repeated concentration. The procedure for determining the activity of small molecule inhibitors is exactly the same as that for solvent calibration.

Affinity Kinetic Data Analysis

Solvent Calibration

(1) Establish a solvent calibration curve. Use BIAevaluation software to open the solvent calibration result file and select Rpoint table. Use the Data/filter command in the opened table, select Fc1 in the channel column data, select Binding1 in the Id column data, then copy the data in the RelResp column to a new table, and name the column Fc1. Go back to the original table and select Fc2-1 in the channel column, then copy the data in the RelResp column to the newly created table and name the column Fc2-1. Use the Data/Generate Calibration command in the new table, set X to Fc2-1, Y column to Fc1, select the Line command in the Other drop-down menu and confirm to obtain the solvent calibration standard curve.

(2) The influence of solvent correction is removed from the sample detection results. After the solvent calibration curve is established, directly open the result file of the small molecule test and select the Rpoint table method to open it. Use the Data/filter command in the opened table, select Fc1 in the channel column data, select Binding1 in the Id column data, then copy the data in the RelResp column to a new table, and name the column Fc1, Then copy Fc2-1 according to the same method, and name it Fc2-1, then use the Data/Calculate values command, select the solvent correction curve just obtained in the pop-up dialog box, click next, and set Y to Fc1, that is The corresponding solvent correction factor can be obtained, and the solvent correction factor column is named corr, and the actual value of Fc2-1 can be obtained by subtracting the corresponding solvent correction factor from the data of Fc2-1 in the next column, and the column is named as True.

The establishment of sample small molecule curve and the calculation of equilibrium dissociation constant

Copy the obtained response value (True column) data for removing the solvent effect to a new table, and copy the small molecule sample concentration corresponding to each data to the next column and name the column Conc. Note that the data in the Conc column cannot contain There are units. Then use the Create Curve command, set X as the Conc column, set Y as the True column, and confirm to get the relationship curve points between the response value and the concentration, and then use the fitting function in the software, using the Fit/general command, In the pop-up dialog box, select steady state affinity to fit the curve data, and the equilibrium dissociation constant K _D of the small molecule compound can be obtained. The meaning of this constant is the ligand concentration required to make half of the total protein bind to the ligand. The smaller the value, the stronger the binding ability of the compound to the protein.

Target compound and NS5B affinity assay results

Table 1 The results of affinity experiments between target compounds and NS5B

Active compounds are in bold.

^a K _D : represents the equilibrium dissociation constant between the compound and the protein, the smaller the value, the stronger the affinity between the compound and the protein.

in conclusion:

It can be seen from Table 1 that the substituted indole derivatives of the present invention are a series of novel non-nucleoside HCV inhibitors with different affinities to NS5B. Among them, compounds a02, a03, a11 and b05 all have affinity comparable to or even slightly better than the positive control compound. Especially compounds a10 (K _D =2.8μM) and b13 (K _D =2.3μM), their affinity to NS5B was 32 times and 39 times that of the positive control compound 121 (K _D =90.8μM), far exceeding the positive As a reference compound, this kind of substituted indole derivatives has value for further research and development, and can be used as a lead compound against HCV.

Claims (7)

1. A substituted indole derivative, or a pharmaceutically acceptable salt, ester or prodrug thereof, characterized by having a structure shown in general formula I:

wherein,

R₁、R₂each independently selected from substituted benzene ring, substituted naphthalene ring, various substituted six-membered heterocyclic rings, various substituted five-membered heterocyclic rings,Various substituted six-membered and five-membered heterocyclic rings, various substituted six-membered and six-membered heterocyclic rings, various substituted five-membered and five-membered heterocyclic rings, various substituted benzo five-membered heterocyclic rings or various substituted benzo six-membered heterocyclic rings, various aliphatic rings, various saturated aliphatic chains or various unsaturated aliphatic chains;

R₃are various carboxylic acids, carboxylic esters or amides.

2. The substituted indole derivative of claim 1, wherein in formula I, R is₁、R₂Each independently selected from phenyl, p-methylphenyl, p-methoxyphenyl, 2-furyl, 3-thienyl, m-methylphenyl, m-fluorophenyl or p-trifluoromethylphenyl; r₃Is carboxyl or carbomethoxy.

3. The substituted indole derivative according to claim 1 or 2, which is one of the following specific compounds:

4. the process for preparing substituted indole derivatives according to claim 1 or 2, comprising one of the following steps:

route one: indole-6-methyl formate 1 is used as an initial raw material, and is subjected to affinity substitution reaction with chloroacetyl morpholine in an N, N-dimethylformamide solution to generate an intermediate compound 2; then the intermediate compound 2 reacts with N-bromosuccinimide in a dichloromethane solution to generate an intermediate compound 3; then, the intermediate compound 3 reacts with various substituted boric acids in dioxane solution to generate a target product a, and then different target products a react with sodium hydroxide solution in methanol solution to obtain a target product b;

the synthesis route one is as follows:

wherein R is₁、R₂The general formula I is shown in the specification;

reagents and conditions: (i) chloroacetyl morpholine, sodium hydride, N-dimethylformamide, room temperature; (ii) dichloromethane, N-bromosuccinimide; (iii) boronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate, dioxane, 90 ℃; (iv) refluxing methanol and sodium hydroxide water solution;

the substituted boric acid is phenylboronic acid, p-methoxyphenylboronic acid, m-fluorophenylboronic acid, 3-furanboronic acid, p-methylphenylboronic acid, m-methylphenylboronic acid, 2, 4-dimethylphenylboronic acid, 2-thiopheneboronic acid, 2, 3-dimethylphenylboronic acid or 2-fluoro-3-methoxyphenylboronic acid;

and a second route: taking an intermediate compound 3 as a raw material, reacting the intermediate compound with phenylboronic acid in a dioxane solution to generate an intermediate compound 4, and then reacting the intermediate compound with different substituted boric acids to generate a target product a; then the compound a reacts with a sodium hydroxide aqueous solution in a methanol solution to generate a target product b;

the second synthetic route is as follows:

wherein R is₁The general formula I is shown in the specification;

reagents and conditions: (i) phenylboronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate, dioxane, 90 ℃; (ii) boronic acid, tetrakis (triphenylphosphine) palladium, potassium phosphate, dioxane, 90 ℃; (iii) refluxing methanol and sodium hydroxide water solution;

the substituted boric acid is phenylboronic acid, p-methoxyphenylboronic acid or 3-furanboronic acid.

5. The process for preparing substituted indoles derivative of claim 4, comprising the step of one of the following schemes:

route one:

(1) weighing 5.26g of raw material 6-methyl indole carboxylate, placing the raw material in a 100mL flask, adding 20mL of N, N-dimethylformamide for dissolving, adding 791.9mg of sodium hydride, stirring at room temperature for 30 minutes, then adding 3.90mL of chloroacetyl morpholine, stirring at room temperature for reaction for 12 hours, performing thin-layer detection reaction, then performing spin-drying on the solvent, adding 20mL of water and 20mL of dichloromethane for extraction, separating an organic phase, washing with 20mL of water twice, drying with anhydrous sodium sulfate, performing spin-drying on the solvent to obtain a white solid, and performing vacuum drying to obtain an intermediate compound 2;

(2) weighing 4.53g of intermediate compound 2, placing the intermediate compound 2 in a 250mL flask, adding 50mL of dichloromethane for dissolving, then adding 5.61g N-bromosuccinimide in batches, stirring at room temperature for reaction for 12 hours, adding 30mL of water for quenching reaction after the thin-layer detection reaction is completed, washing an organic phase by 30mL of water twice, drying the organic phase by anhydrous sodium sulfate, carrying out Flash column chromatography after solvent is dried in a spinning mode to obtain a white solid, and carrying out vacuum drying to obtain an intermediate 3;

(3) weighing 92.0mg of intermediate compound 3, placing the intermediate compound in a 25mL microwave reactor, adding 10mL of redistilled dioxane for dissolution, and adding 420 mu mol of different substituted phenylboronic acid, 11.6mg of tetrakis (triphenylphosphine) palladium and 89.2mg of tripotassium phosphate; reacting for 30 minutes at 150 ℃ in a microwave reactor, detecting the reaction through a thin layer, filtering the reaction liquid by using kieselguhr after the reaction is completed, spin-drying the solvent, dissolving the solid by using 10mL of dichloromethane, washing the solid twice by using 10mL of water, drying the anhydrous sodium sulfate, spin-drying the solvent, carrying out Flash column chromatography, and recrystallizing the chromatographic product by using ethanol to obtain a target product a 01;

(4) weighing 200.00 mu mol of compound a01 in a 25mL flask, adding 15mL of methanol for dissolving, heating for refluxing, slowly dropwise adding 1M of sodium hydroxide solution into the reaction solution, monitoring the pH value of the solution in real time until the pH value is stabilized above 10, stopping dropwise adding the sodium hydroxide solution, detecting the reaction completion by using a thin-layer plate, adjusting the pH value to 1 by using 1M of hydrochloric acid, then spin-drying the solvent, adding 10mL of ethyl acetate for dissolving, washing twice by using 10mL of water, drying by using anhydrous sodium sulfate, spin-drying the solvent, and recrystallizing by using ethanol to obtain a target product b 01;

and a second route:

(1) weighing 92.0mg of intermediate 3 into a 50mL microwave reactor, adding 10mL of redistilled dioxane to dissolve, and adding 24.4mg of phenylboronic acid, 11.6mg of tetrakis (triphenylphosphine) palladium and 46.7mg of tripotassium phosphate; reacting for 30 minutes at 150 ℃ in a microwave reactor to obtain an intermediate 4;

(2) 220.00 mu mol of p-methoxyphenylboronic acid, 11.6mg of tetrakis (triphenylphosphine) palladium and 46.7mg of tripotassium phosphate are added into the reaction solution of the previous reaction; reacting in a microwave reactor at 150 ℃ for 30 minutes, detecting by TLC (thin layer chromatography) to complete the reaction, filtering the reaction liquid by using kieselguhr, spin-drying the solvent, dissolving the solid by using 20mL of ethyl acetate, washing by using 20mL of water twice, drying by using anhydrous sodium sulfate, spin-drying the solvent, carrying out Flash column chromatography, and recrystallizing the chromatographic product by using ethanol to obtain a target product a 02;

(3) weighing 200.00 mu mol of compound a02 in a 25mL flask, adding 15mL of methanol for dissolving, heating for refluxing, slowly dropwise adding 1M of sodium hydroxide solution into the reaction solution, monitoring the pH value of the solution in real time until the pH value is stabilized above 10, stopping dropwise adding the sodium hydroxide solution, detecting the reaction completion by using a thin-layer plate, adjusting the pH value to 1 by using 1M of hydrochloric acid, then spin-drying the solvent, dissolving in 10mL of ethyl acetate, washing twice by using 10mL of water, drying by using anhydrous sodium sulfate, spin-drying the solvent, and recrystallizing by using ethanol to obtain the target product b 02.

6. Substituted indole derivatives as claimed in any of claims 1 to 3 for use as HCV inhibitors for the preparation of anti-hepatitis c medicaments.

7. An anti-HCV pharmaceutical composition comprising a substituted indole derivative according to any of claims 1 to 3 and one or more pharmaceutically acceptable carriers or excipients.