CN107915726B - Novel 3, 5-disubstituted 1H-indole derivative and synthesis and application thereof - Google Patents

Abstract

The invention discloses a novel 3,5-disubstituted 1H-indole derivative and its synthesis and application. Its structural formula is shown in formula 11, wherein, R is selected from halogen, C ₁ -C ₆ methyl, C ₁ -C ₆ methoxy, -OH, -COOH, -SO ₃ H or -SO ₂ NH ₂ . The present invention also provides a method for preparing the compound, which consists of eight steps, the reaction yield of each step is higher than 60%, and the reaction yield of four steps is higher than 90%, and the purity of all compounds of the present invention is higher than 95% , in line with the purity required for the later anti-tumor cell activity test. The compound 11a-11h prepared by the present invention has good inhibitory activity on pancreatic cancer cell line BxPC-3, wherein the compound represented by formula 11e has the best activity, IC ₅₀ is as high as 2.28 μmol/L, and the compound is normal to human Cells have almost no toxic side effects.

Description

Novel 3,5-disubstituted 1H-indole derivatives and their synthesis and application

technical field

The present invention relates to a novel 3,5-disubstituted 1H-indole derivative and its synthesis and application.

Background technique

Cancer is the second leading cause of death after cardiovascular disease, accounting for nearly one in six deaths globally. In recent years, the incidence and mortality of cancer have continued to rise. In 2015, there were approximately 4.292 million new cases of malignant tumors and 2.814 million deaths in my country, accounting for nearly 22% of new cases and 27% of deaths in the world, respectively.

At present, the commonly used drugs for clinical treatment of malignant tumors mainly include traditional cytotoxic drugs, such as doxorubicin, 5-fluorouracil, cyclophosphamide, etc., as well as targeted drugs, such as sorafenib, gefitinib, crizotitinib Ni et al. Although these drugs have good clinical efficacy in the treatment of various tumors, cytotoxic drugs are generally limited in clinical use due to their high toxicity and poor patient tolerance. Targeted drugs are usually due to individual differences. The lack of large and serious drug resistance can not achieve a very satisfactory clinical effect.

In view of this, the development of new anti-tumor drug research is still one of the key tasks of global new drug development tasks.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a novel 3,5-disubstituted 1H-indole derivative and its synthesis and application.

The present invention provides a compound represented by formula 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof:

In the formula, R is selected from halogen, C ₁ -C ₆ methyl, C ₁ -C ₆ methoxy, -OH, -COOH, -SO ₃ H or -SO ₂ NH ₂ .

Preferably, the position of the R substituent is 3-position substitution or 4-position substitution of the compound represented by formula 11.

Preferably, the R is selected from halogen, C ₁ -C ₄ methyl or C ₁ -C ₄ methoxy.

Further preferably, the structure of the compound is:

The present invention provides a method for preparing the aforementioned compound, which comprises the following steps:

(1) adding POCl ₃ to DMF, stirring for 0.5h, adding the compound shown in formula 1 to obtain the compound shown in formula 2;

(2) adding the compound shown in formula 2, di-tert-butyl dicarbonate and 4-dimethylaminopyridine into tetrahydrofuran to obtain the compound shown in formula 3;

(3) adding the compound shown in formula ₃ and NaBH to methanol successively, and quenching to obtain the compound shown in formula 4;

(4) sequentially adding the compound shown in formula 4 and thionyl chloride to DCM, reacting for 1.5h, and quenching to obtain the compound shown in formula 5;

(5) adding the compound represented by formula 5, the compound represented by formula 6 and K ₂ CO ₃ into DMF to obtain the compound represented by formula 7;

(6) adding the compound shown in formula 7, iron powder and NH ₄ Cl into absolute ethanol and water to obtain the compound shown in formula 8;

(7) adding the compound represented by formula 8, the compound represented by formula 9, HOBT, EDCI and DIEA into DCM in turn to obtain the compound represented by formula 10;

(8) The compound represented by formula 10 and trifluoroacetic acid were added to DCM to prepare the target compound 11.

Preferably, in step (1), the temperature at which the POCl ₃ is added to the DMF is 0° C.; the molar ratio of the compound shown in formula 1, POCl ₃ , and DMF is 1:8:10; The compound shown is obtained by adjusting the pH of the obtained reaction solution after the reaction to 7, extracting and concentrating in turn; the pH adjustment is to adjust the pH by using 6N NaOH; the extraction is to extract 3 times by using ethyl acetate;

In step (2), the reaction temperature is 25±2°C; the reaction time is 3h; the feed ratio of the compound shown in formula 2, di-tert-butyl dicarbonate, 4-dimethylaminopyridine and tetrahydrofuran is: 18.9:20.8:1:76.9mmol/mmol/mmol/ml; The compound formula shown in the formula 3 is obtained by successively concentrating and recrystallizing the reacted reaction solution;

In step (3), the reaction temperature is 25±2°C; the reaction time is 0.5h; the feed ratio of the compound shown in formula 3, NaBH ₄ , and methanol is 1:2.16:5.26mmol/mmol/ml The quenching is to utilize acetone to quench the reaction; the compound shown in the formula 4 is obtained by concentration and column chromatography; the column chromatography is to take PE:EA=3:1 as the eluent;

In step (4), the reaction temperature is 25±2°C; the feed ratio of the compound shown in formula 4, thionyl chloride and DCM is 1:4:4.2mmol/mmol/ml; the quenching is Add water to quench the reaction; the compound shown in formula 5 is obtained by extraction, drying, concentration, and column chromatography; the extraction is to extract 3 times with DCM; the drying is to use anhydrous magnesium sulfate to dry; the column layer The analysis is based on PE:EA=4:1 as the eluent;

In step (5), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in formula 5, K ₂ CO ₃ , the compound shown in formula 6, and DMF is 1:2 : 1.23:3.85mmol/mmol/mmol/ml; The compound shown in the formula 7 is obtained by dilution, extraction, washing, drying, concentration, column chromatography; The dilution is to add 10 times of water dilution; The extraction is Utilize ethyl acetate extraction 3 times; Described washing utilizes saturated brine to wash; Described drying utilizes anhydrous magnesium sulfate to dry; Described column chromatography uses PE:EA=4:1 as eluent;

In step (6), described reaction temperature is 80 ± 2 ℃; Described reaction time is 0.5h; Described compound shown in formula 7, iron powder, NH ₄ Cl, dehydrated alcohol, the feed ratio of water is 1: 5:0.48:12.9:6.45mmol/mmol/mmol/ml/ml; the compound shown in the formula 8 is obtained by filtering, concentrating, diluting, extracting, washing, drying, concentrating, and column chromatography; the dilution is adding water Dilution; the extraction is to use ethyl acetate to extract 3 times; the washing is to use saturated brine to wash; the drying is to use anhydrous magnesium sulfate to dry; the column chromatography is PE:EA=1:1 eluent;

In step (7), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in the formula 8, the compound shown in the formula 9, HOBT, EDCI, DIEA, DCM is 0.69: 1:1:1:2.13:7.5mmol/mmol/mmol/mmol/mmol/ml; the compound shown in the formula 10 is obtained by concentrating, washing, extracting, washing, drying, concentrating, and column chromatography; the washing It is washed with saturated sodium bicarbonate; the extraction is with ethyl acetate for 3 times; the washing is with saturated brine; the drying is with anhydrous magnesium sulfate; the column chromatography is with DCM: MeOH=60:1 is the eluent;

In step (8), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in formula 10, trifluoroacetic acid and DCM is 1:0.94:9.4mmol/ml/ml ; Described target compound 11 is obtained by adjusting pH to be 8, extraction, drying, concentration, column chromatography; Described adjusting pH is to utilize saturated sodium bicarbonate to regulate; Described extraction utilizes ethyl acetate extraction 3 times; Described Drying was over anhydrous magnesium sulfate; the column chromatography was eluent with DCM:MeOH=20:1.

Use of the compound represented by Formula 11, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolite, in the preparation of a medicament for treating pancreatic cancer.

The present invention discloses a pharmaceutical combination, which uses the aforementioned compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolite as an active ingredient , and pharmaceutically acceptable auxiliary materials or auxiliary components are added to prepare a commonly used pharmaceutical preparation.

In the present invention:

DCM is dichloromethane.

DMF is dimethylformamide.

HOBT is 1-hydroxybenzotriazole.

EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

DIEA is N,N-diisopropylethylamine.

The "room temperature" in the present invention is 25±2°C.

The "overnight" in the present invention is 12±1h.

The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The terms "salts" and "pharmaceutically acceptable salts" refer to the above-mentioned compounds or their stereoisomers, acid and/or base salts with inorganic and/or organic acids and bases, and also zwitterionic salts (internal). salts), also including quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (for example, an equivalent amount). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

The present invention provides a novel 3,5-disubstituted 1H-indole derivative, and also provides a method for preparing the compound, which involves Vilsmeier-Haack reaction (Vilsmeier-Haack), tert-butyl amine of secondary amines Oxycarbonyl (Boc) protection, reduction of aldehyde, condensation of carboxylic acid and amine and other eight-step reactions, the yield of each step is higher than 60%, and the yield of four-step reaction is higher than 90%, all compounds of the present invention The purity is greater than 95%, which is in line with the purity required for the later anti-tumor cell activity test. The compounds 11a-11h prepared by the present invention all have good inhibitory activity on the pancreatic cancer cell line BxPC-3, among which the compound represented by the formula 11e has the best activity, and the IC ₅₀ is as high as 2.28 μmol/L, and the compound is normal to human beings. Cells have almost no toxic side effects.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Detailed ways

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.

The synthesis of embodiment 1 target compound

1. Synthesis of Intermediate 2

At 0 °C, POCl ₃ (9.2 ml, 98.5 mmol) was added dropwise to DMF (9.5 ml, 123.1 mmol), and after stirring for 0.5 h, 5-nitro-1H-indole (compound 1, 2.0 g , 12.3 mmol) was added to the reaction solution, followed by reaction at room temperature for 1.5 h. Then the reaction solution was poured into a large amount of ice water and adjusted to pH 7 with 6N NaOH, extracted with ethyl acetate (×3), and concentrated to obtain 2.34 g of a gray solid with a yield of 98% and a purity of 95.2%. LCMS m/z: 191.0 [M+H] ⁺ .

2. Synthesis of Intermediate 3

Compound 2 (2.34g, 12.3mmol), di-tert-butyl dicarbonate (2.95g, 13.5mmol), 4-dimethylaminopyridine (75.8mg, 0.62mmol) were added to 50ml of tetrahydrofuran, and reacted at room temperature 3h, then concentrated, and recrystallized from ethyl acetate to obtain 3.3 g of a pale yellow solid with a yield of 92% and a purity of 97.0%. LCMS m/z: 291.1 [M+H] ⁺ .

3. Synthesis of Intermediate 4

In an ice bath, compound 3 (3.3 g, 11.4 mmol) was added to 60 ml of methanol, then NaBH ₄ (0.93 g, 24.6 mmol) was added, and the reaction was moved to room temperature for 0.5 h. Finally, the reaction was quenched with acetone, concentrated, and separated on a silica gel column (PE:EA=3:1) to obtain 3.03 g of a brown-yellow solid with a yield of 91% and a purity of 96.57%. LCMS m/z: 293.0 [M+H] ⁺ .

4. Synthesis of Intermediate 5

Compound 4 (2.78g, 9.5mmol) was added to 40ml DCM, then at 0°C, thionyl chloride (2.75ml, 37.8mmol) was added to the above reaction solution, and the reaction solution was moved to room temperature to react for 1.5h . Finally, water was added to quench the reaction, extracted with DCM (×3), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by silica gel column (PE:EA=4:1) to obtain 2.0 g of white solid with a yield of 67% and a purity of 95.7 %. LCMS m/z: 311.1 [M+H] ⁺ .

5. Synthesis of Intermediate 7

1) Synthesis of intermediate 7e

Compound 5 (400 mg, 1.3 mmol), K ₂ CO ₃ (357 mg, 2.6 mmol) and p-fluorothiophenol (Compound 6, 165 μl, 1.6 mmol) were added to 5 ml of DMF, respectively, and reacted at room temperature overnight. Then it was diluted with 10 times of water, extracted with ethyl acetate (×3), washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated, and separated by silica gel column (PE:EA=4:1) to obtain 476 mg of solid with a yield of 92%, The purity was 96.0%. LCMS m/z: 403.1 [M+H] ⁺ .

2) Synthesis of intermediates 7a-7j

The method for synthesizing intermediates 7a-7j is the same as the method for synthesizing intermediate 7e, wherein 6a-6j correspond to 6e, as shown in Table 1, intermediates 7a-7j are finally obtained.

The yield and purity of raw materials and products used in the synthesis of intermediates 7a to 7f in Table 1

6. Synthesis of Intermediate 8

1) Synthesis of intermediate 8e

Compound 7e (250 mg, 0.62 mmol), iron powder (174 mg, 3.1 mmol) and NH ₄ Cl (17 mg, 0.3 mmol) were added to absolute ethanol (8 ml) and water (4 ml), respectively, and reacted at 80 ° C for 0.5 h. After the reaction was completed, filter while hot, concentrated, diluted with water, extracted with ethyl acetate (×3), washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated, and separated by silica gel column (PE:EA=1:1), 196 mg of transparent viscous liquid was obtained with a yield of 85% and a purity of 95.3%. LCMS m/z: 373.1 [M+H] ⁺ .

2) Synthesis of intermediates 8a～8j

The method for synthesizing intermediates 8a-8j is the same as that for synthesizing intermediate 8e, wherein 7a-7j correspond to 7e, as shown in Table 2, intermediates 8a-8j are finally obtained.

The yield and purity of raw materials and products used in the synthesis of intermediates 8a～8f of Table 2

7. Synthesis of Intermediate 10

1) Synthesis of intermediate 10e

1-Methyl-1H-pyrazole-4-carboxylic acid (9, 101 mg, 0.8 mmol), compound 8e (200 mg, 0.55 mmol), HOBT (117 mg, 0.8 mmol), EDCI (154 mg, 0.8 mmol) and DIEA ( 266 μl, 1.7 mmol) was added to 6 ml of DCM under ice bath, and then the reaction solution was moved to room temperature to react overnight. After the reaction was completed, it was concentrated, washed with saturated sodium bicarbonate, extracted with ethyl acetate (×3), washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated, and separated by silica gel column (DCM:MeOH=60:1) to obtain 225 mg of white Solid, 87% yield, 98.1% purity. LCMS m/z: 463.2 [M+H] ⁺ .

2) Synthesis of intermediates 10a-10j

The method for synthesizing intermediates 10a-10j is the same as that for synthesizing intermediate 10e, wherein 8a-8j correspond to 8e, as shown in Table 3, intermediates 10a-10j are finally obtained.

The yield and purity of raw materials and products of table 3 synthetic intermediates 10a～10f

8. Synthesis of target compound 11

1) Synthesis of target compound 11e

Compound 10e (154 mg, 0.32 mmol) and trifluoroacetic acid (300 μl, 10%) were added to 3 ml of DCM and then reacted at room temperature overnight. After the reaction was completed, adjust pH=8 with saturated sodium bicarbonate, extract with ethyl acetate (×3), dry over anhydrous magnesium sulfate, concentrate, and separate on silica gel column (DCM:MeOH=20:1) to obtain 82 mg of white solid, with a yield of 60 %, the purity is 96.7%.

It is characterized by:

LCMS m/z: 363.1 [M+H] ⁺ .

¹ H NMR(400MHz, DMSO)δ10.91(s,1H), 9.73(s,1H), 8.30(s,1H), 8.05(s,1H), 7.97(s,1H), 7.48-7.36(m ,3H),7.32(d,J＝8.4Hz,1H),7.24(d,J＝2.0Hz,1H),7.16(t,J＝8.8Hz,2H),4.37(s,2H),3.91(s , 3H)ppm.

¹³ C NMR (100 MHz, DMSO) δ 162.4, 160.7, 139.2, 133.7, 132.9, 132.8, 131.6, 131.5, 131.4, 126.8, 125.7, 119.4, 116.8, 116.5, 116.3, 111.8, 111.0, 30.3, 109.7, 39 ppm

96.7% HPLC purity.

2) Synthesis of target compound 11a

The synthetic method of target compound 11a is the same as that of target compound 11e, wherein the amount of 10a corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) yields a white solid with a yield of 63% and a purity of 97.6%.

It is characterized by:

LCMS m/z: 363.1[M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.96(s,1H), 9.73(s,1H), 8.30(s,1H), 8.04(s,1H), 7.98(s,1H), 7.44(d,J =8.8Hz,1H),7.34(t,J=12.0Hz,3H),7.24(d,J=10.0Hz,1H),7.19(d,J=8.4Hz,1H),6.99(td,J=8.8 ,2.4Hz,1H),4.46(s,2H),3.91(s,3H)ppm;

¹³ C NMR (100MHz, DMSO) δ164.0, 160.7, 140.7, 139.2, 133.7, 132.8, 131.4, 131.1, 126.8, 125.9, 123.8, 119.4, 116.8, 114.4, 112.5, 111.8, 111.0, 109.1, 39 ppm;

97.6% HPLC purity.

3) Synthesis of target compound 11b

The synthetic method of target compound 11b is the same as that of target compound 11e, wherein the amount of 10b corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) gives a white solid with a yield of 65% and a purity of 98.1%.

It is characterized by:

LCMS m/z: 397.1 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.95(s,1H), 9.72(s,1H), 8.30(s,1H), 8.04(s,1H), 7.98(s,1H), 7.44(d,J =9.2Hz,2H),7.36-7.28(m,4H),7.26-7.18(m,1H),4.46(s,2H),3.91(s,3H)ppm;

¹³ C NMR (100MHz, DMSO) δ160.7, 140.5, 139.2, 134.0, 133.7, 132.8, 131.4, 130.9, 127.1, 126.8, 126.6, 125.9, 125.6, 119.4, 116.9, 111.8, 111.0, 109.1, 39 ppm;

98.1% HPLC purity.

4) Synthesis of target compound 11c

The synthetic method of target compound 11c is the same as that of target compound 11e, wherein the amount of 10c corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) yields a white solid with a yield of 65% and a purity of 97%.

It is characterized by:

LCMS m/z: 377.1 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.92(s,1H), 9.73(s,1H), 8.30(s,1H), 8.04(s,1H), 7.97(s,1H), 7.44(d,J =8.8Hz,1H),7.33-7.30(m,2H),7.22-7.16(m,3H),6.98(d,J=6.8Hz,1H),4.39(s,2H),3.91(s,3H) ,2.29(s,3H)ppm;

¹³ C NMR (100MHz, DMSO)δ160.7,139.2,138.7,137.6,133.7,132.8,131.4,129.2,128.8,126.9,126.6,125.7,125.4,119.4,116.8,111.7,111.0,109.7,39ppm ;

97.3% HPLC purity.

5) Synthesis of target compound 11d

The synthetic method of target compound 11d is the same as that of target compound 11e, wherein the amount of 10d corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) gives a white solid with a yield of 60% and a purity of 97%.

It is characterized by:

LCMS m/z: 393.1 [M+H] ⁺ ;

¹ H NMR(400MHz,DMSO)δ10.93(s,1H), 9.72(s,1H), 8.29(s,1H), 8.04(s,1H), 7.98(s,1H), 7.44(d,J ＝8.8Hz,1H),7.32(d,J＝9.2Hz,2H),7.23(t,J＝8.0Hz,1H),6.94(d,J＝7.6Hz,1H),6.90(s,1H), 6.74(d,J=10.0Hz,1H),4.41(s,2H),3.91(s,3H),3.74(s,3H)ppm;

¹³ C NMR (100MHz, DMSO) δ 160.7, 160.0, 139.2, 139.1, 133.7, 132.8, 131.4, 130.2, 126.9, 125.7, 120.3, 119.4, 116.8, 113.4, 111.7, 111.0, 109.6, 55.5, 39 ppm;

97.0% HPLC purity.

6) Synthesis of target compound 11f

The synthetic method of target compound 11f is the same as that of target compound 11e, wherein the amount of 10f corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) yields a white solid with a yield of 65% and a purity of 97.8%.

It is characterized by:

LCMS m/z: 397.1 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.93(s,1H), 9.71(s,1H), 8.29(s,1H), 8.02(s,1H), 7.95(s,1H), 7.44-7.27(m ,7H),4.40(s,2H),3.90(s,3H)ppm;

97.8% HPLC purity.

7) Synthesis of target compound 11g

The synthetic method of target compound 11g is the same as that of target compound 11e, wherein the amount of 10g corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) gives a white solid with a yield of 66% and a purity of 98.2%.

It is characterized by:

LCMS m/z: 377.1 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO)δ10.93(s,1H), 9.71(s,1H), 8.29(s,1H), 8.02(s,1H), 7.95(s,1H), 7.51-7.45(m ,2H),7.42(dd,J=8.8,1.6Hz,1H),7.35-7.27(m,4H),4.40(s,2H),3.90(s,3H),3.35(s,3H)ppm;

98.2% HPLC purity.

8) Synthesis of target compound 11h

The synthetic method of target compound 11h is the same as that of target compound 11e, wherein the amount of 10h corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) yields a white solid with a yield of 63% and a purity of 97.6%.

It is characterized by:

LCMS m/z: 393.1 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.87(s,1H), 9.73(s,1H), 8.31(s,1H), 8.05(s,1H), 7.96(s,1H), 7.44(d,J ＝8.8Hz,1H),7.31(d,J＝8.8Hz,3H),7.16(d,J＝2.4Hz,1H),6.90(d,J＝8.8Hz,2H),4.27(s,2H), 3.91(s,3H),3.74(s,3H)ppm;

¹³ C NMR (100MHz, DMSO)δ160.7,158.7,139.2,133.7,132.8,132.4,131.3,127.4,126.8,125.6,119.5,116.8,115.1,111.7,111.1,110.3,55.6,39.3,31.1ppm;

97.6% HPLC purity.

9) Synthesis of target compound 11i

The synthetic method of target compound 11i is the same as that of target compound 11e, wherein the amount of 10i corresponds to 10e, and silica gel column separation (DCM:MeOH=20:1) yields a white solid with a yield of 62% and a purity of 97.5%.

It is characterized by:

LCMS m/z: 441.1 [M+H] ⁺ ;

¹ H NMR(400MHz,DMSO)δ10.97(s,1H),9.74(s,1H),8.30(s,1H),8.05(s,1H),7.99(s,1H),7.55(s,1H) ), 7.45(d, J=8.8Hz, 1H), 7.40-7.30(m, 4H), 7.26(t, J=8.0Hz, 1H), 4.46(s, 2H), 3.91(s, 3H) ppm;

¹³ C NMR (100MHz, DMSO) δ160.7, 140.8, 139.2, 133.7, 132.8, 131.4, 131.2, 130.0, 128.5, 127.0, 126.8, 125.9, 122.6, 119.4, 116.9, 111.8, 111.0, 109.1, 39 ppm;

97.8% HPLC purity.

10) Synthesis of target compound 11j

LCMS m/z: 441.1 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO)δ10.89(s,1H),9.71(s,1H),8.29(s,1H),8.03(s,1H),7.94(d,J=1.6Hz,1H), 7.43(dd,J=8.8,2.0Hz,1H),7.33-7.21(m,4H),7.12(d,J=8.0Hz,2H),4.33(s,2H),3.90(s,3H)ppm;

97.9% HPLC purity.

The beneficial effects of the present invention are described below by means of experimental examples:

Experimental example 1

1. Experimental materials

DMEM: DMEM medium

10% FBS: Fetal Bovine Serum

DMSO: Dimethyl Sulfoxide

Experimental tumor cell line: pancreatic cancer BxPC-3 cell line, human normal hepatocyte HL-7702

2. Preparation and processing of cells

First, the cell line was inoculated into a 96-well plate containing DMEM+10% FBS medium, and cultured overnight at 37°C, 5% CO ₂ . The next day, the compound to be tested at a gradient concentration diluted in the medium and the blank control were added to the culture plate for 72 hours, and then 20 μl of MTT reagent with a concentration of 5 mg/mL was added to each well, and the culture was continued for 2-4 hours. After the formazan was formed, the supernatant was aspirated, and 150 μL of DMSO was added to each well, and the formed formazan was fully dissolved by shaking.

Finally, according to the inhibition rate=(OD570 of blank control group-OD570 of experimental group)/OD570 of blank control group, the inhibition rate of the compound on various cells was calculated, and the _IC50 value was obtained by fitting with Graphpad software.

3. Experimental results

3.1 Inhibition of tumor cell activity

We selected the BxPC-3 pancreatic cancer cell line and tested the proliferation inhibitory activity of 10 target compounds on these two groups of cell lines. The test results are shown in Table 4.

Table 4 Inhibitory activity of target compounds 11a-11j on pancreatic cancer cells

It can be seen from Table 4 that the target compounds 11a-11j have good proliferation inhibitory activity (IC ₅₀ <10 μmol/L) on the BxPC-3 cell line, and the phenyl 4-position substitution in these compounds is higher than that of the phenyl 3- The suppression effect of position substitution is better (11e is better than 11a, 11f is better than 11b, 11g is better than 11c, 11h is better than 11d, 11j is better than 11i,).

Among all 10 target compounds, 11e had the best proliferation inhibitory activity on BxPC-3 cell line, with IC ₅₀ as high as 2.28μmol/L.

3.2 Compound toxicity

In order to verify whether the target compound prepared in the present invention is a cytotoxic compound and preliminarily evaluate the toxicity of the compound, we tested the inhibitory activity of the target compound 11e on various tumor cells and human normal hepatocytes HL-7702 at a concentration of 50 μmol/L , and the test results are shown in Table 5.

Table 5 Inhibitory activity of 11e in target compounds on various tumor cells and human normal hepatocytes HL-7702

It can be seen from Table 5 that compound 11e only has an inhibitory rate of up to 91±5% on pancreatic cancer cell line BxPC-3 at 50 μmol/L, while on other tumor cells such as breast cancer cells MDA-MB-231, mantle cells Lymphoma cells JeKo-1, lung cancer cells PC-9 and liver cancer cells HUH7 have almost no inhibitory activity. It was shown that the target compound 11e specifically inhibited the proliferation of pancreatic cancer cell line BxPC-3, rather than the inhibition of cell proliferation caused by cytotoxicity. In addition, the inhibitory rate of 11e on human normal hepatocytes HL-7702 at a concentration of 50 μmol/L was only 17±2%, indicating that the compound has almost no toxic and side effects on human normal cells. In view of the above results, the target compound 11e is a better anti-pancreatic cancer hit compound.

In conclusion, the present invention provides a novel 3,5-disubstituted 1H-indole derivative, and also provides a method for preparing the compound, which involves Vilsmeier-Haack reaction, secondary amine The eight-step reaction of tert-butoxycarbonyl (Boc) protection, reduction of aldehyde, condensation of carboxylic acid and amine, etc., the yield of each step is higher than 60%, and the yield of four-step reaction is higher than 90%. The purity of all compounds in the invention is greater than 95%, which meets the purity required for the later anti-tumor cell activity test. The compounds 11a-11h prepared by the present invention all have good inhibitory activity on the pancreatic cancer cell line BxPC-3, among which the compound represented by the formula 11e has the best activity, and the IC ₅₀ is as high as 2.28 μmol/L, and the compound is normal to human beings. Cells have almost no toxic side effects.

Claims (8)

1. A compound represented by formula 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

In the formula, R is selected from halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, -OH, -COOH, -SO ₃ H or -SO ₂ NH ₂ . 2 . The compound according to claim 1 , wherein the position of the R substituent is 3-position substitution or 4-position substitution of the compound represented by formula 11. 3 . 3 . The compound according to claim 1 or 2 , wherein the R is selected from halogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy. 4 . 4. The compound according to claim 3, wherein the structure of the compound is:

5. A method for preparing the compound according to any one of claims 1 to 4, characterized in that: it comprises the following steps:

(1) adding POCl ₃ to DMF, stirring for 0.5h, adding the compound shown in formula 1 to obtain the compound shown in formula 2; (2) adding the compound shown in formula 2, di-tert-butyl dicarbonate and 4-dimethylaminopyridine into tetrahydrofuran to obtain the compound shown in formula 3; (3) adding the compound shown in formula ₃ and NaBH to methanol successively, and quenching to obtain the compound shown in formula 4; (4) sequentially adding the compound shown in formula 4 and thionyl chloride to DCM, reacting for 1.5h, and quenching to obtain the compound shown in formula 5; (5) adding the compound represented by formula 5, the compound represented by formula 6 and K ₂ CO ₃ into DMF to obtain the compound represented by formula 7; (6) adding the compound shown in formula 7, iron powder and NH ₄ Cl into absolute ethanol and water to obtain the compound shown in formula 8; (7) adding the compound represented by formula 8, the compound represented by formula 9, HOBT, EDCI and DIEA into DCM in turn to prepare the compound represented by formula 10; (8) The compound represented by formula 10 and trifluoroacetic acid were added to DCM to prepare the target compound 11. 6. preparation method according to claim 5, is characterized in that: In step (1), the temperature at which the POCl ₃ is added to the DMF is 0° C.; the molar ratio of the compound shown in formula 1, POCl ₃ , and DMF is 1:8:10; the compound shown in formula 2 is It is obtained by adjusting the pH of the reaction solution obtained after the reaction to 7, extracting and concentrating in turn; the pH adjustment is to adjust the pH by using 6N NaOH; the extraction is to use ethyl acetate to extract 3 times; In step (2), the reaction temperature is 25±2°C; the reaction time is 3h; the feed ratio of the compound shown in formula 2, di-tert-butyl dicarbonate, 4-dimethylaminopyridine and tetrahydrofuran is: 18.9:20.8:1:76.9mmol/mmol/mmol/ml; The compound formula shown in the formula 3 is obtained by successively concentrating and recrystallizing the reacted reaction solution; In step (3), the reaction temperature is 25±2°C; the reaction time is 0.5h; the feed ratio of the compound shown in formula 3, NaBH ₄ , and methanol is 1:2.16:5.26mmol/mmol/ml The quenching is to utilize acetone to quench the reaction; the compound shown in the formula 4 is obtained by concentration and column chromatography; the column chromatography is to take PE:EA=3:1 as the eluent; In step (4), the reaction temperature is 25±2°C; the feed ratio of the compound shown in formula 4, thionyl chloride and DCM is 1:4:4.2mmol/mmol/ml; the quenching is Add water to quench the reaction; the compound shown in formula 5 is obtained by extraction, drying, concentration, and column chromatography; the extraction is to extract 3 times with DCM; the drying is to use anhydrous magnesium sulfate to dry; the column layer The analysis is based on PE:EA=4:1 as the eluent; In step (5), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in formula 5, K ₂ CO ₃ , the compound shown in formula 6, and DMF is 1:2 : 1.23:3.85mmol/mmol/mmol/ml; The compound shown in the formula 7 is obtained by dilution, extraction, washing, drying, concentration, column chromatography; The dilution is to add 10 times of water dilution; The extraction is Utilize ethyl acetate extraction 3 times; Described washing utilizes saturated brine to wash; Described drying utilizes anhydrous magnesium sulfate to dry; Described column chromatography uses PE:EA=4:1 as eluent; In step (6), described reaction temperature is 80 ± 2 ℃; Described reaction time is 0.5h; Described compound shown in formula 7, iron powder, NH ₄ Cl, dehydrated alcohol, the feed ratio of water is 1: 5:0.48:12.9:6.45mmol/mmol/mmol/ml/ml; the compound shown in the formula 8 is obtained by filtering, concentrating, diluting, extracting, washing, drying, concentrating, and column chromatography; the dilution is adding water Dilution; the extraction is to use ethyl acetate to extract 3 times; the washing is to use saturated brine to wash; the drying is to use anhydrous magnesium sulfate to dry; the column chromatography is PE:EA=1:1 eluent; In step (7), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in the formula 8, the compound shown in the formula 9, HOBT, EDCI, DIEA, DCM is 0.69: 1:1:1:2.13:7.5mmol/mmol/mmol/mmol/mmol/ml; the compound shown in the formula 10 is obtained by concentrating, washing, extracting, washing, drying, concentrating, and column chromatography; the washing It is washed with saturated sodium bicarbonate; the extraction is with ethyl acetate for 3 times; the washing is with saturated brine; the drying is with anhydrous magnesium sulfate; the column chromatography is with DCM: MeOH=60:1 is the eluent; In step (8), the reaction temperature is 25±2°C; the reaction time is 12h; the feed ratio of the compound shown in formula 10, trifluoroacetic acid and DCM is 1:0.94:9.4mmol/ml/ml ; Described target compound 11 is obtained by adjusting pH to be 8, extraction, drying, concentration, column chromatography; Described adjusting pH is to utilize saturated sodium bicarbonate to regulate; Described extraction utilizes ethyl acetate extraction 3 times; Described Drying was over anhydrous magnesium sulfate; the column chromatography was eluent with DCM:MeOH=20:1. 7. Use of the compound represented by formula 11 according to any one of claims 1 to 4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating pancreatic cancer. 8. A pharmaceutical combination, characterized in that: it uses the compound described in any one of claims 1 to 4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, plus a pharmaceutically acceptable compound. A formulation prepared from an accepted accessory ingredient.