CN114751910B - Compound capable of inducing cell megalophage death and preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound capable of inducing apoptosis of macrovacuoles as well as a preparation method and application thereof, wherein the compound is 2-chloro-N- (4- (pyrazole [1,5-a ] pyrimidine-6-yl) phenyl) acetamide. The 2-chloro-N- (4- (pyrazole [1,5-a ] pyrimidine-6-yl) phenyl) acetamide of the invention has the effect of inducing tumor cell death, and the cell death is caused by the formation of a plurality of huge vacuoles in cells induced by the compound and is called macrovesicular death. The compound has higher clinical application value and good development prospect, and can be used for treating tumor diseases. The preparation method is simple and convenient and is suitable for synthesizing the compound in large quantity.

Description

A compound capable of inducing cell macrovesicle death and its preparation method and application

technical field

The invention belongs to the technical field of medicinal chemistry, and specifically relates to a compound capable of inducing macrovesicle death of cells, a preparation method and application thereof.

Background technique

Malignant tumors are major diseases that seriously threaten human life and health. At present, most chemotherapeutic drugs kill tumor cells through the mechanism of apoptosis to achieve therapeutic effects. Evolution makes itself continuously adapt to the external environment and gradually develop resistance to chemotherapy drugs, which restricts the clinical application of chemotherapy drugs in tumor treatment. Methuosis is a new form of non-apoptotic cell death discovered in recent years. Its main feature is the accumulation of a large number of transparent vacuoles in the cytoplasm, but it does not have the general characteristics of apoptosis. Therefore, the development of antitumor drugs that induce cell macrovesicle death has important scientific significance and clinical value for improving the chemotherapy effect of malignant tumors.

Contents of the invention

Aiming at the above prior art, the present invention provides a compound capable of inducing cell macrovesicle death and its preparation method and application, so as to solve the problem of drug resistance caused by chemotherapy drugs that kill tumor cells by the existing apoptosis mechanism.

In order to achieve the above object, the technical scheme adopted in the present invention is to provide a compound capable of inducing macrovesicle death, which is 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidine-6 -base) phenyl) acetamide, its chemical formula is

The present invention also provides a preparation method of the above-mentioned compound capable of inducing macrovesicle death, comprising the following steps:

(1) In an ice-water bath, add 2-bromomalondialdehyde and 5-aminopyrazole to ethanol in sequence, and stir for 4 to 6 minutes, then add concentrated hydrochloric acid drop by drop, and react at room temperature for 5 to 7 hours , then filtered, washed the filter cake, and dried to obtain a light brown solid;

(2) After mixing the light brown solid obtained in step (1), 4-aminophenylboronic acid pinacol ester, tetrakis (triphenylphosphine) palladium and potassium carbonate, add dioxane/water mixed solution, under inert gas Under protection, react at 70-90°C for 7-9 hours, then evaporate to remove the solvent, and the residue is extracted, washed, dried, and then purified to obtain a yellow-brown solid;

(3) Dissolve the yellow-brown solid obtained in step (2) in dichloromethane, then add N,N-diisopropylethylamine and cool in an ice-water bath for 8-12 minutes, then add chloroacetyl chloride dropwise, and in an inert Under the protection of gas, react at room temperature for 1.5-2.5 hours, and then obtain it after extraction, washing, drying and purification.

On the basis of the above technical solutions, the present invention can also be improved as follows.

黄棕色固体的化学式为

Further, the chemical formula of the light brown solid is

The chemical formula of the yellow-brown solid is

Further, the dosage ratio of 2-bromomalondialdehyde, 5-aminopyrazole, ethanol and concentrated hydrochloric acid is 30mol:30mol:36L:4ml, wherein the concentration of concentrated hydrochloric acid is 12 mol/liter; light brown solid, 4-aminobenzene The consumption ratio of boric acid pinacol ester, tetrakis (triphenylphosphine) palladium, potassium carbonate and dioxane/water mixed solution is 5mol:5mol:5mol:7.5mol:12ml, wherein dioxane/water mixed solution The volume ratio of dioxane to water is 5:1; the dosage ratio of yellow-brown solid, dichloromethane, N,N-diisopropylethylamine and chloroacetyl chloride is 5mol:40L:6.5mol:6mol.

Further, in step (1), washing is sequentially washing with saturated sodium bicarbonate solution, water and ethanol; drying is vacuum drying at 50-70° C. for 10-14 hours.

Further, the extractant extracted in step (2) is a mixture of methanol and dichloromethane in a volume ratio of 1:9; washing is washing with saturated sodium bicarbonate solution and saturated brine; drying is drying with anhydrous sodium sulfate ; Purified by column chromatography, the solvent used was a mixture of ethyl acetate and n-hexane in a volume ratio of 1:3.

Further, in step (3), the extracted extractant is dichloromethane; washing is washing with saturated sodium bicarbonate solution and saturated brine; drying is drying with anhydrous sodium sulfate; purification is column chromatography, and the solvent used is methanol Mixed liquid with dichloromethane in a volume ratio of 1:49.

The present invention also provides the application of the above-mentioned compound capable of inducing cell macrobubble death in the preparation of a drug for treating tumors.

Further, tumors include colon cancer, lung adenocarcinoma, cervical cancer, liver cancer, breast cancer and glioma cells.

The beneficial effects of the present invention are:

The 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide of the present invention has the effect of inducing tumor cell death, and this cell death is induced by the compound The formation of many huge vacuoles in the cells results in macrovesicular death. The compound has high clinical application value and good development prospect, and can be used for the treatment of tumor diseases. The present invention also provides a method that is convenient and suitable for large-scale synthesis of the compound (2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide).

Description of drawings

Figure 1 shows that 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide induces macrovesicular death of various tumor cells and its effect on HFC of normal cells Figure (in which tumor cells include: colon cancer cells HCT116, lung adenocarcinoma cells H1975, cervical cancer cells Hela, liver cancer cells Hep3B, breast cancer cells MCF-7, glioma cells U87MG and U251; COPPA is 2-chloro-N -(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide);

Fig. 2 is 2-chloro-N-(4-(pyrazol [1,5-a] pyrimidin-6-yl) phenyl) acetamide (COPPA) suppresses the cell proliferation figure of breast cancer cell MDA-MB-231;

Figure 3 shows that 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) inhibits the growth of subcutaneous tumor of breast cancer cell MDA-MB-231 and its effect on The influence of animal body weight (wherein figure (A) is that COPPA inhibits breast cancer cell MDA-MB-231 subcutaneous tumor photo; Figure (B) is that COPPA inhibits breast cancer cell MDA-MB-231 subcutaneous tumor weight statistical figure; Figure (C) is COPPA on the impact figure of BALB/c-nu mouse body weight);

Figure 4 is a flow chart for the preparation of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA);

Figures 5 to 6 are the mass spectrograms in steps (2) and (3) in the embodiment of the present invention in turn;

Figures 7 to 9 are the NMR spectra of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide.

Detailed ways

The specific embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1

The preparation method of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide comprises the following steps:

(1) In an ice-water bath, add 30mmol 2-bromomalonaldehyde and 30mmol 5-aminopyrazole in 36ml ethanol successively, and stir for 5min, then add 4ml 12mmol/ml concentrated hydrochloric acid dropwise, and then React at room temperature for 6 hours, then filter, then wash the filter cake with 20ml of saturated sodium bicarbonate solution, 20ml of water, and 6ml of ethanol in sequence and repeat once, then vacuum-dry at 60°C for 12h to obtain 5.70g of a light brown solid (yield: 96%).

(2) After mixing 5 mmol of the light brown solid obtained in step (1), 5 mmol of 4-aminophenylboronic acid pinacol ester, 5 mmol of tetrakis(triphenylphosphine) palladium and 7.5 mmol of potassium carbonate, add 12 ml of dioxane/water The mixed solution (the volume ratio of dioxane to water is 5:1), then reacted at 80°C for 8h under the protection of nitrogen, then evaporated (40°C, 0.5h) to remove the solvent, and the residue was washed with 80ml methanol/dichloro Methane mixture (wherein the volume ratio of methanol to dichloromethane is 1:9) extraction, 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine washing, 6g of anhydrous sodium sulfate after drying, and then through column chromatography (solvent is Ethyl acetate and n-hexane mixed solution 1.2L) was purified to obtain 0.86g of yellow-brown solid (yield: 82%); ¹ H NMR (400MHz, DMSO) δ9.21 (d, J =2.2Hz,1H),8.84(d,J=2.2Hz,1H),8.17(d,J=2.3Hz,1H),7.60(d,J=2.7Hz,1H),7.51(d,J=8.4 Hz,2H),6.68(d,J＝8.8Hz,2H),5.38(s,2H)ppm.MS(ESI,m/z):211[M+H] ⁺ ,252[M+CH ₃ CN+ H] ⁺ .

(3) Dissolve 5 mmol of the yellow-brown solid obtained in step (2) in 40 ml of dichloromethane, then add 6.5 mmol of N,N-diisopropylethylamine and cool in an ice-water bath for 10 minutes, then add 6 mmol of chloroacetyl chloride dropwise, And reacted at room temperature under the protection of nitrogen for 2 hours, then extracted with 80ml of dichloromethane, washed with 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine, dried with 6g of anhydrous sodium sulfate, and then subjected to column chromatography (solvents were methanol and dichloromethane) After purification of the mixed solution (1.5 L) of methane mixed at a volume ratio of 1:49, 1.26 g of light yellow solid was obtained (yield: 88%). ¹ H NMR (400MHz,DMSO)δ10.48(s,1H),9.44(s,1H),8.94(s,1H),8.25(s,1H),7.84(d,J=8.2Hz,2H), 7.73(d, J=8.2Hz, 2H), 6.77(s, 1H), 4.30(s, 2H) ppm. ¹³ C NMR (101MHz, DMSO) δ164.87, 149.19, 146.93, 145.30, 138.65, 131.98, 128.88, 127.39 , 120.91, 119.88, 96.12, 43.65 ppm. MS (ESI, m/z): 287[M+H] ⁺ , 328[M+CH ₃ CN+H] ⁺ .

Example 2

The preparation method of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide comprises the following steps:

(1) In an ice-water bath, add 30mmol 2-bromomalondialdehyde and 30mmol 5-aminopyrazole in 36ml ethanol successively, and stir for 4min, then add 4ml 12mmol/ml concentrated hydrochloric acid dropwise, and then React at room temperature for 5 hours, then filter, then wash the filter cake with 20ml of saturated sodium bicarbonate solution, 20ml of water, and 6ml of ethanol in sequence and repeat once, then vacuum-dry at 50°C for 14 hours to obtain a light brown solid;

(2) After mixing 5 mmol of the light brown solid obtained in step (1), 5 mmol of 4-aminophenylboronic acid pinacol ester, 5 mmol of tetrakis(triphenylphosphine) palladium and 7.5 mmol of potassium carbonate, add 12 ml of dioxane/water The mixed solution (the volume ratio of dioxane to water is 5:1), and then reacted at 70°C for 9h under the protection of nitrogen, then evaporated (40°C, 0.5h) to remove the solvent, and the residue was washed with 80ml methanol/dichloro Methane mixture (wherein the volume ratio of methanol to dichloromethane is 1:9) extraction, 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine washing, 6g of anhydrous sodium sulfate after drying, and then through column chromatography (solvent is Ethyl acetate and n-hexane mixed solution (1.2L) in a volume ratio of 1:3) was purified to obtain a yellow-brown solid;

(3) Dissolve 5 mmol of the yellow-brown solid obtained in step (2) in 40 ml of dichloromethane, then add 6.5 mmol of N,N-diisopropylethylamine and cool in an ice-water bath for 8 minutes, then add 6 mmol of chloroacetyl chloride dropwise, And reacted at room temperature under the protection of nitrogen for 1.5h, then extracted with 80ml of dichloromethane, washed with 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine, dried with 6g of anhydrous sodium sulfate, and then subjected to column chromatography (the solvent was methanol and di Chloromethane was mixed at a volume ratio of 1:49 (1.5 L) to obtain a light yellow solid after purification.

Example 3

The preparation method of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide comprises the following steps:

(1) In an ice-water bath, add 30mmol 2-bromomalonaldehyde and 30mmol 5-aminopyrazole to 36ml ethanol successively, and stir for 6min, then add 4ml 12mmol/ml concentrated hydrochloric acid dropwise, and then React at room temperature for 7 hours, then filter, then wash the filter cake with 20ml of saturated sodium bicarbonate solution, 20ml of water, and 6ml of ethanol in sequence and repeat once, then vacuum-dry at 70°C for 10 hours to obtain a light brown solid;

(2) After mixing 5 mmol of the light brown solid obtained in step (1), 5 mmol of 4-aminophenylboronic acid pinacol ester, 5 mmol of tetrakis(triphenylphosphine) palladium and 7.5 mmol of potassium carbonate, add 12 ml of dioxane/water The mixed solution (the volume ratio of dioxane to water is 5:1), then reacted at 90°C for 7h under the protection of nitrogen, then evaporated (40°C, 0.5h) to remove the solvent, and the residue was washed with 80ml methanol/dichloro Methane mixture (wherein the volume ratio of methanol to dichloromethane is 1:9) extraction, 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine washing, 6g of anhydrous sodium sulfate after drying, and then through column chromatography (solvent is Ethyl acetate and n-hexane mixed solution (1.2L) in a volume ratio of 1:3) was purified to obtain a yellow-brown solid;

(3) Dissolve 5 mmol of the yellow-brown solid obtained in step (2) in 40 ml of dichloromethane, then add 6.5 mmol of N,N-diisopropylethylamine and cool in an ice-water bath for 12 minutes, then add 6 mmol of chloroacetyl chloride dropwise, And reacted at room temperature under the protection of nitrogen for 2.5h, then extracted with 80ml of dichloromethane, washed with 80ml of saturated sodium bicarbonate solution and 80ml of saturated brine, dried with 6g of anhydrous sodium sulfate, and then subjected to column chromatography (solvent is methanol and di Chloromethane was mixed at a volume ratio of 1:49 (1.5 L) to obtain a light yellow solid after purification.

Experimental example 1

2-Chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) induces macrovesicle death in tumor cells:

Cultured colon cancer cells HCT116, lung adenocarcinoma cells H1975, cervical cancer cells Hela, liver cancer cells Hep3B, breast cancer cells MCF-7, glioma cells U87MG and U251, and normal colon cells FHC were used at 3000 cells/well. The cells were seeded in 96-well plates, and the cells were cultured overnight in a CO ₂ incubator (37° C., 5% CO ₂ ). Then, COPPA was added to different tumor cells so that the final concentration was 1 μM, an equal volume of DMSO was added to the control wells, and the cells were further cultured for 12 hours. Finally, a high-content camera system was used to take pictures to observe the formation of intracellular vacuoles and cell death. The results are shown in Figure 1, the red arrows indicate the huge vacuoles formed in the cells. 1μM COPPA induced the formation of a large number of vacuoles in tumor cells and induced macrovesicular cell death; it had no effect on normal colonic FHC cells.

Experimental example 2

2-Chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) inhibits the proliferation of breast cancer cell MDA-MB-231:

Breast cancer cells MDA-MB-231 were seeded in 96-well plates at 3000 cells/well, and the cells were cultured overnight in a CO ₂ incubator (37° C., 5% CO ₂ ). In the experimental group, 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) was added to make the final concentration of 0.5, 1, 2, 5, 10 , 20, 40 μM, the corresponding volume of DMSO was added to the control group. After continuing to cultivate for 48 hours, add 10 μL of MTT solution to each well and continue to incubate for 4 hours, discard the medium and add 200 μL of DMSO reagent, shake the table (shake at 300 rpm for 10 min); use a microplate reader (BioTek Cytation5) to read the OD value at 570 nm, Cell growth inhibition rate=(1-OD experiment/OD control)×100%, and IC50 value was calculated by Graphpad Prism 8. The results showed that the IC50 of 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) on breast cancer cell line MDA-MB-231 was treated for 48 hours. The value was 6.4±0.3 μM.

Experimental example 3

2-Chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) inhibits subcutaneous tumor growth of breast cancer cell line MDA-MB-231:

4-week-old BALB/c-nu female mice were kept in an SPF animal room, and 10 ⁷ MDA-MB-231 cells were subcutaneously inoculated on the left upper forelimb of the mice. When the tumor size was about 100 mm ³ , the small Rats were divided into: control group, COPPA 20mg/kg group and COPPA 40mg/kg group, and administered. Administration method: intraperitoneal administration; administration volume: 100 μL; administration frequency: 3 days/time; administration frequency: 10 times. And at the same time of administration, the body weight of the mice was recorded. Finally, the mice were sacrificed, and the tumors were taken out, photographed, and weighed. The experimental results are shown in Figure 3. COPPA can inhibit the growth of MDA-MB-231 subcutaneous tumors, and has significant anti-tumor activity. As shown by body weight, COPPA has no obvious side effects.

Although the specific implementation manner of the present invention has been described in detail in conjunction with the accompanying drawings, it should not be construed as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative work still belong to the protection scope of this patent.

Claims (9)

1. A compound that can induce cell macrovesicle death, characterized in that: the compound is 2-chloro-N-(4-(pyrazol[1,5-a]pyrimidin-6-yl)phenyl) Acetamide, whose chemical formula is

2. The preparation method of the compound capable of inducing macrovesicle death according to claim 1, characterized in that, comprising the following steps: (1) In an ice-water bath, add 2-bromomalondialdehyde and 5-aminopyrazole to ethanol in sequence, and stir for 4 to 6 minutes, then add concentrated hydrochloric acid dropwise, and then react at room temperature for 5 to 7 hours. Then filter, wash the filter cake, and dry to obtain a light brown solid; (2) After mixing the light brown solid obtained in step (1), 4-aminophenylboronic acid pinacol ester, tetrakis(triphenylphosphine) palladium and potassium carbonate, add dioxane/water mixed solution, and then Under gas protection, react at 70-90°C for 7-9 hours, then evaporate to remove the solvent, and the residue is extracted, washed, dried, and then purified to obtain a yellow-brown solid; (3) Dissolve the yellow-brown solid obtained in step (2) in dichloromethane, then add N,N-diisopropylethylamine and cool in an ice-water bath for 8-12 minutes, then add chloroacetyl chloride dropwise, and in an inert Under the protection of gas, react at room temperature for 1.5-2.5 hours, and then obtain it after extraction, washing, drying and purification. 3. preparation method according to claim 2 is characterized in that: the chemical formula of described light brown solid is

The chemical formula of the yellow-brown solid is

4. preparation method according to claim 2 is characterized in that: the consumption ratio of described 2-bromomalondialdehyde, 5-aminopyrazole, ethanol and concentrated hydrochloric acid is 30mol:30mol:36L:4ml, wherein concentrated hydrochloric acid The concentration is 12 moles/liter; The consumption ratio of described light brown solid, 4-aminophenylboronic acid pinacol ester, tetrakis (triphenylphosphine) palladium, salt of wormwood and dioxane/water mixed solution is 5mol: 5mol:5mol:7.5mol:12ml, wherein the volume ratio of dioxane to water in the dioxane/water mixture is 5:1; the yellow-brown solid, dichloromethane, N,N-diisopropyl The consumption ratio of base ethylamine and chloroacetyl chloride is 5mol:40L:6.5mol:6mol. 5. The preparation method according to claim 2, characterized in that: in step (1), the washing is sequential washing with saturated sodium bicarbonate solution, water, and ethanol; the drying is vacuum drying at 50-70°C 10～14h. 6. preparation method according to claim 2 is characterized in that: in step (2), the extraction agent of described extraction is the mixed solution that methanol and dichloromethane mix by volume ratio 1:9; Described washing is with Washing with saturated sodium bicarbonate solution and saturated brine; the drying is drying with anhydrous sodium sulfate; the purification is column chromatography, and the solvent used is a mixture of ethyl acetate and n-hexane in a volume ratio of 1:3. 7. preparation method according to claim 2 is characterized in that: in step (3), the extraction agent of described extraction is dichloromethane; Described washing is washing with saturated sodium bicarbonate solution and saturated saline; The drying is drying with anhydrous sodium sulfate; the purification is column chromatography, and the solvent used is a mixture of methanol and dichloromethane at a volume ratio of 1:49. 8. Use of the compound capable of inducing cell macrovesicle death according to claim 1 in the preparation of a drug for treating tumors. 9. The application according to claim 8, characterized in that: said tumors include colon cancer, lung adenocarcinoma, cervical cancer, liver cancer, breast cancer and glioma cells.

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