CN114751910A - A compound capable of inducing macrovesicular cell death and its preparation method and application - Google Patents

Abstract

The invention discloses a compound capable of inducing macrovesicular cell death, a preparation method and application thereof, and the compound is 2-chloro-N-(4-(pyrazo[1,5-a]pyrimidin-6-yl) phenyl)acetamide. The 2-chloro-N-(4-(pyrazolo[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 giant vacuoles in cells is called macrovesicular death. The compound has high clinical application value and good development prospect, and can be used for the treatment of tumor diseases. The preparation method of the present invention is simple and suitable for mass synthesis of the compound.

Description

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

technical field

The invention belongs to the technical field of medicinal chemistry, and in particular relates to a compound that can induce macrovesicular death of cells and a preparation method and application thereof.

Background technique

Malignant tumor is a major disease that seriously threatens human life and health. At present, most chemotherapeutic drugs in clinical use kill tumor cells through apoptosis mechanism to achieve therapeutic effect. Evolution makes itself constantly adapt to the external environment and gradually develops drug resistance to chemotherapeutic drugs, thus restricting the clinical application of chemotherapeutic 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 clear vacuoles in the cytoplasm, but it does not have the general characteristics of apoptosis. Therefore, the development of antitumor drugs that induce macrobubble death has important scientific significance and clinical value for improving the chemotherapy effect of malignant tumors.

SUMMARY OF THE INVENTION

In view of the above-mentioned prior art, the present invention provides a compound that can induce macrovesicular cell death, a preparation method and an application thereof, so as to solve the drug resistance problem caused by chemotherapy drugs that kill tumor cells by an existing apoptosis mechanism.

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

The present invention also provides a method for preparing the above-mentioned compound capable of inducing macrovesicular cell death, comprising the following steps:

(1) In an ice-water bath, 2-bromomalonaldehyde and 5-aminopyrazole were added to ethanol in turn, and the reaction was stirred for 4 to 6 minutes, then concentrated hydrochloric acid was added dropwise, and the reaction was carried out 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, place in inert gas Under the protection, react at 70～90℃ for 7～9h, then evaporate the solvent, the residue is extracted, washed, dried, and then purified to obtain a yellow-brown solid;

(3) Dissolve the yellowish-brown solid obtained in step (2) in dichloromethane, add N,N-diisopropylethylamine and cool it in an ice-water bath for 8-12min, dropwise add chloroacetyl chloride, and keep inert Under the protection of gas, react at room temperature for 1.5-2.5 hours, and then extract, wash, dry and purify to obtain the product.

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 consumption ratio of 2-bromomalonaldehyde, 5-aminopyrazole, ethanol and concentrated hydrochloric acid is 30mol:30mol:36L:4ml, and wherein the concentration of concentrated hydrochloric acid is 12 mol/liter; light brown solid, 4-aminobenzene The consumption ratio of pinacol borate, 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 middle 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 washing with saturated sodium bicarbonate solution, water and ethanol in sequence; drying is vacuum drying at 50-70° C. for 10-14 hours.

Further, the extractant extracted in step (2) is a mixed solution 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 is a mixed solution of ethyl acetate and n-hexane in a volume ratio of 1:3.

Further, in step (3), the extractant for extraction 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 with dichloromethane in a volume ratio of 1:49.

The present invention also provides the application of the above-mentioned compound capable of inducing macrovesicular cell death in the preparation of a medicament 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-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide of the present invention has the effect of inducing tumor cell death, which is induced by the compound The formation of many giant vacuoles in cells is called 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 simple and suitable method for the large-scale synthesis of the compound (2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide).

Description of drawings

Figure 1 shows that 2-chloro-N-(4-(pyrazolo[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-(Pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide);

Figure 2 is a graph showing that 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) inhibits the cell proliferation of breast cancer cell MDA-MB-231;

Figure 3 shows that 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) inhibited the subcutaneous tumor growth of breast cancer cell MDA-MB-231 and its effect on The effect of animal body weight (in which Figure (A) is a photo of COPPA inhibiting breast cancer cell MDA-MB-231 subcutaneous tumor; Figure (B) is a statistical chart of COPPA inhibiting breast cancer cell MDA-MB-231 subcutaneous tumor weight; Figure (C) is the effect of COPPA on the body weight of BALB/c-nu mice);

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

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

7 to 9 are nuclear magnetic spectra of 2-chloro-N-(4-(pyrazo[1,5-a]pyrimidin-6-yl)phenyl)acetamide.

Detailed ways

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

Example 1

A method for preparing 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide, comprising the following steps:

(1) in ice-water bath, 30mmol 2-bromomalonaldehyde and 30mmol 5-aminopyrazole are added successively in 36ml ethanol, and after stirring reaction 5min, then dropwise add the concentrated hydrochloric acid of 4ml 12mmol/ml, then in The reaction was performed at room temperature for 6 h, followed by filtration, and the filter cake was washed with 20 ml of saturated sodium bicarbonate solution, 20 ml of water, and 6 ml of ethanol in turn and repeated once, and then vacuum-dried at 60 ° C for 12 h to obtain 5.70 g of light brown solid (yield: 96%).

(2) After mixing 5mmol of the light brown solid obtained in step (1), 5mmol of 4-aminophenylboronic acid pinacol ester, 5mmol of tetrakis(triphenylphosphine)palladium and 7.5mmol of potassium carbonate, 12ml of dioxane/water was added The mixed solution (wherein the volume ratio of dioxane and water is 5:1) was then reacted under nitrogen protection at 80°C for 8h, then evaporated (40°C, 0.5h) to remove the solvent, and the residue was washed with 80ml methanol/dichloromethane Methane mixed solution (wherein the volume ratio of methanol and dichloromethane is 1:9) extraction, 80ml saturated sodium bicarbonate solution and 80ml saturated brine washing, after 6g anhydrous sodium sulfate drying, then through column chromatography (solvent is 1.2L of the mixture of ethyl acetate and n-hexane in a volume ratio of 1:3) was purified to obtain a yellow-brown solid 0.86g (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) 5mmol of the yellowish-brown solid obtained in step (2) was dissolved in 40ml of dichloromethane, then 6.5mmol of N,N-diisopropylethylamine was added, and after cooling in an ice-water bath for 10min, 6mmol of chloroacetyl chloride was added dropwise, and reacted at room temperature for 2 h under nitrogen protection, then extracted with 80 ml of dichloromethane, washed with 80 ml of saturated sodium bicarbonate solution and 80 ml of saturated brine, dried with 6 g of anhydrous sodium sulfate, and then subjected to column chromatography (solvents are methanol and dichloromethane). 1.5L of the mixed solution of methane in a volume ratio of 1:49) was purified to obtain 1.26 g of a light yellow solid (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) δ 164.87, 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

A method for preparing 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide, comprising the following steps:

(1) in ice-water bath, 30mmol 2-bromomalonaldehyde and 30mmol 5-aminopyrazole are added successively in 36ml ethanol, and after stirring reaction 4min, then dropwise add the concentrated hydrochloric acid of 4ml 12mmol/ml, then in The reaction was carried out at room temperature for 5 hours, then filtered, and the filter cake was washed with 20 ml of saturated sodium bicarbonate solution, 20 ml of water, and 6 ml of ethanol in turn and repeated once, and then vacuum-dried at 50 °C for 14 hours to obtain a light brown solid;

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

(3) 5mmol of the yellow-brown solid obtained in step (2) was dissolved in 40ml of dichloromethane, then 6.5mmol of N,N-diisopropylethylamine was added, and after cooling in an ice-water bath for 8min, 6mmol of chloroacetyl chloride was added dropwise, And reacted at room temperature for 1.5h under nitrogen protection, 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: methanol and dichloromethane). After purifying the mixed solution of chloromethane in a volume ratio of 1:49 (1.5L), a pale yellow solid was obtained.

Example 3

A method for preparing 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide, comprising the following steps:

(1) in ice-water bath, 30mmol 2-bromomalonaldehyde and 30mmol 5-aminopyrazole are added successively in 36ml ethanol, and after stirring reaction 6min, the concentrated hydrochloric acid of 4ml 12mmol/ml is added dropwise thereafter, and then in The reaction was carried out at room temperature for 7 hours, then filtered, and the filter cake was washed with 20 ml of saturated sodium bicarbonate solution, 20 ml of water, and 6 ml of ethanol in turn and repeated once, and then vacuum-dried at 70 °C for 10 hours to obtain a light brown solid;

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

(3) 5mmol of the yellow-brown solid obtained in step (2) was dissolved in 40ml of dichloromethane, then 6.5mmol of N,N-diisopropylethylamine was added, and after cooling in an ice-water bath for 12min, 6mmol of chloroacetyl chloride was added dropwise, And reacted at room temperature for 2.5h under nitrogen protection, 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: methanol and dichloromethane). After purifying the mixed solution of chloromethane in a volume ratio of 1:49 (1.5L), a pale yellow solid was obtained.

Experimental example 1

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

The 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 collected at 3000 cells/well. The cells were seeded in 96-well plates and cultured overnight in a CO ₂ incubator (37° C., 5% CO ₂ ). Then, COPPA was added to different tumor cells at a final concentration of 1 μM, an equal volume of DMSO was added to control wells, and the cells were further cultured for 12 hours. Finally, take pictures with a high-content camera system to observe the formation of intracellular vacuoles and cell death. The results are shown in Fig. 1, the red arrows indicate the giant vacuoles formed inside the cells. 1μM COPPA induced a large number of intracellular vacuoles in tumor cells and induced macrovesicular cell death; but had no effect on normal colonic FHC cells.

Experimental example 2

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

Breast cancer cells MDA-MB-231 were seeded in a 96-well plate 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-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) was added to the final concentration of 0.5, 1, 2, 5, 10 , 20, 40 μM, and the control group was added with the corresponding volume of DMSO. After culturing for 48 hours, add 10 μL of MTT solution to each well and continue to incubate for 4 hours. Aspirate the medium and add 200 μL of DMSO reagent. Shaker (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 using Graphpad Prism 8. The results showed that the IC50 of 2-chloro-N-(4-(pyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetamide (COPPA) for 48h on breast cancer cells MDA-MB-231 The value was 6.4±0.3 μM.

Experimental example 3

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

4-week-old BALB/c-nu female mice were housed in SPF animal rooms, and 10 ⁷ MDA-MB-231 cells were subcutaneously inoculated into the left upper forelimb ^of the mice. When the tumor size was about 100 mm, the small Mice were divided into: control group, COPPA 20mg/kg group and COPPA 40mg/kg group, and were administered. Administration mode: 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 mice was recorded. Finally, the mice were sacrificed, and the tumors were removed, photographed, and weighed. The experimental results are shown in Figure 3. COPPA can inhibit the growth of MDA-MB-231 subcutaneous tumor, and has significant anti-tumor activity. It is shown by body weight that COPPA has no obvious toxic and side effects.

Although the specific embodiments of the present invention have been described in detail with reference to the accompanying drawings, they should not be construed as limiting the protection scope of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative efforts still belong to the protection scope of this patent.

Claims (9)

1. A compound capable of inducing macrovesicular cell death, wherein the compound is 2-chloro-N-(4-(pyrazo[1,5-a]pyrimidin-6-yl)phenyl) Acetamide, its chemical formula is

2. the preparation method of the compound of inducible macrovesicular death according to claim 1, is characterized in that, comprises the following steps: (1) In an ice-water bath, 2-bromomalondialdehyde and 5-aminopyrazole were added to ethanol in turn, and the reaction was stirred for 4-6 min, then concentrated hydrochloric acid was added dropwise, and then the reaction was carried out at room temperature for 5-7 h, Then filter, wash the filter cake, and dry to obtain 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 inert Under gas protection, react at 70～90℃ for 7～9h, then evaporate the solvent to remove the solvent. After the residue is extracted, washed, dried, and then purified, a yellow-brown solid is obtained; (3) Dissolve the yellowish-brown solid obtained in step (2) in dichloromethane, add N,N-diisopropylethylamine and cool it in an ice-water bath for 8-12min, dropwise add chloroacetyl chloride, and keep inert Under the protection of gas, react at room temperature for 1.5-2.5 hours, and then extract, wash, dry and purify to obtain the product. 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 mol/liter; The consumption ratio of described light brown solid, 4-aminophenylboronic acid pinacol ester, tetrakis (triphenylphosphine) palladium, potassium carbonate and dioxane/water mixed solution is 5mol: 5mol:5mol:7.5mol:12ml, wherein the volume ratio of dioxane and water in the dioxane/water mixed solution is 5:1; the yellow-brown solid, dichloromethane, N,N-diisopropyl The dosage ratio of ethyl ethylamine and chloroacetyl chloride is 5mol:40L:6.5mol:6mol. 5. preparation method according to claim 2 is characterized in that: in step (1), described washing is to wash successively with saturated sodium bicarbonate solution, water, ethanol; Described drying is vacuum drying at 50～70 ℃ 10～14h. 6. preparation method according to claim 2 is characterized in that: in step (2), the extractant of described extraction is the mixed solution that methanol and dichloromethane mix by volume ratio 1:9; Washed 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 mixed solution 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 extractant of described extraction is methylene dichloride; Described washing is to wash 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 mixed solution of methanol and dichloromethane in a volume ratio of 1:49. 8. The application of the compound capable of inducing macrovesicular cell death according to claim 1 in the preparation of a medicament for treating tumors. 9. The use according to claim 8, wherein the tumor comprises colon cancer, lung adenocarcinoma, cervical cancer, liver cancer, breast cancer and glioma cells.

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