CN114409721B - Pentacyclic triterpene derivative containing electrophilic warhead, and preparation method and application thereof - Google Patents

Pentacyclic triterpene derivative containing electrophilic warhead, and preparation method and application thereof Download PDF

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CN114409721B
CN114409721B CN202210076983.1A CN202210076983A CN114409721B CN 114409721 B CN114409721 B CN 114409721B CN 202210076983 A CN202210076983 A CN 202210076983A CN 114409721 B CN114409721 B CN 114409721B
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王世盛
杨东雪
郭修晗
蔡森
张巧丽
李广哲
赵伟杰
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Dalian University of Technology
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Abstract

The invention relates to the field of medicines, in particular to a series of novel pentacyclic triterpene derivatives containing electrophilic warheads, and also discloses a preparation method, structural characteristics and medical application of the compounds, in particular to application in preventing and/or treating tumor diseases.

Description

Pentacyclic triterpene derivative containing electrophilic warhead, and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, in particular to a series of novel pentacyclic triterpene derivatives containing electrophilic warheads, and also discloses a preparation method, structural characteristics and medical application of the compounds, in particular to application in preventing and/or treating tumor diseases.
Background
Chemical antitumor drugs have entered the molecular targeted drug era through development for over half a century. With the wide application of small molecule kinase inhibitors as antitumor drugs in recent years, the inhibitors face the problem of drug resistance caused by the mutation of residues at binding sites, so the development of novel kinase inhibitors capable of overcoming tumor drug resistance is a problem to be solved in the research of antitumor drugs at present. Covalent kinase inhibitors containing electrophilic groups have been a hotspot in the field of antitumor drug research in the last decade due to their better resistance.
In recent years, natural products have received widespread attention as effective drugs for the treatment of various diseases. Pentacyclic triterpene compounds are used as one of natural products, have wide distribution, and have various unique biological activities. Therefore, researchers have generated great interest in pentacyclic triterpene compounds and derivatives thereof, and synthesized thousands of pentacyclic triterpene derivatives having different backbone modifications.
Oleanolic acid is the most widely studied pentacyclic triterpene compound, which is found mainly in a variety of edible and medicinal plants. CDDO-Me (2-cyano-3, 12-oleanane-1, 9 (11) -diene-28-carboxylic acid methyl ester) is an oleanolic acid derivative with potential anti-tumor activity, and has been found to have good biological activity through intensive research, and has entered clinical research stages of various diseases such as leukemia, chronic kidney disease, solid tumor and the like.
Based on the above, if the covalent inhibitor design strategy is applied to the structural modification of natural products, the high activity of covalent reactive groups and the biocompatibility of natural product frameworks are utilized, so that the method has great significance in discovering efficient and safe targeted covalent kinase inhibitors.
Disclosure of Invention
The invention aims to provide a series of synthesis of novel pentacyclic triterpene derivatives containing electrophilic warheads.
Another object of the present invention is to provide a process for preparing the above derivatives of pentacyclic triterpene natural products.
It is still another object of the present invention to provide the use of the above derivatives of pentacyclic triterpene natural products in the preparation of antitumor drugs.
The technical scheme of the invention is as follows:
the derivatives of pentacyclic triterpene natural products have the structures shown in the general formulas I-III:
wherein: r is R 1 Represents independent hydrogen, NHR 8 、OR 8 ，R 2 Represents independent hydrogen, NHR 8 、OR 8 Or R 1 And R is 2 Together represent oxygen; wherein R is 3 Represents independent hydrogen, NHR 8 、OR 8 ；R 4 Represents independent hydrogen, NHR 8 、OR 8 The method comprises the steps of carrying out a first treatment on the surface of the And R is 1 、R 2 、R 3 And R is 4 At least one of them is NHR 8 OR 8 The method comprises the steps of carrying out a first treatment on the surface of the And R is 1 And R is 2 Not simultaneously NHR 8 、OR 8 And R is 1 If it is NHR 8 、OR 8 R is then 2 Is hydrogen, R 2 If it is NHR 8 、OR 8 R is then 1 Is hydrogen; and R is 3 And R is 4 Not simultaneously NHR 8 、OR 8 And R is 3 If it is NHR 8 、OR 8 R is then 4 Is hydrogen, R 4 If it is NHR 8 、OR 8 R is then 3 Is hydrogen;
R 5 represents hydrogen or methyl, R 6 Represents hydrogen or methyl, and R 5 R when representing hydrogen 6 Represents methyl only, and R 6 R when representing hydrogen 5 Represents only methyl;
R 7 represents CH 2 OR 9 、COOR 9 、CONHR 9 、COR 10 ；
R 8 Represents COCH 2 X、COCHX 2 、COCX 3 、SO 2 CH 2 X、COCR 11 ＝CH 2 、COCH＝CHR 11 ；
X represents F, cl, br, I, and when multiple atoms are substituted, the X is the same element or different elements;
R 9 represents unsubstituted, linear or branched alkanes, alkenes, alkynes, phenyl, benzyl, naphthyl of 1 to 10 carbon atoms;
or R is 9 Y-substituted linear or branched alkanes, alkenes, alkynes, phenyl, benzyl, naphthyl, representing 1 to 10 carbon atoms; y represents H, X, CF 3 、SH、OH、CN、NO 2 、NH 2 、、COOH、OCH 3 、OC 2 H 5 COOCH 3 、COOC 2 H 5 Linear or branched alkanes, alkenes, alkynes, phenyl, benzyl, naphthyl of 1 to 10 carbon atoms;
R 10 represents piperazine, piperidine, pyrrole, methylpiperazine, morpholine, tert-butyloxycarbonyl piperazine;
R 11 representative H, X, CN, CF 3 Methyl, ethyl, phenyl, benzyl, CH 2 OH、CH 2 OMe、CH 2 NMe 2 、CH 2 NEt 2 、CH 2 R 10 。
The derivatives of pentacyclic triterpene natural products are used for forming pharmaceutically acceptable salts, including sodium salt, potassium salt, ammonium salt, organic amine salt, or basic amino acid of the compounds shown in the general formulas I-III; also included are acid addition salts of compounds of formulae I-III with: hydrochloric acid, hydrobromic acid, sulfuric acid, carbonic acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid.
The preparation method of the derivatives of the pentacyclic triterpene natural products comprises the following steps:
(1)
(a) At the temperature of 0-70 ℃, the volume ratio is 1:1, and the oleanolic acid reacts with the Jones reagent for 1-6 hours to obtain a compound A1, wherein the mol ratio of the oleanolic acid to the Jones reagent is 1:1.3 to 1:5, a step of;
(b) Under the temperature of 0-90 ℃, tertiary butanol is taken as a solvent, and the compound A1 reacts with isoamyl nitrite for 8-24 hours under the alkaline condition to obtain a compound A2; wherein the molar ratio of the compound A1 to the isoamyl nitrite is 1:1.1 to 1:15; the base used is potassium tert-butoxide and the molar ratio of compound A1 to potassium tert-butoxide is 1: 3-1: 15;
(c) Under the temperature of 0-90 ℃, methanol and concentrated hydrochloric acid with the volume ratio of 20:1-5:1 are used as solvents, and the compound A2 is subjected to palladium-carbon catalytic hydrogenation reduction reaction for 8-24 hours to obtain a compound A3; wherein the mass ratio of palladium carbon to the compound A2 is 1:10-1:5;
(d) The method comprises the following steps: under the temperature of 0-90 ℃, under the alkaline condition, the compound A3 reacts with acyl chloride or anhydride for 4-12 hours to obtain a compound A4; wherein the solvent comprises one or more of dichloromethane, tetrahydrofuran and acetonitrile, the alkali comprises potassium carbonate, sodium bicarbonate, triethylamine and N, N-diisopropylethylamine, and the molar ratio of the compound A3 to the alkali is 1:1 to 1:5, a step of; the molar ratio of the compound A3 to the acid chloride or the acid anhydride is 1:1 to 1:10;
the second method is as follows: under the temperature of 0-90 ℃, the compound A3 reacts with carboxylic acid or carboxylate through condensation reaction for 6-24 hours to obtain a compound A4; wherein, the solvent used in the reaction system is one or more than two of dichloromethane, tetrahydrofuran, acetonitrile and N, N-dimethylformamide; the base comprises potassium carbonate, sodium bicarbonate, triethylamine and N, N-diisopropylethylamine, and the molar ratio of the compound A3 to the base is 1:1 to 1:5, a step of; condensing agent used in the reaction system comprises one or more than two of EDCI, DCC, hoBt, HBTU and organic phosphorus; the molar ratio of the compound A3 to the condensing agent is 1:1 to 1:5, a step of; the molar ratio of compound A3 to carboxylic acid or carboxylate is 1:1.1 to 1:4, a step of;
(2)
(a) At the temperature of 0-70 ℃, the volume ratio is 1:1, and the oleanolic acid reacts with the Jones reagent for 1-6 hours to obtain a compound B1, wherein the mol ratio of the oleanolic acid to the Jones reagent is 1:1.3-1:5, a step of;
(b) At the temperature of 0-90 ℃, the volume ratio is 40:1-15:1, ethanol and water are used as solvents, and the compound B1 reacts with hydroxylamine hydrochloride and anhydrous sodium acetate for 6-24 hours to obtain a compound B2; wherein, the mol ratio of the compound B2 to hydroxylamine hydrochloride is 1:1.1 to 1:15; the molar ratio of the compound B2 to the anhydrous sodium acetate is 1: 4-1: 20, a step of;
(c) Under the temperature of 0-90 ℃, methanol and methylene dichloride with the volume ratio of 1:1 are used as solvents, and the compound B2 reacts with a cyano sodium borohydride reducing agent for 8-24 hours to obtain a compound B3; the molar ratio of the compound B2 to the sodium cyanoborohydride is 1:1.1 to 1:30;
(d) The method comprises the following steps: under the temperature of 0-90 ℃, under the alkaline condition, the compound B3 reacts with acyl chloride or anhydride for 4-12 hours to obtain a compound B4; wherein the solvent comprises one or more of dichloromethane, tetrahydrofuran and acetonitrile; the base comprises potassium carbonate, sodium hydroxide, triethylamine and N, N-diisopropylethylamine, and the molar ratio of the compound B3 to the base is 1:1 to 1:5, a step of; the molar ratio of the compound B3 to the acid chloride or the acid anhydride is 1:1 to 1:10;
the second method is as follows: under the temperature of 0-90 ℃, the compound B3 reacts with carboxylic acid or carboxylate through condensation reaction for 6-24 hours to obtain a compound B4; the solvent used in the reaction system comprises one or more of dichloromethane, tetrahydrofuran, acetonitrile and N, N-dimethylformamide; the base comprises potassium carbonate, sodium hydroxide, triethylamine and N, N-diisopropylethylamine, and the molar ratio of the compound B3 to the base is 1:1 to 1:5, a step of; condensing agent used in the reaction system comprises one or more than two of EDCI, DCC, hoBt, HBTU and organic phosphorus, and the molar ratio of the compound B3 to the condensing agent is 1:1 to 1:5, a step of; the molar ratio of compound B3 to carboxylic acid or carboxylate is 1:1.1 to 1:4.
the use of derivatives of pentacyclic triterpene natural products in the preparation of medicaments for preventing and/or treating tumor diseases. The pentacyclic triterpene natural product derivative is prepared by the preparation method.
A pharmaceutical composition comprises a compound of formula I-III and a pharmaceutically acceptable carrier therefor.
The invention has the beneficial effects that: the invention provides a series of novel pentacyclic triterpene derivatives containing electrophilic warhead and application thereof. The analogue of the series of pentacyclic triterpene natural products provided by the invention has obvious antiproliferative activity on tumor cells, and has potential for further research.
Detailed Description
The invention is further illustrated by the following experimental examples, which are not intended to limit the invention. The structure of the compound prepared by the invention is confirmed by analysis means such as nuclear magnetism, mass spectrum and the like.
Example 1
Preparation of 3-hydroxy-oleanane-12-ene-28-carboxylic acid benzyl ester
Oleanolic acid (4.567 g,10mmoL,1.0 eq.) was dissolved in 30mL of anhydrous DMF and added without waterWater K 2 CO 3 (2.764 g,20mmol,2.0 eq.). Benzyl bromide (1.46 mL,12mmol,1.2 eq.) was slowly added dropwise under nitrogen, reacted at room temperature, monitored by TLC and allowed to react for 6 h. Pouring the reaction solution into ice water to precipitate a large amount of white solid, fully precipitating the solid, filtering, washing a filter cake with a large amount of water, and drying in vacuum at 40 ℃. 5.198g of white solid is obtained in 95% yield.
Example 2
Preparation of 3-oxo-oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 01 (1.15 g,2.1mmol,1.0 eq.) was weighed into a 100mL reaction flask and dissolved by adding 40mL of a mixed solvent of acetone and dichloromethane (Ace: dcm=1:1). Under nitrogen protection, an ice-water bath, freshly prepared agar reagent (chromium trioxide 490mg,4.9mmol,2.3 eq.) was added dropwise, followed by reaction at room temperature by TLC monitoring until reaction was complete. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, and NaSO 4 Drying, and concentrating by rotary evaporation. PE/EA=10:1 column chromatography, yielding 1.02g of a white solid in 89% yield.
1 H NMR(400MHz,CDCl3)δ7.38-7.28(m,5H),5.31(t,J＝3.6Hz,1H),5.10(d,J＝12.5Hz,1H),5.05(d,J＝12.5Hz,1H),2.91(dd,J＝13.8,4.1Hz,1H),2.62–2.46(m,1H),2.40–2.29(m,1H),1.13(s,3H),1.08(s,3H),1.03(s,3H),1.01(s,3H),0.92(s,3H),0.90(s,3H),0.65(s,3H).
Example 3
Preparation of 3-hydroxyimino-olean-12-ene-28-carboxylic acid benzyl ester
Compound 02 (500 mg,0.91mmol,1.0 eq.) hydroxylamine hydrochloride (254 mg,3.68mmol,4.0 eq.) and anhydrous sodium acetate (302 mg,3.68mmol,4.0 eq.) were weighed into a 100mL reaction flask and most of the starting materials were dissolved by adding 20mL of ethanol and 1mL of water. After 12 hours at room temperature, a large amount of white solid was precipitated, and the reaction was completed by TLC. The solvent was removed by rotary evaporation, the solid was dissolved with ethyl acetate and water, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried, and concentrated to give 473mg of white powder in 92% yield.
Example 4
Preparation of 3-amino-oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 03 (200 mg,0.136mmol,1.0 eq.) was weighed out in a mixed solution of methylene chloride and methanol (1 ml:1 ml), and ammonium acetate (157 mg,2.72mmol,15.0 eq.) was added under the protection of nitrogen in an ice-water bath. Sodium cyanoborohydride (171 mg,2.72mmol,20.0 eq.) was weighed quickly and dissolved well in methanol solution, followed by slow dropwise addition to the reaction flask. The reaction temperature was maintained, and a titanium trichloride solution (352. Mu.L, 3.0 eq.) was added dropwise, and the reaction solution turned purple and then grey. After the completion of the dropwise addition, the reaction was carried out at room temperature for 12 hours, the reaction solution became colorless, and a large amount of white solid was precipitated, and the completion of the reaction was monitored by TLC. 2N sodium hydroxide solution to adjust ph=10, dichloromethane extraction, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. D/m=10: 1 to give 169.6mg of a white solid in 87% yield.
MS m/z:546.52[M+H] +
Example 5
Preparation of 3-acrylamido-oleanane-12-alkene-28-carboxylic acid benzyl ester
Compound 04 (45 mg,0.083mmol,1.0 eq.) was weighed, dissolved in 2mL of tetrahydrofuran solution, nitrogen protected, and added dropwise under ice-water bath with 1.27NAqueous sodium hydroxide (65 μl,0.083mmol,1.0 eq.) and acryloyl chloride (7.5 μl,0.091mmol,1.1 eq.). The reaction was carried out for 5h and tlc monitored the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=3:1 column chromatography, yielding 38.6mg of a white solid in 78% yield.
1 H NMR(600MHz,CDCl 3 )δ7.36–7.26(m,5H),6.27(dd,J＝16.9,0.9Hz,1H),6.08(dd,J＝16.9,10.3Hz,1H),5.63(dd,J＝10.3,0.9Hz,1H),5.37(d,J＝9.8Hz,1H),5.29(t,1H),5.09(d,J＝12.6Hz,1H),5.05(d,J＝12.5Hz,1H),3.83–3.71(m,1H),2.90(d,J＝10.8Hz,1H),1.13(s,3H),0.92(s,3H),0.90(s,6H),0.88(s,3H),0.79(s,3H),0.61(s,3H).
Example 6
Preparation of 3-chloroacetamido-oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 04 (50 mg,0.092mmol,1.0 eq.) is weighed out and dissolved in 1mL of tetrahydrofuran. Under nitrogen protection, an ice-water bath was added dropwise 1.27N aqueous sodium hydroxide solution (108. Mu.L, 0.138mmoL,1.5 eq.) and chloroacetyl chloride (15. Mu.L, 0.183mmoL,2.0 eq.) at room temperature for 12 hours, and TLC monitored the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=5:1 column chromatography, yielding 52.4mg of white solid in 92% yield.
MS m/z:622.42[M+H] +
1 H NMR(600MHz,CDCl 3 )δ7.28–7.22(m,5H),6.40(d,J＝9.9Hz,1H),5.21(t,1H),5.01(d,J＝12.5Hz,1H),4.97(d,J＝12.6Hz,1H),3.99(s,2H),3.73(dd,J＝9.6,3.0Hz,1H),3.66–3.47(m,1H),2.83(dd,J＝13.7,4.4Hz,1H),1.05(s,3H),0.84(s,3H),0.82(s,3H),0.81(s,6H),0.74(s,3H),0.53(s,3H).
Example 7
Preparation of 3-bromoacetamido-oleanane-12-alkene-28-carboxylic acid benzyl ester
Compound 04 (50 mg,0.092mmol,1.0 eq.) is weighed out and dissolved in 1mL of tetrahydrofuran. Under nitrogen protection, an ice-water bath was added dropwise 1.27N aqueous sodium hydroxide (108. Mu.L, 0.138mmoL,1.5 eq.) bromoacetyl bromide (16. Mu.L, 0.183mmoL,2.0 eq.) at room temperature for 12 hours, and TLC monitored the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=10:1 column chromatography, yielding 43.4mg of a white solid in 71% yield.
MS m/z:666.42[M+H] +
1 H NMR(400MHz,CDCl 3 )δ7.36–7.28(m,5H),6.38(d,J＝9.8Hz,1H),5.29(t,J＝3.6Hz,1H),5.09(d,J＝12.5Hz,1H),5.04(d,J＝12.6Hz,1H),3.91(s,2H),3.68–3.57(m,1H),2.90(dd,J＝13.7,3.9Hz,1H),1.12(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,6H),0.82(s,3H),0.61(s,3H).
Example 8
Preparation of 3- (dichloroacetamido) -oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 04 (50 mg,0.092mmol,1.0 eq.) is weighed out and dissolved in 1mL of tetrahydrofuran. Under nitrogen protection, an ice-water bath was added dropwise 1.27N aqueous sodium hydroxide solution (108. Mu.L, 0.138 mmole, 1.5 eq.) and dichloroacetyl chloride (17. Mu.L, 0.183 mmole, 2.0 eq.) at room temperature for 12 hours, and TLC monitored the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Water-solubleWashing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=10:1 column chromatography, yielding 51.8mg of a white solid in 86% yield.
MS m/z:656.37[M+H] +
1 H NMR(600MHz,CDCl 3 )δ7.39–7.26(m,5H),6.32(d,J＝9.9Hz,1H),5.92(s,1H),5.29(t,J＝3.5Hz,1H),5.09(d,J＝12.5Hz,1H),5.05(d,J＝12.5Hz,1H),3.68–3.55(m,1H),2.91(dd,J＝13.6,4.5Hz,1H),1.13(s,3H),0.92(s,3H),0.91(s,3H),0.90(s,3H),0.89(s,3H),0.84(s,3H),0.61(s,3H).
Example 9
Preparation of 3- (difluorochloroacetamido) -oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 04 (50 mg,0.09mmol,1.0 eq.) was weighed out and dissolved in 2mL of dichloromethane. EDCI (21 mg,0.25mmoL,1.2 eq.) and HOBt (15 mg,0.11mmoL,1.2 eq.) were added under nitrogen protection in an ice-water bath. After 1h of reaction, sodium difluorochloroacetate (17 mg,0.11mmol,1.2 eq.) was added and the reaction was continued for 8h, and the reaction was completed by tlc. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=5:1 column chromatography, separated to give 60.3mg of a white solid in 93% yield.
MS m/z:658.24[M+H] +
1 H NMR(400MHz,CDCl 3 )δ7.41–7.29(m,5H),6.03(d,J＝9.8Hz,1H),5.29(t,1H),5.10(d,J＝12.6Hz,1H),5.04(d,J＝12.5Hz,1H),3.76–3.60(m,1H),2.91(dd,J＝13.7,3.7Hz,1H),1.13(s,3H),0.92(s,3H),0.91(s,3H),0.90(s,3H),0.89(s,3H),0.82(s,3H),0.61(s,3H).
Example 10
Preparation of the Compound 3- (trans-4-dimethylamino-but-2-en-amide) -oleanane-12-en-28-carboxylic acid benzyl ester
Compound 04 (100 mg,0.183mmol,1.0 eq.) is weighed out and dissolved in 2mL of DMF. EDCI (38 mg,0.2mmol,1.1 eq.) was added, HOBt (27 mg,0.2mmol,1.1 eq.) and trans-4-dimethylaminocrotonic acid hydrochloride (30 mg,0.183mmoL,1.0 eq.) were added under nitrogen in an ice-water bath, the reaction was continued for 8h, and TLC was monitored to finish. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=5:1 column chromatography, separating 99mg of white solid with 82% yield
MS m/z:657.63[M+H] +
1 H NMR(400MHz,CDCl 3 )δ7.35–7.28(m,5H),6.87–6.69(m,J＝14.8,6.0Hz,1H),6.18(d,J＝15.3Hz,1H),5.97(d,J＝9.4Hz,1H),5.29(t,J＝2.5Hz,1H),5.09(d,J＝12.6Hz,1H),5.04(d,J＝12.6Hz,1H),3.99–3.85(m,J＝9.4Hz,1H),3.28(d,J＝5.6Hz,2H),2.91(d,J＝13.4Hz,1H),2.43(s,6H),1.18(s,3H),0.96(s,3H),0.92(s,6H),0.90(s,3H),0.85(s,3H),0.61(s,3H).
1 H NMR(400MHz,CDCl 3 )δ7.35–7.28(m,5H),6.87–6.69(m,J＝14.8,6.0Hz,1H),6.18(d,J＝15.3Hz,1H),5.97(d,J＝9.4Hz,1H),5.29(t,J＝2.5Hz,1H),5.09(d,J＝12.6Hz,1H),5.04(d,J＝12.6Hz,1H),3.99–3.85(m,J＝9.4Hz,1H),3.28(d,J＝5.6Hz,2H),2.91(d,J＝13.4Hz,1H),2.43(s,6H),1.18(s,3H),0.96(s,3H),0.92(s,6H),0.90(s,3H),0.85(s,3H),0.61(s,3H).
Example 11
Preparation of the Compound 3- (E-4- (piperidin-1-yl) but-2-en-amide) -oleanane-12-en-28-carboxylic acid benzyl ester
Compound 04 (100 mg,0.18mmol,1.0 eq.) is weighed out and dissolved in 2mL of DMF. Nitrogen protection under ice water bathEDCI (38 mg,0.2mmol,1.1 eq.) HOBt (27 mg,0.2mmol,1.1 eq.) E-4- (piperidin-1-yl) but-2-ene hydrochloride (32 mg,0.18mmoL,1.0 eq.) was added and the reaction continued for 8h with TLC monitoring the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=5:1 column chromatography, separated to give 91mg of white solid in 72% yield.
MS m/z:697.25[M+H] +
1 H NMR(400MHz,CDCl 3 ) Delta 7.37-7.28 (m, 5H), 6.92-6.67 (m, 1H), 6.08 (d, J=15.4 Hz, 1H), 5.50 (d, J=10.0 Hz, 1H), 5.29 (s, 1H), 5.09 (d, J=12.6 Hz, 1H), 5.04 (d, J=12.6 Hz, 1H), 3.84-3.70 (m, 1H), 3.29 (s, 2H), 2.90 (dd, J=13.7, 3.6Hz, 1H), 2.63 (s, 4H), 1.13 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H), example 12
Preparation of 3- (4-dimethylamino-butyramide) -oleanane-12-ene-28-carboxylic acid benzyl ester
Compound 04 (100 mg,0.183mmol,1.0 eq.) is weighed out and dissolved in 2mL of DMF. EDCI (38 mg,0.2mmol,1.1 eq.) was added, HOBt (27 mg,0.2mmol,1.1 eq.) 4- (dimethylamino) butyrate (36 mg,0.183mmoL,1.2 eq.) and DIPEA (64. Mu.L, 0.37mmoL,2.0 eq.) were added under nitrogen and ice water bath and the reaction was continued for 8h with TLC monitoring. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating.
DCM/meoh=10:1 column chromatography, isolated 87.1mg of white solid in 73% yield.
MS m/z:659.23[M+H] +
1 H NMR(600MHz,CDCl 3 )δ7.36–7.27(m,5H),7.18(d,J＝8.2Hz,1H),5.26(t,1H),5.08(d,J＝12.6Hz,1H),5.03(d,J＝12.6Hz,1H),3.88–3.77(m,J＝8.5Hz,1H),3.00(d,J＝6.8Hz,2H),2.88(d,J＝10.9Hz,1H),2.75(s,6H),2.67–2.54(m,2H),1.23(s,3H),0.93(s,3H),0.90(s,6H),0.87(s,6H),0.60(s,3H).
1 H NMR(600MHz,CDCl 3 )δ7.42–7.27(m,5H),6.43(d,J＝7.7Hz,1H),5.28(t,1H),5.08(d,J＝12.7Hz,1H),5.04(d,J＝12.7Hz,1H),3.65(s,1H),2.89(d,J＝12.0Hz,1H),2.81(s,2H),2.60(s,6H),2.45(s,2H),1.12(s,3H),0.91(s,3H),0.89(s,3H),0.86(s,6H),0.80(s,3H),0.60(s,3H).
Example 13
Preparation of 2-hydroxyimino-3-oxo-olean-12-ene-28-carboxylic acid methyl ester
Compound 13 (50 mg,0.11mmoL,1.0 eq.) was dissolved in 2mL of t-butanol in a 25mL reaction flask, followed by potassium t-butoxide (56 mg,0.5mmoL,4.5 eq.). Under nitrogen protection, the reaction was allowed to proceed for half an hour, isoamyl nitrite (78 μl,4.5 eq.) was added dropwise, and the reaction was continued for 12 hours, with TLC monitoring the end of the reaction. Removing reaction solution by rotary evaporation, adding ethyl acetate and 1% potassium hydroxide solution for dissolving and transferring, extracting with ethyl acetate, washing with 1% potassium hydroxide solution, and saturating with NH 4 Washing with Cl solution, washing with saturated NaCl solution, drying, and concentrating. D/m=20:1 separation gave 44.8mg of a yellowish solid in 87% yield.
MS m/z:484.30[M+H] +
Example 14
Preparation of 2-amino-3-oxo-oleanane-12-ene-28-carboxylic acid methyl ester
Compound 14 (50 mg,0.1mmol,1.0 eq.) was added to a 25mL reaction flask and replaced three times with nitrogen by adding 2.5mL of concentrated hydrochloric acid in methanol (conc.hcl: meoh=1:9). Under the protection of nitrogen, 5mg of 10% Pd/C was added, the nitrogen was replaced three times, and the hydrogen was replaced three times. The reaction was completed by TLC under hydrogen atmosphere for 10 hours. The palladium on carbon was filtered off through celite and the yellowish solid was concentrated 42.7mg, 89% yield.
MS m/z:470.47[M+H] +
Example 15
Preparation of 2-acrylamide-3-oxo-oleanane-12-ene-28-carboxylic acid methyl ester
Compound 15 (120 mg,0.248mmol,1.0 eq.) was weighed, dissolved in 2mL of tetrahydrofuran solution, and under nitrogen protection, an aqueous 1.27N sodium hydroxide solution (167 μl,0.213mmol,1.0 eq.) and acryloyl chloride (21 μl,0.26mmol,1.1 eq.) were added dropwise under ice-water bath. The reaction was carried out for 5h and tlc monitored the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. PE/EA=3:1 column chromatography, yielding 98.7mg of a white solid in 74% yield.
MS m/z:538.43[M+H] +
1 H NMR(600MHz,CDCl 3 )δ6.66(d,J＝5.8Hz,1H),6.28(d,J＝17.0Hz,1H),6.15(dd,J＝17.0,10.3Hz,1H),5.66(d,J＝10.3Hz,1H),5.27(s,1H),4.98–4.84(m,1H),3.61(s,3H),2.85(dd,J＝13.6,3.4Hz,1H),2.67(t,J＝12.4Hz,1H),1.17(s,3H),1.14(s,3H),1.11(s,3H),0.92(s,3H),0.91(s,3H),0.80(s,3H),0.72(s,3H).
Example 16
Preparation of 2- (trans-4-dimethylamino-but-2-en-amide) -3-oxo-oleanane-12-en-28-carboxylic acid methyl ester
Compound 15 (100 mg,0.21mmol,1.0 eq.) is weighed out in 2mL of dichloromethane and 2 drops of DMF are added dropwise. EDCI (48 mg,0.25mmoL,1.2 eq.) and HOBt (34 mg,0.25mmoL,1.2 eq.) were added under nitrogen protection in an ice-water bath. Reaction1h, trans-4-dimethylaminocrotonic acid hydrochloride, DIPEA (80. Mu.L, 0.46mmoL,2.2 eq.) was added, the reaction was continued for 8h, and TLC monitored for the end of the reaction. The solvent was removed by rotary evaporation, dichloromethane was added to dissolve the solid, and the solid was transferred to a separatory funnel. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating. DCM/eOH =10:1 column chromatography, isolated as a white solid 100.8mg, 82% yield.
MS m/z:595.53[M+H] +
1 H NMR(400MHz,CDCl 3 )δ6.95–6.76(m,1H),6.64(d,J＝5.3Hz,1H),6.06(d,J＝15.0Hz,1H),5.27(t,1H),5.03–4.82(m,1H),3.61(s,3H),3.17(d,J＝5.1Hz,2H),2.85(d,J＝12.3Hz,1H),2.34(s,6H),1.16(s,4H),1.13(s,3H),1.11(s,3H),0.92(s,7H),0.79(s,3H),0.72(s,3H).
Example 17
3 beta- (trans-4-dimethylamino-but-2-en-amide) oleanane-12-oxo-9 (11) -en-28-carboxylic acid benzyl ester
Compound 18 (100 mg,0.183mmol,1.0 eq.) was weighed out and dissolved in 2mL of DMF. EDCI (38 mg,0.2mmol,1.1 eq.) was added, HOBt (27 mg,0.2mmol,1.1 eq.) 4- (dimethylamino) butyrate (36 mg,0.183mmoL,1.2 eq.) and DIPEA (64. Mu.L, 0.36mmoL,2.0 eq.) were added under nitrogen and ice water bath and the reaction was continued for 8h with TLC monitoring. Extraction with dichloromethane, saturated NaHCO 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, drying, and concentrating.
DCM/meoh=10:1 column chromatography, isolated 126.2mg of white solid in 91% yield.
1 H NMR(600MHz,CDCl 3 )δ7.38–7.26(m,5H),6.83–6.72(m,1H),6.16(d,J＝15.1Hz,1H),5.79(d,J＝9.0Hz,1H),5.69(s,1H),5.14(d,J＝12.3Hz,1H),5.11(d,J＝12.4Hz,1H),3.78–3.69(m,J＝15.6,6.4Hz,1H),3.34(d,J＝3.6Hz,2H),3.05(d,J＝13.5Hz,1H),2.73(d,J＝4.3Hz,1H),2.47(s,6H),1.12(s,3H),0.99(s,3H),0.95(s,3H),0.90(s,6H),0.88(s,3H),0.80(s,3H).
The analogue of pentacyclic triterpene natural products of the invention has antiproliferative effect on tumor cells
To evaluate the antitumor activity of the synthesized OA derivatives, we tested their inhibitory effects on the proliferation of MCF-7, hepG2, heLa, MDA-MB-468, MDA-MB-231 and JIMT-1 by MTT method.
Experimental principle: MTT is commercially available as thiazole blue, yellow dye. Succinate dehydrogenase in live cell mitochondria can reduce MTT, and under the action of cytochrome C, blue-violet formazan is generated and precipitated in cells, formazan can be dissolved in DMSO, and the amount of formazan can be detected by absorbance. Therefore, the number of living cells can be estimated from the absorbance value.
The operation steps are as follows: the experimental 96-well plate, glove, centrifuge tube, etc. were wiped with 75% alcohol, placed in an ultra clean bench, irradiated with ultraviolet lamp for 30min, and the culture solution, PBS, drug, etc. were removed from the refrigerator and warmed to 37deg.C. Taking cells in logarithmic growth phase, obtaining a cell suspension by the method of passage, counting by a cell counting plate, preparing the cell suspension by a culture solution, adding 100 mu L of the cell suspension into each hole of a 96-well plate, and placing the cells in an incubator for culturing for 24 hours. All the medicines to be tested are provided with 6 concentration gradients and 4 auxiliary holes, the medicine stock solution is diluted to the required concentration by the culture solution, the 96-hole plate is taken out, and 100 mu L of medicine is added into each hole. After 48h incubation, 20 μLMTT (5 mg/mL) solution was added to each well, after 4h incubation, the broth was aspirated, 200 μLDMSO was added to each well to dissolve formazan, the microplate was shaken for 5min after heating the microplate reader to 37℃and absorbance at 570nm was measured.
The data was processed through IBM SPSS Statistics software.
The anti-proliferation experimental result of the analogue of the pentacyclic triterpene natural product on tumor cells:
wherein: a and b represent the alpha and beta configurations of the ring A of the compound; c represents the measured value obtained by three experiments; d represents that no activity test was performed
From the above experimental data, it can be seen that the pentacyclic triterpene derivatives having electrophilic active warhead provided by the invention have general antitumor activity against various cancer cells, and some of the compounds such as compound 5, compound 6 and compound 13 have strong antitumor activity. Therefore, the invention can be used for treating tumor diseases, in particular breast cancer, cervical cancer, liver cancer and the like. In addition, the invention creatively introduces the electrophilic active warhead into the natural product structure of pentacyclic triterpene, and provides a thinking for developing novel pentacyclic triterpene derivatives containing electrophilic warhead.

Claims (4)

1. A derivative of pentacyclic triterpene natural products is characterized in that the derivative of the pentacyclic triterpene natural products has 7 b 、8 b The structure shown:
2. the derivative of a pentacyclic triterpene natural product according to claim 1, wherein the derivative of a pentacyclic triterpene natural product is useful for forming a pharmaceutically acceptable salt comprising 7 b 、8 b Acid addition salts of the compounds shown with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, carbonic acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid.
3. Use of a derivative of a pentacyclic triterpene natural product according to claim 1, for the manufacture of a medicament for the prevention and/or treatment of neoplastic diseases.
4. A pharmaceutical composition, wherein the derivative of the pentacyclic triterpene natural product according to claim 1, and a pharmaceutically acceptable carrier thereof.
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