α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives and their preparation methods and applications

Abstract

The present invention belongs to the technical field of biomedicine, and relates to α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives, a preparation method thereof, and applications thereof. The chemical structural formula thereof is as follows: #imgabs0# wherein n is 4 or 5, R1 is a mono-substituted or di-substituted phenyl, and the substitution groups of the phenyl are halogen, trifluoromethyl, methyl, and / or nitro. Research shows that the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives provided by the present invention have high anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives, their preparation methods and applications. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] 18β-glycyrrhetinic acid (18β-GA) belongs to oleanane-type pentacyclic triterpenoid compounds and is the main active ingredient of glycyrrhizic acid in the traditional Chinese medicine licorice. It also has rich pharmacological activities, including anti-inflammatory, antiviral, and hepatoprotective activities including antitumor effects. However, this type of compound itself has defects such as poor water solubility and low bioavailability, which limit its use as an antitumor preparation. In addition, there is still a gap between its biological activity and the drugs used clinically. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives, their preparation methods and applications. Research shows that the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives provided by the present invention have high antitumor activity.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] On the one hand, an α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative, the chemical structural formula of which is as follows:

[0007]

[0008] Among them, n is 4 or 5, and R1 is a mono-substituted or di-substituted phenyl group, and the substituents of the phenyl group are halogen, trifluoromethyl, methyl and / or nitro.

[0009] In some embodiments, R1 is 2-chloro-5-nitrophenyl, 4-fluoro-3-methylphenyl, 3-trifluoromethylphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl or p-iodophenyl.

[0010] In some embodiments, R1 is 2-chloro-5-nitrophenyl, p-trifluoromethylphenyl or p-fluorophenyl.

[0011] In some embodiments, n is 4 and R1 is 2-chloro-5-nitrophenyl; n is 5 and R1 is p-trifluoromethylphenyl or p-fluorophenyl.

[0012] On the other hand, a method for preparing the above-mentioned α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative comprises the steps of reacting 18β-glycyrrhetinic acid (18β-GA) as a raw material according to the following reaction route;

[0013]

[0014] wherein, R1 and n are as described above.

[0015] Specifically, 18β-glycyrrhetinic acid undergoes a hydroxy oxidation reaction with an oxidizing agent to obtain compound 2, the compound undergoes an aldol condensation reaction with substituted benzaldehyde (R1CHO) to obtain compound 3, compound 3 undergoes a substitution reaction with 1,4-dibromobutane or 1,5-dibromopentane to obtain compound 4, and compound 4 undergoes a quaternary phosphonation reaction with triphenylphosphine to obtain compound 5, namely the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative.

[0016] More specifically, the oxidizing agent is pyridinium chlorochromate (PCC).

[0017] More specifically, the temperature of the hydroxy oxidation reaction is from 0 °C to room temperature. The room temperature in the present invention refers to the temperature of the indoor environment, generally 15 - 30 °C.

[0018] More specifically, in the aldol condensation reaction, potassium hydroxide or sodium hydroxide is added and the reaction is carried out at 40 - 50 °C.

[0019] More specifically, the substitution reaction is carried out at room temperature.

[0020] More specifically, the temperature of the quaternary phosphonation reaction is 75 - 85 °C.

[0021] More specifically, the reagents and reaction conditions are as follows: a. pyridinium chlorochromate (PCC), silica gel, CH2Cl2 (DCM), from 0 °C to room temperature, 2 d; b. potassium hydroxide (KOH), substituted benzaldehyde, anhydrous ethanol (dry CH3CH2OH), 46 °C, 1 d; c. 1,4-dibromobutane or 1,5-dibromopentane, DMF, room temperature, 7 h; d. triphenylphosphine (TPP), anhydrous acetonitrile (dry CH3CN), 80 °C, reflux for 3 d.

[0022] In a third aspect, a pharmaceutical composition comprises the above-mentioned α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative or a pharmaceutically acceptable salt thereof.

[0023] The pharmaceutically acceptable salts described in the present invention include hydrochloride, sulfate, benzenesulfonate, acetate, etc.

[0024] Fourth aspect, a pharmaceutical preparation, comprising the above-mentioned α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative or the above-mentioned pharmaceutical composition and pharmaceutically acceptable excipients.

[0025] The pharmaceutically acceptable excipients described in the present invention are excipients and / or carriers, wherein the carriers include serum proteins, polyethylene glycol, buffers, alumina, lecithin, etc.; the excipients include binders, fillers, buffers, pH regulators, emulsifiers, stabilizers, etc.

[0026] Fifth aspect, an application of the above-mentioned α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative, pharmaceutical composition or pharmaceutical preparation in the preparation of anti-tumor drugs.

[0027] Specifically, the tumors include liver cancer, colon cancer, and neuroblastoma. More specifically, the tumor cells are HepG2, HCT-116, and SH-SY5Y.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention uses 18β-GA as the parent nucleus for structural modification, and introduces an exocyclic α,β-unsaturated carbonyl structure at the C-2 position of 18β-GA through an aldol condensation reaction. At the same time, an alkyltriphenylphosphonium cation group (Triphenylphosphonium cation, TPP+) targeting the mitochondria of tumor cells is introduced at the carboxyl group at the C-30 position. On the one hand, benzaldehydes with various substituents are combined with the 18β-GA parent nucleus through an aldol condensation reaction to form an exocyclic α,β-unsaturated carbonyl structure; on the other hand, the introduction of a lipophilic cation, namely the alkyl TPP+ in this study, can further improve the selectivity of the derivative and reduce the toxic and side effects on normal somatic cells. Experiments have shown that the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative provided by the present invention has high anti-tumor activity. Among them, the growth inhibitory activities of some α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivatives against HepG2, HCT-116, and SH-SY5Y are more significant, and the IC50 values are in the ranges of 1.52-3.46 μM, 1.58-1.66 μM, and 1.60-2.18 μM, respectively. Detailed implementation manners

[0030] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the following will detail the technical solutions of the present invention in combination with specific examples.

[0031] Examples

[0032] Compound structure and synthesis route

[0033] Sixteen 18β-GA derivatives modified with α,β-unsaturated carbonyl groups from 5a to 5p were designed and synthesized. The related structures and synthetic routes are shown below.

[0034]

[0035] Reagents and reaction conditions: a. Pyridinium chlorochromate (PCC), silica gel, CH2Cl2 (DCM), 0 °C to room temperature, 2 d; b. Potassium hydroxide (KOH), substituted benzaldehyde, dry ethanol (dry CH3CH2OH), 46 °C, 1 d; c. 1,4-Dibromobutane or 1,5-dibromopentane, DMF, room temperature, 7 h; d. Triphenylphosphine (TPP), dry acetonitrile (dry CH3CN), 80 °C, reflux for 3 d.

[0036] Synthesis steps:

[0037] (1) Synthesis of 18β-GA derivative 2:

[0038] Add 18β-GA (5 mmol), silica gel of 200 - 300 mesh (weigh the same mass as PCC), and 30 mL of DCM into a 150 mL round-bottom flask. Under stirring at 0 °C, slowly pour PCC (7.5 mmol) into the reaction solution through an addition funnel. Stir the reaction for 2 d at room temperature and monitor the reaction progress by TLC. Filter off the silica gel solid in the system by vacuum filtration. Dilute the reaction solution with DCM, wash the organic phase 3 times with deionized water and 3 times with saturated brine. Treat the organic phase with anhydrous sodium sulfate and let it stand for 10 min. Filter by vacuum filtration, rotary evaporate, transfer and concentrate the crude product. Purify by normal-phase silica gel column chromatography, with the mobile phase of n-hexane:ethyl acetate = 6:1 → 4:1. Add glacial acetic acid to the mobile phase of this column chromatography, with the ratio of 1 mL of glacial acetic acid added to every 100 mL of the mobile phase.

[0039] (2) Synthesis of 18β-GA derivatives 3a - 3p

[0040] Compound 2 (1 mmol), potassium hydroxide (2 mmol), and 25 mL of absolute ethanol solvent were added to a 50 mL round-bottom flask and stirred at 46 °C for 1 h for activation. Then, the substituted benzaldehyde (2 mmol) was added to the reaction system, and the reaction was stirred at 46 °C for 1 day, and the reaction progress was monitored by TLC. After the reaction was completed, dilute hydrochloric acid solution was added dropwise under stirring to adjust the pH value of the reaction solution to 5 - 6. At this time, white solids precipitated in the originally clear and homogeneous solution. The reaction solution was diluted with ethyl acetate, and the organic phase was washed 3 times with deionized water and 3 times with saturated brine. The organic phase was treated with anhydrous sodium sulfate and allowed to stand for 10 min. Filtration was carried out under reduced pressure and rotary evaporation was performed, and the crude product was transferred and concentrated. Purification was carried out by normal-phase silica gel column chromatography, and the mobile phase was n-hexane:ethyl acetate = 60:1 → 40:1 → 20:1 or 10:1. Glacial acetic acid was also added to this mobile phase in the same proportion as above (1).

[0041] Among them, the substituted benzaldehydes were 2-chloro-5-nitrobenzaldehyde, 4-fluoro-3-methylbenzaldehyde, 4-trifluoromethylbenzaldehyde, 3-trifluoromethylbenzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, and 4-iodobenzaldehyde respectively.

[0042] (3) Synthesis of 18β-GA derivatives 4a - 4p

[0043] Compound 3a - 3p (1 mmol), anhydrous potassium carbonate (6 mmol), and 6 mL of DMF were added to a 50 mL round-bottom flask, and the reaction was stirred at room temperature for 30 min for activation. Then, 1,4-dibromobutane or 1,5-dibromopentane was added to the reaction system, and the reaction was stirred at room temperature for 7 h, and the reaction progress was monitored by TLC. The reaction solution was diluted with DCM, and the organic phase was washed 3 times with deionized water and 3 times with saturated brine to remove DMF and water-soluble impurities. The organic phase was treated with anhydrous sodium sulfate and allowed to stand for 10 min. Filtration was carried out under reduced pressure and rotary evaporation was performed, and the crude product was transferred and concentrated. Purification was carried out by normal-phase silica gel column chromatography, and the mobile phase was n-hexane:ethyl acetate = 20:1 → 10:1.

[0044] (4) Synthesis of 18β-GA derivatives 5a - 5p

[0045] Compound 4a - 4p (1 mmol), triphenylphosphine (4 mmol), and 10 mL of anhydrous acetonitrile were added to a 25 mL round-bottom flask, and the reaction was refluxed and stirred at 80 °C for 3 days, and the reaction progress was monitored by TLC. The reaction solution did not require post-treatment and was directly concentrated by rotary evaporation under reduced pressure to obtain the crude product. Purification was carried out by normal-phase silica gel column chromatography, and the mobile phase was dichloromethane:methanol = 100:1 → 80:1 → 50:1 → 30:1 → 20:1.

[0046] Nomenclature and structural characterization of derivatives 5a - 5p

[0047] 2-((2-chloro-5-nitrophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphos-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0048] Compound 5a: Melting point: 157.0–158.2 °C; Yield: 36%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ8.18–7.97(m,2H,Ph-H at C-2),7.82–7.63(m,15H,PPh3-H),7.57–7.49(m,2H,Ph-Hat C-2and C=CH at C-2),5.44(s,1H,H-12),4.10(m,2H,COOCH2),3.84(dt,2H,Ph3PCH2,J=16.3,5.2Hz),2.42(s,1H,H-9),2.30–0.81(m,41H,CH and CH2 in pentacyclicskeleton or alkyl chains),1.33(s,3H,CH3),1.17(s,9H,3×CH3),1.09(s,3H,CH3),0.92(s,3H,CH3),0.70(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ206.05,198.56,175.86,169.95,145.75,137.65,135.72,134.75,133.40(d,J c,p =9.9Hz),131.60,130.20(d,J c,p =12.7Hz),127.97,124.70,123.53,117.79(d,J c,p =86.3Hz),58.67,53.51,47.95,45.69,43.08,40.75,37.37,36.33,31.47,31.20,30.62,29.38,28.91,28.35,27.97,26.14,25.98,22.98,22.50,21.62,19.14,18.93,17.84,14.93.

[0049] 2-((2-chloro-5-nitrophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0050] Compound 5b: Melting point: 142.0–143.3 °C; Yield: 35%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.80–7.60(m,15H,PPh3-H),7.31(s,1H,C=CH at C-2),7.06–6.91(m,1H,Ph-H atC-2),6.61(d,2H,Ph-H at C-2,J=19.8Hz),5.52(s,1H,H-12),4.09(t,2H,COOCH2,J=6.0Hz),3.81–3.75(m,2H,Ph3PCH2),2.42(s,1H,H-9),2.23–0.72(m,31H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.31(s,3H,CH3),1.12(s,9H,3×CH3),1.09(s,3H,CH3),0.92(s,3H,CH3),0.69(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.11,198.68,175.97,169.91,156.43,136.04,135.87,134.73(d,J c,p =2.8Hz),133.29(d,J c,p =10.5Hz),130.18(d,J c,p =12.6Hz),128.72,127.93,117.69(d,J c,p =86.0Hz),115.49,106.10,100.88,77.05,62.74,59.06,56.26,53.09,47.75,45.47,44.62,43.56,42.97,40.75,37.41,36.14,31.41,30.54,28.83,28.28,27.93,26.09,25.89,23.07,22.35,19.08,17.74,15.32.

[0051] 2-((4-Fluoro-3-methylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0052] Compound 5c: Melting point: 150.1–151.5 °C; Yield: 38%; Product state: White solid; 1 1H NMR (400 MHz, CDCl3) δ 7.84–7.57 (m, 15H, PPh3-H), 7.33 (s, 1H, C=CH at C-2), 7.21 (d, 2H, Ph-H at C-2, J=6.6 Hz), 6.83 (t, 1H, Ph-H at C-2, J=9.2 Hz), 5.52 (s, 1H, H-12), 4.12–4.05 (m, 2H, COOCH2), 3.85–3.73 (m, 2H, Ph3PCH2), 2.46 (s, 1H, H-9), 2.18 (s, 3H, CH3), 2.08–0.81 (m, 23H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.33 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.07 (s, 9H, 3×CH3), 0.93 (s, 3H, CH3), 0.69 (s, 3H, CH3). 13 13C NMR (101 MHz, CDCl3) δ 207.14, 198.86, 175.88, 169.92, 135.86, 134.74 (d, J c,p =2.8 Hz), 133.86, 133.31 (d, J c,p =10.1 Hz), 130.17 (d, J c,p =12.7 Hz), 128.71, 128.63, 128.02, 124.59, 117.68 (d, J c,p =86.0 Hz), 114.73, 77.05, 62.73, 58.95, 52.94, 47.91, 45.04, 44.62, 43.93, 43.58, 43.00, 40.76, 37.37, 35.91, 31.43, 31.06, 30.55, 29.29, 28.31, 27.93, 26.10, 25.92, 22.94, 22.21, 19.19, 18.94, 17.69, 15.02, 14.25.

[0053] 2-((4-Fluoro-3-methylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0054] Compound 5d: Melting point: 128.9–130.7 °C; Yield: 10%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.93–7.59(m,17H,PPh3-H and Ph-H at C-2),7.39(s,1H,C=CH at C-2),6.89(td,1H,Ph-H at C-2,J=11.4,10.4,6.7Hz),5.62(s,1H,H-12),4.13–3.98(m,2H,COOCH2),3.90–3.77(m,2H,Ph3PCH2),2.54(s,1H,H-9),2.23(s,3H,CH3),2.10–1.42(m,38H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.40(s,3H,CH3),1.19(s,3H,CH3),1.14(s,3H,CH3),1.13(s,3H,CH3),1.11(s,3H,CH3),1.08(s,3H,CH3),0.80(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.34,199.16,176.11,135.98,134.76(d,J c,p =2.7Hz),134.36,133.68,133.51(d,J c,p =10.0Hz),131.60,130.23(d,J c,p =12.6Hz),128.14,118.08(d,J c,p =86.0Hz),77.05,63.80,59.11,53.07,48.44,45.13,44.83,43.80,43.23,41.09,36.06,31.21,29.49,28.18,26.17,23.07,22.31,19.40,17.87.

[0055] 2-((3-Trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0056] Compound 5e: Melting point: 145.7–147.0 °C; Yield: 35%; Product state: White solid; 1 HNMR (400 MHz, CDCl3) δ 7.82–7.57 (m, 17H, PPh3-H and Ph-H at C-2), 7.51–7.36 (m, 3H, Ph-H at C-2 and C=CH at C-2), 5.54 (s, 1H, H-12), 4.16–4.07 (m, 2H, COOCH2), 3.86–3.74 (m, 2H, Ph3PCH2), 2.47 (s, 1H, H-9), 2.43–0.76 (m, 27H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.34 (s, 3H, CH3), 1.17–1.07 (s, 12H, 4×CH3), 0.94 (s, 3H, CH3), 0.70 (s, 3H, CH3). 13 C NMR (101 MHz, CDCl3) δ 207.01, 198.77, 175.96, 169.97, 136.30, 135.49, 134.77 (d, J c,p =2.8 Hz), 133.35 (d, J c,p =10.1 Hz), 132.31, 130.21 (d, J c,p =12.6 Hz), 128.63, 117.74 (d, J c,p =85.9 Hz), 77.05, 62.76, 58.89, 53.07, 47.92, 45.24, 44.65, 43.61, 37.41, 36.03, 31.47, 31.09, 29.22, 28.35, 27.95, 22.97, 22.27, 19.21, 17.73, 15.09, 15.09.

[0057] 2-((3-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0058] Compound 5f: Melting point: 136.5–137.6 °C; Yield: 39%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.83–7.77(m,1H,Ph-H at C-2),7.74–7.52(m,15H,PPh3-H),7.47–7.28(m,4H,Ph-Hat C-2and C=CH at C-2),5.54(s,1H,H-12),4.03–3.88(m,2H,COOCH2),3.80–3.50(m,2H,Ph3PCH2),2.49(s,1H,H-9),2.45–0.76(m,28H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.36(s,3H,CH3),1.14(s,3H,CH3),1.09(s,3H,CH3),1.07(s,3H,CH3),1.05(s,3H,CH3),1.02(s,3H,CH3),0.73(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ206.95,198.90,175.93,170.47,136.10,135.34,134.68(d,J c,p =3.3Hz),133.42,133.22(d,J c,p =10.0Hz),132.24,130.42,130.10(d,J c,p =12.4Hz),128.81,127.81,127.12,117.74(d,J c,p =86.0Hz),77.05,63.51,58.79,52.87,48.35,45.06,44.62,44.07,43.62,43.07,40.84,37.22,35.86,31.46,30.97,30.64,29.27,28.33,28.04,27.95,26.14,25.93,22.86,22.14,19.17,17.67,14.99.

[0059] 2-((4-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphos-phonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0060] Compound 5g: Melting point: 158.2–159.7 °C; Yield: 40%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.81–7.59(m,15H,PPh3-H),7.54–7.44(m,4H,Ph-H at C-2),7.38(s,1H,C=CH atC-2),5.52(s,1H,H-12),4.14–4.06(m,2H,COOCH2),3.88–3.75(m,2H,Ph3PCH2),2.45(s,1H,H-9),2.39–1.35(m,32H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.32(s,3H,CH3),1.14(s,3H,CH3),1.12–1.06(s,9H,3×CH3),0.92(s,3H,CH3),0.69(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ206.97,198.90,175.82,170.25,139.09,135.88,134.74,133.32(d,J c,p =10.0Hz),130.16(d,J c,p =12.4Hz),129.90,127.94,124.89,117.67(d,J c,p =86.0Hz),77.05,62.66,58.86,53.00,47.95,45.21,44.60,43.94,43.56,43.03,35.94,31.43,31.02,30.54,29.19,28.30,27.92,26.07,25.90,22.93,22.22,19.14,15.03.

[0061] 2-((4-trifluoromethylphenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphos-phonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0062] Compound 5h: Melting point: 148.4–149.7 °C; Yield: 37%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.87–7.60(m,15H,PPh3-H),7.55–7.44(m,4H,Ph-H at C-2),7.39(s,1H,C=CH atC-2),5.61(s,1H,H-12),4.12–3.96(m,2H,COOCH2),3.81–3.71(m,2H,Ph3PCH2),2.51(s,1H,H-9),2.33–1.41(m,32H,CH and CH2 in pentacyclic skeleton or alkyl chains),1.37(s,3H,CH3),1.18(s,3H,CH3),1.12(s,6H,2×CH3),1.07(s,6H,2×CH3),0.77(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.26,199.15,176.00,170.75,139.08,136.06,134.75(d,J c,p =2.6Hz),134.65,133.53,133.37(d,J c,p =10.2Hz),130.19(d,J c,p =12.4Hz),128.38,127.99,125.03,124.99,117.89(d,J c,p =86.1Hz),77.05,63.68,58.96,52.98,48.30,45.21,44.71,43.70,43.18,40.92,37.35,36.00,31.55,31.07,30.73,29.34,28.43,28.09,26.88,26.22,26.04,22.99,22.20,19.26,17.76,15.15.

[0063] 2-((4-fluorophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0064] Compound 5i: Melting point: 150.2–151.4 °C; Yield: 32%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.91–7.58(m,15H,PPh3-H),7.51–7.32(m,3H,Ph-H at C-2and C=CH at C-2),7.05–6.83(m,2H,Ph-H at C-2),5.53(s,1H,H-12),4.16–4.04(m,2H,COOCH2),3.83(td,2H,Ph3PCH2,J=12.4,5.7Hz),2.47(s,1H,H-9),2.26–0.81(m,25H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.33(s,3H,CH3),1.17–1.04(s,12H,4×CH3),0.94(s,3H,CH3),0.70(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.11,198.95,175.88,170.10,135.54,134.77,134.75,133.36(d,J c,p =9.9Hz),131.95,131.87,130.19(d,J c,p =12.5Hz),128.02,117.74(d,J c,p =85.8Hz),115.21,114.99,77.05,62.77,58.99,52.92,45.05,44.64,44.09,43.60,43.05,40.80,37.37,35.90,31.47,31.09,30.58,29.34,28.33,27.95,26.12,25.95,22.95,22.22,19.21,18.99,17.70,15.05.

[0065] 2-((4-fluorophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0066] Compound 5j: Melting point: 134.5–135.8 °C; Yield: 41%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.85–7.59(m,16H,PPh3-H and Ph-H at C-2),7.44–7.40(m,2H,Ph-H at C-2and C=CH at C-2),7.00–6.93(m,2H,Ph-H at C-2),5.62(s,1H,H-12),4.10–3.97(m,2H,COOCH2),3.90–3.73(m,2H,Ph3PCH2),2.54(s,1H,H-9),2.32–1.45(m,34H,CH and CH2 inpentacyclic skeleton or alkyl chains),1.40(s,3H,CH3),1.19(s,3H,CH3),1.14(s,3H,CH3),1.12(s,3H,CH3),1.10(s,3H,CH3),1.07(s,3H,CH3),0.79(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.32,199.22,176.06,135.56,134.75(d,J c,p =2.9Hz),133.48(d,J c,p =10.0Hz),132.22,132.13,130.21(d,J c,p =12.5Hz),128.07,118.00(d,J c,p =86.0Hz),115.41,115.19,63.79,59.09,52.95,48.48,45.08,44.79,44.44,43.78,43.22,41.04,37.38,35.98,31.62,31.15,30.81,29.53,28.49,28.16,26.31,26.12,23.03,22.25,19.36,17.83,15.16.

[0067] 2-((4-chlorophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0068] Compound 5k: Melting point: 141.4–143.2 °C; Yield: 33%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.85–7.62(m,15H,PPh3-H),7.41–7.31(m,3H,Ph-H at C-2and C=CH at C-2),7.21(d,2H,Ph-H,J=8.3Hz),5.53(s,1H,H-12),4.15–4.08(m,2H,COOCH2),3.95–3.81(m,2H,Ph3PCH2),2.47(s,1H,H-9),2.29–0.84(m,33H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.35(s,3H,CH3),1.16(s,3H,CH3),1.10(s,9H,3×CH3),0.96(s,3H,CH3),0.72(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.05,198.93,175.91,170.15,135.39,134.77(d,J c,p =2.3Hz),134.32,134.11,133.87,133.46(d,J c,p =10.0Hz),131.26,130.23(d,J c,p =12.5Hz),128.32,117.84(d,J c,p =85.6Hz),77.05,59.02,53.04,48.07,45.17,44.14,43.66,43.11,40.88,37.41,35.98,31.53,31.15,29.38,28.39,28.03,26.01,23.01,22.29,19.26,19.04,17.76,15.11.

[0069] 2-((4-chlorophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0070] Compound 5l: Melting point: 148.0–149.7 °C; Yield: 21%; Product state: White solid; 1 HNMR(400MHz,CDCl3)δ7.84–7.60(m,15H,PPh3-H),7.40–7.32(m,3H,Ph-H at C-2and C=CH at C-2),7.23(d,2H,Ph-H,J=8.4Hz),5.61(s,1H,H-12),4.09–3.95(m,2H,COOCH2),3.88–3.64(m,2H,Ph3PCH2),2.53(s,1H,H-9),2.49–0.81(m,38H,CH and CH2 in pentacyclic skeletonor alkyl chains),1.39(s,3H,CH3),1.18(s,3H,CH3),1.12(s,6H,2×CH3),1.09(s,3H,CH3),1.06(s,3H,CH3),0.78(s,3H,CH3). 13 C NMR(101MHz,CDCl3)δ207.27,199.20,176.04,170.71,135.26,134.77,134.75,134.30,134.03,133.41(d,J c,p =10.1Hz),131.44,130.20(d,J c,p =12.5Hz),128.41,128.03,117.91(d,J c,p =85.6Hz),63.76,59.03,52.91,48.42,45.09,44.75,43.74,43.20,40.99,37.36,35.96,31.58,31.10,30.77,29.46,28.46,28.14,26.26,26.08,23.01,22.22,19.32,17.79,15.16.

[0071] 2-((4-bromophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0072] Compound 5m: Melting point: 125.0–126.9 °C; Yield: 34%; Product state: white solid; 1 H NMR (400 MHz, CDCl3) δ 7.85–7.57 (m, 15H, PPh3-H), 7.39–7.20 (m, 4H, Ph-H at C-2 and C=CH at C-2), 7.05–6.88 (m, 1H, Ph-H at C-2), 5.51 (s, 1H, H-12), 4.10 (dd, 2H, COOCH2, J = 11.4, 5.4 Hz), 3.83–3.73 (m, 2H, Ph3PCH2), 2.45 (s, 1H, H-9), 2.22–0.92 (m, 28H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.32 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.10 (s, 9H, 3×CH3), 0.94 (s, 3H, CH3), 0.70 (s, 3H, CH3). 13 C NMR (101 MHz, CDCl3) δ 207.06, 198.95, 178.54, 170.19, 137.28, 134.78, 133.54 (d, J c,p =10.2 Hz), 131.63, 130.26 (d, J c,p =12.6 Hz), 117.50, 62.90, 48.12, 45.24, 43.14, 40.89, 37.44, 36.03, 31.57, 30.69, 29.38, 28.43, 26.24, 26.07, 23.06, 22.34, 17.81, 15.15.

[0073] 2-((4-bromophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0074] Compound 5n: Melting point: 145.6–147.3 °C; Yield: 24%; Product state: White solid; 1 HNMR (400 MHz, CDCl3) δ 7.86–7.58 (m, 15H, PPh3-H), 7.40–7.25 (m, 5H, Ph-H at C-2 and C=CH at C-2), 5.60 (s, 1H, H-12), 4.06–3.93 (m, 2H, COOCH2), 3.79–3.64 (m, 2H, Ph3PCH2), 2.51 (s, 1H, H-9), 2.29–1.41 (m, 32H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.37 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.10 (s, 6H, 2×CH3), 1.07 (s, 3H, CH3), 1.04 (s, 3H, CH3), 0.76 (s, 3H, CH3). 13 C NMR (101 MHz, CDCl3) δ 207.24, 119.15, 175.99, 170.71, 134.76 (d, J c,p =2.9 Hz), 134.42, 134.40, 133.34 (d, J c,p =10.0 Hz), 131.62, 131.31, 130.18 (d, J c,p =12.6 Hz), 127.95, 122.35, 117.81 (d, J c,p =86.1 Hz), 77.05, 63.70, 58.98, 52.84, 48.36, 45.05, 44.71, 43.69, 43.15, 40.93, 31.53, 31.04, 29.41, 28.42, 28.07, 26.21, 26.03, 22.97, 22.17, 19.26, 17.75, 15.12.

[0075] 2-((4-Iodophenyl)methylene)-3,11-dioxo-12-en-30-(4-(triphenylphosphonio)butoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0076] Compound 5o: Melting point: 142.3–143.9 °C; Yield: 20%; Product state: White solid; 1HNMR(400MHz, CDCl3) δ 7.83–7.61 (m, 15H, PPh3-H), 7.57–7.51 (m, 2H, Ph-H at C-2), 7.30 (s, 1H, C=CH at C-2), 7.14–7.07 (m, 2H, Ph-H at C-2), 5.50 (s, 1H, H-12), 4.12–4.07 (m, 2H, COOCH2), 3.91–3.75 (m, 2H, Ph3PCH2), 2.45 (s, 1H, H-9), 2.22–1.36 (m, 30H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.32 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.09 (s, 6H, 2×CH3), 1.05 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.70 (s, 3H, CH3). 13 C NMR(101MHz, CDCl3) δ 206.97, 198.86, 175.82, 170.14, 137.15, 136.83, 135.42, 135.01, 134.75 (d, J c,p =3.0Hz), 134.57, 133.39 (d, J c,p =10.2Hz), 130.18 (d, J c,p =12.5Hz), 130.01, 127.96, 117.71 (d, J c,p =86.1Hz), 62.70, 58.93, 52.96, 48.02, 45.11, 44.62, 44.06, 43.59, 43.04, 40.80, 37.34, 35.91, 31.46, 31.06, 30.58, 29.28, 28.33, 27.97, 26.10, 25.94, 22.96, 22.23, 19.18, 18.97, 17.69, 15.04.

[0077] 2-((4-Iodophenyl)methylene)-3,11-dioxo-12-en-30-(5-(triphenylphosphonio)

[0078] pentoxycarbonyl)-18β-glycyrrhetinic acid bromide

[0079] Compound 5p: Melting point: 138.8–140.1 °C; Yield: 31%; Product state: White solid; 11H NMR (400 MHz, CDCl3) δ 7.80–7.54 (m, 17H, PPh3-H and Ph-H at C-2), 7.28 (s, 1H, C=CH at C-2), 7.11 (d, 2H, Ph-H at C-2, J=8.0 Hz), 5.58 (s, 1H, H-12), 4.04–3.94 (m, 2H, COOCH2), 3.78–3.66 (m, 2H, Ph3PCH2), 2.49 (s, 1H, H-9), 2.44–0.78 (m, 34H, CH and CH2 in pentacyclic skeleton or alkyl chains), 1.35 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.12–1.05 (s, 9H, 3×CH3), 1.02 (s, 3H, CH3), 0.75 (s, 3H, CH3). 13 13C NMR (101 MHz, CDCl3) δ 207.20, 199.10, 175.94, 170.69, 137.22, 135.24, 134.90, 134.74 (d, J c,p =2.8 Hz), 134.58, 133.31 (d, J c,p =10.0 Hz), 131.68, 130.16 (d, J c,p =12.5 Hz), 129.97, 127.91, 117.76 (d, J c,p =85.9 Hz), 63.66, 58.94, 52.78, 48.30, 45.00, 44.66, 44.38, 43.65, 43.11, 40.89, 37.27, 35.87, 31.48, 30.99, 30.68, 29.37, 28.39, 28.05, 26.16, 25.99, 22.95, 22.12, 19.21, 17.70, 15.09.

[0080] In vitro cytotoxic activity assay:

[0081] Cell types, cell culture and test procedures:

[0082] The test cells included 3 human cancer cells and 1 human normal liver cell, namely human colon cancer cell HCT-116, human neuroblastoma cell SH-SY5Y, human liver cancer cell HepG2 and human normal liver cell QSG-7701. The test compounds included 16 α,β-unsaturated carbonyl modified 18β-GA derivatives and the positive control drug doxorubicin.

[0083] The MTT assay was used to detect the cytotoxic activity of the compounds. All cell lines were seeded in 96-well plates at a density of 3 - 4×10 3 cells / well and incubated overnight at 37 °C and 5% CO2. When the cells adhered and were in good growth condition, they were treated with the test compound at a designated concentration for 48 hours, and three parallel groups with the same concentration were set up simultaneously. The negative control group was added with the same concentration of DMSO. Subsequently, 20 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 hours. The supernatant was aspirated and discarded using a pipette pump, and 150 μL of DMSO was added. Next, the optical density value (OD) was measured at 570 nm using a microplate reader. The cell viability was analyzed using IC 50 software (Prism 5.0), and the average value of three independent parallel experiments was calculated. The results of the cytotoxic activity test by the MTT method are shown in Table 1.

[0084] Table 1 Results of the cytotoxic activity test by the MTT method

[0085]

[0086]

[0087]

[0088] 1. IC 50 value: that is, the half-maximal inhibitory concentration; in this experiment, it refers to the concentration of the corresponding compound when 50% of the test cells are inhibited; all test results are mean ± SD (n = 3).

[0089] 2. Doxorubicin, a positive control drug.

[0090] In this example, the anti-proliferative activities of 16 18β-GA derivatives against 3 human cancer cell lines, namely human colon cancer cell line HCT-116, human neuroblastoma cell line SH-SY5Y, and human hepatoma cell line HepG2, as well as 1 human normal liver cell line QSG-7701, were tested using the MTT method. The results showed that all relevant derivatives exhibited high cytotoxic activity, and the IC 50 values ranged from 1.52 to 5.21 μM. However, the cytotoxic activity of all derivatives was slightly inferior to or lower than that of the positive control drug doxorubicin. Among them, compound 5a showed the best cytotoxic activity, with an IC 50 value of 1.52 μM against HCT-116 cells, which may indicate that when the substituent on the benzene ring at the C-2 position of 18β-GA is 2-Cl-5-NO2, it is beneficial to improve the cytotoxic activity of the derivatives.

[0091] The results of this example showed that alkyl TPP in the derivatives of the present invention +The number of carbon atoms in the side chain affects the activity of the derivatives. Within a certain range, the more carbon atoms there are, the higher the cytotoxic activity of the derivatives. For example, for compounds 5g and 5h, and the alkyl TPP of compounds 5i and 5j + The number of carbon atoms in the side chain has a significant impact on the cytotoxic activity. When n = 5, generally lower IC 50 values are shown.

[0092] In addition, the effect of the benzene ring substituents outside the C-2 position of the derivative on the cytotoxic activity. Generally speaking, when there are electron-withdrawing substituents on the benzene ring, it is beneficial to the cytotoxic activity of the compound. For example, for 5-NO2 and 4-CF3 substituents, the IC 50 value ranges of compounds 5a and 5h are 1.52 - 3.46 μM and 1.58 - 1.66 μM respectively. At the same time, the stronger the electron-withdrawing ability of the group, the higher the cytotoxic activity of the compound. For example, the activity of the halogen substituents at the 4-position is generally lower than that of compounds 5a and 5h. Similarly, the compounds substituted with halogen at the 4-position generally conform to this rule, and the activity is roughly 4-F > 4-Cl > 4-Br > 4-I. Among them, the compound 5j with 4-F substituent has the best activity, and the IC 50 value range is 1.60 - 2.18 μM.

[0093] Summary

[0094] In this example, an active group with α,β-unsaturated carbonyl was introduced into the structural nucleus of 18β-GA, and 16 novel α,β-unsaturated carbonyl-modified triphenylphosphine 18β-GA derivatives were designed and synthesized. The cytotoxic activities of the above derivatives against 3 human cancer cell lines, namely HepG2, HCT-116, SH-SY5Y and 1 human normal liver cell line QSG-7701 were tested by the MTT method. The results showed that the growth inhibitory activities of derivatives 5a, 5h and 5j against HCT-116, SH-SY5Y and HepG2 were more significant, and the IC 50 value ranges were 1.52 - 3.46 μM, 1.58 - 1.66 μM and 1.60 - 2.18 μM respectively. The structure-activity relationship showed that introducing an active group with α,β-unsaturated carbonyl outside the C-2 position of 18β-GA could significantly improve the inhibitory activity. In addition, the substituents on the benzene ring at the C-2 position were crucial for the activity of the derivatives, that is, the stronger the electron-withdrawing effect of the substituents and the more the number of electron-withdrawing groups, the higher the activity of the derivatives.

[0095] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An α,β-unsaturated carbonyl class of 18β-glycyrrhetinic acid derivatives, characterized in that, Its chemical structural formula is as follows: Wherein, n is 4 or 5, and R1 is a mono-substituted or di-substituted phenyl group, and the substituents of the phenyl group are halogen, trifluoromethyl, methyl and / or nitro group.

2. The α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative according to claim 1, characterized in that, R1 is 2-chloro-5-nitrophenyl, 4-fluoro-3-methylphenyl, 3-trifluoromethylphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, p-bromophenyl or p-iodophenyl.

3. The α,β-unsaturated carbonyl class of 18β-glycyrrhetinic acid derivatives according to claim 1, characterized in that, R1 is 2-chloro-5-nitrophenyl, p-trifluoromethylphenyl or p-fluorophenyl.

4. The α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative according to claim 1, characterized in that, When n is 4, R1 is 2-chloro-5-nitrophenyl; when n is 5, R1 is p-trifluoromethylphenyl or p-fluorophenyl.

5. A method for preparing the α,β-unsaturated carbonyl class of 18β-glycyrrhetinic acid derivatives as claimed in claim 1, characterized in that, The step of reacting with 18β-glycyrrhetinic acid as a raw material according to the following reaction route; Wherein, R1 and n are as described in claim 1.

6. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative as described in claim 5, characterized in that, 18β-glycyrrhetinic acid undergoes a hydroxyl oxidation reaction with an oxidant to obtain compound 2, the compound undergoes an aldol condensation reaction with R1CHO to obtain compound 3, compound 3 undergoes a substitution reaction with 1,4-dibromobutane or 1,5-dibromopentane to obtain compound 4, and compound 4 undergoes a quaternary phosphonation reaction with triphenylphosphine to obtain compound 5, namely α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative.

7. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative according to claim 6, characterized in that, The oxidant is pyridinium chlorochromate.

8. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative as described in claim 6, characterized in that, The temperature of the hydroxyl oxidation reaction is from 0 °C to room temperature.

9. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative as claimed in claim 6, characterized in that, In the aldol condensation reaction, potassium hydroxide or sodium hydroxide is added and the reaction is carried out at 40-50 °C.

10. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative as claimed in claim 6, characterized in that, The substitution reaction is carried out at room temperature.

11. The preparation method of the α,β-unsaturated carbonyl class 18β-glycyrrhetinic acid derivative according to claim 6, characterized in that, The temperature of the quaternary phosphonation reaction is 75-85 °C.

12. A pharmaceutical composition, characterized in that, It includes the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical preparation, characterized in that, It includes the α,β-unsaturated carbonyl 18β-glycyrrhetinic acid derivative according to any one of claims 1 to 4 or the pharmaceutical composition and pharmaceutically acceptable excipients according to claim 12.

14. Use of the α,β-unsaturated carbonyl class of 18β-glycyrrhetinic acid derivatives according to any one of claims 1 to 4, the pharmaceutical composition according to claim 12, or the pharmaceutical preparation according to claim 13 in the preparation of an anti-tumor drug, characterized in that, The tumor is liver cancer, colon cancer, neuroblastoma.

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