CN104926912A - Synthesis and purpose of glycyrrhetinic acid derivative - Google Patents

Abstract

The present invention designs and synthesizes a glycyrrhetinic acid derivative as a carrier, which is connected with norcantharidin and its derivatives by an ester bond, and expects to obtain a lead compound that has liver targeting effect, prolongs the action time, reduces toxic and side effects, and increases the curative effect against liver cancer. , to provide model compounds for the study of liver-targeted anti-liver cancer drugs. Firstly, the two parts of glycyrrhetinic acid molecule, C11 and C30, are respectively reduced and chemically modified into methyl esters to form glycyrrhetinic acid (GA) and 18α-glycyrrhetinic acid. Acid and its derivatives, using it as the backbone molecule, condensed with norcantharidin (NCTD) and its derivatives through a series of reactions to form prodrugs, and carried out cell activity screening tests on 5 target compounds, and synthesized 3 A series of 13 target compounds were characterized by carbon spectrum, hydrogen spectrum, and mass spectrometry; MTT method was used to investigate the effects of different concentrations of 5 target compounds on the proliferation of liver cancer HepG2 cells, and the results showed that the inhibitory effect was time- and dose-dependent. mL inhibition rate was the highest.

Description

Synthesis and application of glycyrrhetinic acid derivatives

technical field

The invention relates to a preparation method and application of a glycyrrhetinic acid derivative, in particular to a liver-targeting anticancer drug of norcantharidin with glycyrrhetinic acid as a carrier.

Background technique

Licorice (Glycyrrhiza root) belongs to leguminous plants, distributed in western my country and European countries such as Russia, and widely used medicinally in many countries. Its main pharmacologically active substances are glycyrrhizic acid (glycyrrhizic acid, GL for short) and its aglycone glycyrrhetinic acid (glycyrrhetinic acid, GA for short), etc. (Figure 1). Glycyrrhizic acid drugs are mainly hydrolyzed by gastric acid or decomposed into glycyrrhetinic acid by β-glucuronidase in the liver in the human body, and then partly generate 3-epi-glycyrrhetinic acid and a small amount of 3- dehydroglycyrrhetinic acid for pharmacological activity. Therefore, the effect of glycyrrhizic acid drugs is essentially the effect of glycyrrhetinic acid. GA has various functions such as antibacterial, anti-inflammatory, anti-virus, anti-tumor, anti-oxidation, anti-arrhythmia and immune regulation, so it has been widely studied.

Negishi et al. confirmed that the membrane fraction of rat liver cells contains a large number of GA-specific binding sites, and the binding of GA to this site is saturable and highly specific, and the site has protein properties. In recent years, glycyrrhetinic acid derivatives have become a research hotspot as a good liver-targeting material. Shiro et al. reported that the carrier involved in the active transport of glycyrrhizic acid and glycyrrhetinic acid may be organic anion transporting polypeptide (OATP). The derivatization of the 3-hydroxyl or 30-carboxyl group in the GA molecule may be beneficial to the affinity of the receptor and improve the liver-targeting performance of the carrier. Jin Hui et al. synthesized the 30-amide derivatives of glycyrrhetinic acid and 11-deoxyglycyrrhetinic acid (DGA), and conducted experiments on the uptake of GA by Wistar isolated rat liver cells and the inhibition of GA derivatives on the uptake of GA by liver cells. The uptake of GA by isolated rat hepatocytes was studied, and the results showed that the synthetic GA derivatives could competitively inhibit the uptake of GA by isolated rat hepatocytes, indicating that the 30-amide derivatives of GA and DGA competed with GA. The same receptors on the cell membrane, so they can all be used as liver targeting carriers; in addition, the results of the inhibition constants of the derivatives on GA uptake showed that the compounds with amino acid esters attached to the 30-carboxyl group and amino acids attached to the 30-carboxyl group did not significantly inhibit the uptake of GA. The difference indicates that in the GA molecule, whether there is an anionic group near the 30-carboxyl group has little effect on the transport of GA, and the bond should be formed at the 30-carboxyl group when connecting other molecules. Ma Shuyan et al. linked the 3-hydroxyl groups of 18β- and 18α-methyl glycyrrhetinate with the anticancer active structural fragment of the anticancer drug cyclophosphamide—phosphoryl mustard dichloride to make hepatic liver with anticancer potential. Targeting prodrugs 18β- and 18α-mustine are expected to achieve active liver targeting.

Norcantharidin (NCTD) is a derivative of cantharidin (Figure 2). It is a new type of anti-tumor drug independently developed by my country with strong anti-tumor activity and unique effects of increasing white blood cells, protecting liver cells, and regulating immunity. , NCTD has comprehensive anti-tumor effects. It can inhibit tumor cell proliferation by regulating cell cycle and inhibiting DNA replication; activate mitochondrial pathway, adjust the ratio of pro-apoptotic and anti-apoptotic genes and proteins to induce tumor cell apoptosis, etc. These two aspects work together to achieve anti-tumor effect, and its toxicity is obviously lower than that of cantharidin, while the anti-tumor effect is better than that of cantharidin. It is clinically used for the treatment or adjuvant therapy of liver cancer, showing good curative effect. Norcantharidin (NCTD) is mainly used clinically in tablets and injections. Wei Chunming et al. studied the pharmacokinetics and tissue distribution of norcantharidin in mice. The results showed that the absorption of 3H-norcantharidin in mice was Quickly, the liver distributes less and eliminates quickly, while the kidney distributes more, but eliminates quickly. The results of in vivo distribution indicate that the concentration of norcantharidin in the treatment of liver cancer reaches the target organ is low, so it not only reduces the efficacy of the drug but also increases the toxicity of other organs. In order to better exert its curative effect, reduce toxic and side effects, and prolong the action time.

The liver targeting of drugs is mainly achieved through two ways. One way is to pass the drug through special carriers such as liposomes or nanocarriers, and utilize the phagocytosis of the liver endothelial reticulum system; The bond binds the small molecule drug to the targeting molecule, and uses the receptor on the surface of the stem cell to achieve receptor-mediated endocytosis, such as the prodrug mediated by the asialoglycoprotein receptor.

Fan Feng et al. used norcantharidin as a raw material, modified with various small molecule amino acids, and underwent acylation, hydrogenolysis, glycosidation and deacetylation reactions to synthesize norcantharidin-galactose (NCTD-Gal) derivatives. It is expected to obtain a liver-targeted anticancer prodrug, and a preliminary anti-tumor experiment in mice was carried out on the product a. The results showed that the tumor inhibition rate of the medium and high dose groups of a was significantly higher than that of the NCTD group, indicating that galactosidation has a positive effect on The anti-cancer effect of NCTD has been improved to a certain extent, but the liver-targeting property needs further research. Hu Zhanhong et al. used ethylenediamine as the connecting arm to link norcantharidin and lactobionic acid to synthesize the prodrug lactosyl-norcantharidin (lac-NCTD), expecting to utilize the galactose residue in the molecular structure It can be specifically recognized by AS-GP-R of liver cells in vivo to achieve active liver targeting. Acute toxicity studies showed that the toxicity of Lac-NCTD was far less than that of NCTD, and it was a safe and non-toxic new compound. Wu Chao et al. prepared glycyrrhetinic acid derivatives (stearyl glycyrrhetinate-3-O-galactoside, Gal-GAOSt) modified norcantharidin liposomes by thin film dispersion method, and administered them via the tail vein of mice. Drug, adopt HPLC method to detect the change of the concentration of norcantharidin in mouse liver and kidney over time, compare with the norcantharidin aqueous solution group, and use targeting index TI=(AUC) _NC-SOL /(AUC) _{Gal-GAOStNC-LP} was used to determine the targeting of glycyrrhetinic acid derivative modified norcantharidin liposomes (Gal-GAOStNC-LP) to major organs. The results showed that Gal-GAOStNC-LP has liver targeting, and the modified liposomes have obvious targeting, which can improve drug efficacy and reduce toxic and side effects.

Contents of the invention

The present invention uses glycyrrhetinic acid and glycyrrhetinic acid derivatives as carriers, and synthesizes three series of target compounds (Fig. Targeting effect, prolonging the action time, reducing toxic and side effects, and increasing the anti-hepatic cancer effect of the lead compound, laying the foundation for the pharmacological screening and structure-activity relationship research of liver-targeting prodrugs.

In order to improve fat solubility and cell penetration and reduce the side effects of pseudo-aldosterone in the mother, chemical modification of C ₁₁ and C ₃₀ in the glycyrrhetinic acid molecule, reduction of C ₁₁ -carbonyl and formation of C ₃₀ -COOH Deoxyglycyrrhetinic acid, glycyrrhetinic acid methyl ester and deoxyglycyrrhetinic acid methyl ester are used as prodrug carriers; glycyrrhetinic acid has two optical isomers, and the liver tissue distribution capacity of 18α-glycyrrhetinic acid is higher than that of 18β- Glycyrrhetinic acid is stronger, and the present invention transforms glycyrrhetinic acid (containing 97% 18β-glycyrrhetinic acid) into its optical isomer 18α-glycyrrhetinic acid and its methyl ester as the carrier of the prodrug.

The structure-activity relationship research of cantharidin and norcantharidin shows that the epoxy bridge is its anticancer pharmacophore, and the present invention uses the norcantharidin and its monoethyl ester (3-ethoxyformyl- 7-oxabicyclo[2,2,1]heptane-2-carboxylic acid), cis 7-oxabicyclo[2,2,1]-heptane-5-ene-2,3-dicarboxylic anhydride , as an anticancer functional group; the present invention will synthesize the anticancer functional group with the carrier glycyrrhetinic acid and its methyl ester (M1 series), deoxyglycyrrhetinic acid and its methyl ester (M2 series), 18α-glycyrrhetinic acid Acid and its methyl ester (M3 series) 3-hydroxyl ester to synthesize three series of target compounds M1-3. Among them, M-1 series: glycyrrhetinic acid and its methyl ester as carrier series; M-2 series: deoxyglycyrrhetinic acid and its methyl ester as carrier series; M-3 series: 18α-glycyrrhetinic acid and its methyl ester as carrier series Vector series.

The present invention particularly relates to a class of compounds and stereoisomers thereof, wherein the compounds are selected from Table 1:

Table 1 Structure and name of compounds (M-1~3 series)

The compound of the present invention is confirmed by hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR), carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) and mass spectrometry (MS). After synthesis, the MTT method is used to investigate the effect of the target compound on the cell activity of the tumor cell line HepG2 .

The present invention also relates to a composition comprising the above compound and a pharmaceutically acceptable carrier.

The present invention also relates to the use of the above-mentioned composition in treating liver cancer by administering a therapeutically effective amount of the composition to a patient.

Description of drawings

Figure 1: Chemical structural formulas of glycyrrhizic acid and glycyrrhetinic acid

Figure 2: Structure of norcantharidin

Figure 3: Design of Compounds

Figure 4: Synthetic route of glycyrrhetinic acid derivatives

Figure 5: Synthetic route of 3-ethoxyformyl-7-oxabicyclo[2,2,1]heptane-2-carboxylic acid

Figure 6: Synthetic route of cis 7-oxabicyclo[2,2,1]-heptane-5-ene-2,3-dicarboxylic anhydride

Figure 7: Compound synthesis route

Figure 8: Proton Nuclear Magnetic Resonance Spectrum ( ¹ H-NMR), Carbon Nuclear Magnetic Resonance Spectrum ( ¹³ C-NMR) and Mass Spectrometry (MS) spectrum of the compound

Figure 9: Structural formula of compound M-1-b

Figure 10: 24 hours of different concentrations of five compounds on liver cancer HepG2 cell proliferation inhibition rate curve

Figure 11: Curves of inhibition rate of proliferation of 5 compounds at different concentrations for 48 hours on liver cancer HepG2 cells

Detailed ways

Embodiment 1. compound synthesis experiment

1.1 Experimental materials

1.1.1 Main experimental instruments

XT4A binocular micro melting point apparatus, temperature uncorrected; RE-52A rotary evaporator; CCA-20 circulating water pump; CL-2 magnetic stirrer; ZF-1 ultraviolet analyzer; KQ-250DE numerical control Ultrasonic cleaner; Agilent 1100LC-MSD-Trap-SL mass spectrometer; Bruker 400 nuclear magnetic resonance spectrometer and avanceIII 500 nuclear magnetic resonance spectrometer, the solvent is DMSO-d ₆ or CDCl ₃ , and TMS is the internal standard.

1.1.2 Main reagents and materials

Glycyrrhetinic acid (97% 18β-glycyrrhetinic acid), norcantharidin, tetrahydrofuran (anhydrous), triethylamine (anhydrous), acetic anhydride (anhydrous), N-methylmorpholine, oxalyl chloride, Anhydrous sodium carbonate, DCC (dicyclohexylcarbodiimide), DMAP (4-dimethylaminopyridine), DMF (N,N-dimethylformamide), zinc powder, 4A molecular sieve, VLC: Qingdao Ocean Chemical The factory produces silica gel H 200-300 mesh and 300-400 mesh.

1.2 Experimental steps

1.2.1 Preparation of 18α-glycyrrhetinic acid

Add 2.0g (35.71mmol) KOH and a certain amount of diethylene glycol into the three-necked flask, stir at 100°C for 10min, add 5g (10.64mmol) glycyrrhetinic acid to the above system, and reflux for 2h. Cool to room temperature, add a certain amount of distilled water, adjust the pH value to weak acidity with concentrated hydrochloric acid, filter with suction, wash the filter cake with distilled water, extract with hot acetone after vacuum drying, suction filter while hot, and use absolute ethanol repeatedly after vacuum drying of the filter cake Recrystallization gave 2.3 g (4.89 mmol) of pure white powder, yield 46%, melting point: 325-328 °C.

1.2.2 Synthesis of methyl glycyrrhetinate

In a 1000mL three-necked bottle, add 14.0g (0.03mol) of glycyrrhetinic acid and 700mL of anhydrous methanol, and start stirring. After the glycyrrhetinic acid is completely dissolved, add 21mL of concentrated sulfuric acid dropwise with a dropping funnel. After the addition is complete, heat to reflux for 24h. After the reaction was completed, the reaction solution was naturally cooled, and after crystallization was precipitated, a large amount of solids precipitated after being refrigerated in the refrigerator for half an hour, filtered with suction, washed the filter cake with an appropriate amount of distilled water, drained, and dried to obtain 11.3 g of white solid powder. Yield 88%. Melting point: 254-256°C.

Preparation in the same way: methyl 18-α glycyrrhetinate. After the reaction, the reaction solution was transparent. The solvent was distilled off under reduced pressure to obtain a white solid with a yield of 68%. Melting point: 274-276°C.

1.2.3 Synthesis of deoxyglycyrrhetinic acid

Dissolve 0.88g (0.0019mol) of glycyrrhetinic acid in 200mL of absolute ethanol, add 18g (0.28mol) of zinc powder, heat to reflux, add 160mL of concentrated hydrochloric acid dropwise within 2 hours, and continue to add dropwise of absolute ethanol to The solution was clarified, and continued to reflux for 10 minutes to make it fully reacted. The unreacted zinc powder was removed by hot filtration, and the filtrate was collected, placed at room temperature, and precipitates were precipitated. After standing for 24 hours, suction filtered to obtain 0.65 g of white precipitate (coarse), recrystallized from methanol The pure product was obtained with a yield of 76% and a melting point of 327-329°C.

Preparation by the same method: methyl deoxyglycyrrhetinate, white solid, yield 72%, melting point: 288-291°C.

1.2.4 Synthesis of 3-ethoxyformyl-7-oxabicyclo[2,2,1]heptane-2-carboxylic acid

In a 500mL round bottom flask, add 5g (0.03mol) of norcantharidin and 250mL of absolute ethanol, and reflux for 12h. 88.9%, melting point: 109-111°C.

1.2.5 Synthesis of 3-ethoxyformyl-7-oxabicyclo[2,2,1]heptane-2-carbonyl chloride

Add 3g (0.014mol) monoethyl norcantharidate, 35mL dichloromethane, 2 drops of DMF into a 100mL three-necked flask, slowly add 2.38mL (0.028mol) oxalyl chloride dropwise under ice-salt bath conditions, dropwise, and stir at room temperature After 4 hours, the reaction was completed, and the solvent was distilled off under reduced pressure to obtain 2.02 g of a light yellow solid, with a yield of 62%.

1.2.6 Synthesis of 3β-[-2-formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxyl-glycyrrhetinic acid (M-1-a)

Add 0.94 g (0.002 mol) of glycyrrhetinic acid, 2.00 g (0.012 mol) of norcantharidin, 0.32 g (0.002 mol) of DMAP, and 40 mL of dichloromethane into a 150 mL three-necked flask. Heat to reflux for 20 hours, and monitor the reaction by TLC [developing solvent: V (chloroform): V (methanol)]. After the reaction was completed, it was cooled to room temperature, and the reaction solution was washed three times with 10% citric acid and then concentrated to a yellow oily liquid. Methanol-water recrystallization, suction filtration to obtain a white solid. The white solid was recrystallized from ethanol to obtain a white powder. It was separated by silica gel column chromatography, the eluent was dichloromethane-methanol, gradient elution, and a white needle-like solid was obtained. Melting point: 302°C-305°C. Preparation by the same method: 3β-[-2-formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxyl-glycyrrhetinic acid methyl ester (M-1-b): Separation by silica gel column chromatography, the eluent is petroleum ether - ethyl acetate, gradient elution, to obtain a white powdery solid. Melting point: 194°C-196°C;

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-5-ene-3-carboxy]-oxyl-glycyrrhetinic acid (M-1-e): silica gel Separation by column chromatography, the eluent is dichloromethane-methanol, gradient elution, to obtain a white powdery solid;

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-5-ene-3-carboxy]-oxyl-glycyrrhetinic acid methyl ester (M-1-f): Separation by silica gel column chromatography, the eluent is petroleum ether - ethyl acetate, gradient elution, to obtain a white powdery solid.

3β-[-2-formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxyl-deoxyglycyrrhetinic acid (M-2-g): silica gel column chromatography Separation, the eluent is petroleum ether-ethyl acetate, gradient elution, and a white powdery solid is obtained. Melting point: 287°C-290°C;

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxy-deoxyglycyrrhetinic acid methyl ester (M-2-h): silica gel column Chromatographic separation, the eluent was dichloromethane-methanol, gradient elution, and a white powdery solid was obtained. Melting point: 197-199°C;

3β-[-2-formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxyl-18α-glycyrrhetinic acid (M-3-j): through silica gel column layer Analysis and separation, the eluent was petroleum ether-ethyl acetate, and gradient elution gave a white powdery solid. Melting point: 283°C-288°C;

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-carboxy]-oxy-18α-methyl glycyrrhetinate (M-3-k): silica gel Separation by column chromatography, the eluent is petroleum ether-ethyl acetate, gradient elution, and a white powdery solid is obtained. Melting point: 176°C-179°C;

1.2.7 3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-ethoxyformyl]-oxy-glycyrrhetinic acid (M-1-c) synthesis

Weigh 0.282g (0.006mol) of glycyrrhetinic acid and dissolve it in 12mL of dichloromethane, place in a 50mL single-necked bottle, 0.42g (0.018mol) of monoethyl norcantharidinate monoacyl chloride, 0.4mL of N-methylmorpholine (0.0036mol) was dissolved in 20mL of dichloromethane, placed in the dropping funnel, slowly added dropwise under ice-salt bath conditions, heated to reflux for 10 hours, and TLC monitored the reaction [developing agent: V (chloroform): V (methanol) ], after the reaction was completed, cooled to room temperature, the reaction solution was washed 3 times with 10% citric acid solution, the lower layer (organic layer) was removed, concentrated, methanol-water recrystallized, and suction filtered to obtain a white solid. Separation by silica gel column chromatography, the eluent is dichloromethane-methanol, gradient elution, to obtain a white powdery solid, melting point: 284-287 ° C.

Preparation with the same method: 3β-[-2-formyl-7-oxabicyclo[2,2,1]heptane-3-ethoxyformyl]-oxyl-glycyrrhetinic acid methyl ester (M-1- d): Ethanol recrystallization to obtain a white powdery solid, melting point: 243-245°C.

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-ethoxyformyl]-oxy-deoxyglycyrrhetinic acid methyl ester (M-2-i): Separation by silica gel column chromatography, the eluent is petroleum ether - ethyl acetate, gradient elution, to obtain a white powdery solid.

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-ethoxyformyl]-oxyl-18α-glycyrrhetinic acid (M-3-l): DMAP As a catalyst, it was separated by silica gel column chromatography, the eluent was dichloromethane-methanol, gradient elution, and a white powdery solid was obtained, melting point: 266-269°C.

3β-[-2-Formyl-7-oxabicyclo[2,2,1]heptane-3-ethoxyformyl]-oxy-18α-glycyrrhetinic acid methyl ester (M-3-m) : Separation by silica gel column chromatography, the eluent is petroleum ether-ethyl acetate, gradient elution, to obtain a white powdery solid, melting point: 261-263 ° C.

Embodiment .2 compound pharmacological experiment

2.1 Main reagents and materials

Human liver cancer cell line HepG2 was purchased from Shanghai Cell Bank Center, Chinese Academy of Medical Sciences, DMEM medium, fetal bovine serum (Hyclone, American Hyclone Company), trypsin (Huamei Bioengineering Company), dimethyl sulfoxide (Sigma Company) ), phosphate buffered solution (PBS), 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT), etc.

2.2 Experimental operation

2.2.1 Solution preparation

①MTT solution (5g/L)

Weigh 250mg of MTT, put it into a small beaker, add 50mL of PBS (0.01mol/L, pH7.4) and stir on an electromagnetic stirrer for 30min, filter and sterilize with a 0.22μm microporous membrane, and dispense under aseptic conditions. Store at 4°C. Valid for two weeks.

②10% fetal bovine serum DMEM culture medium

Draw 90mL DMEM culture solution, add 10mL fetal bovine serum, mix well and set aside.

③0.25% trypsin

Weigh 0.25g of trypsin, add it to a volumetric flask, then add 80mL of PBS buffer to dissolve it, put it in a refrigerator at 4°C overnight, use PBS buffer to make up to 100mL the next day, and filter it with a 0.22μm microporous membrane Sterilized by filtration, aliquoted under sterile conditions, and stored at -20°C.

④PBS buffer

The PBS buffer solution was dissolved according to the instructions and then sterilized by high pressure.

⑤75% alcohol

Measure 250mL of distilled water, add it to 750mL of absolute ethanol, mix well, and set aside.

2.2.2. MTT method to detect drugs inhibiting the proliferation of liver cancer HepG2 cells

① Cell recovery and culture: Take out HepG2 cells frozen in liquid nitrogen, place them in a water bath at 37°C to thaw quickly, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 10% inactivated fetal bovine serum DMEM culture medium, cultured in a 37°C, 5% _CO2 incubator.

②Cell subculture and cell counting: After the cells are overgrown with the cell culture flask, subculture, use a pipette to absorb the culture medium in the culture flask, add 3 mL of PBS buffer to wash once, and digest with 0.25% trypsin until most of the cells After rounding, DMEM medium containing serum was added to terminate the digestion. Use a pipette gun to gently blow into a single-cell suspension, centrifuge, add DMEM culture solution to resuspend and pour into a culture bottle.

Cell counting: pipette 20 μL of cell suspension into a 1.5 mL sterile centrifuge tube, add 180 μL of culture medium and mix well, then drop 10 ul of the diluted cell suspension onto a clean cell counting plate for counting, and finally count as 6×10 The density of ⁵ /mL was inoculated into a 96-well cell culture plate (3 wells in parallel), with a volume of 100 μL per well, and placed in a 37° C., 5% CO ₂ incubator for 12 hours.

3. Dosing: The next day, observe that 98% of the cells are in good growth state, and the 96-well cell culture plate is covered, and 100 μL of each well-diluted medicinal solution of different concentrations is added respectively, and the final concentration of the drug is divided into 14 μg/mL, 7μg/mL, 3.5ug/mL, 1.4ug/mL, four dose groups and blank control group, solvent control group (0.1% DMSO) a total of 14 groups, each concentration group set 3 duplicate wells, and then placed at 37 ℃ , 5% CO ₂ incubator.

④ Add MTT for color development: after incubation for 24h and 48h respectively, add 20μL/well MTT (5g/L) and incubate in a 37°C, 5% _CO2 incubator for 4h, remove the original solution from the incubator, and add 150μL/well Pore in DMSO and shake for 3 minutes to fully dissolve the crystals.

⑤ Measuring OD value: Measure the OD value at 490nm on a microplate reader. The average value of 3 replicate experiments was taken. The inhibition rate of cell growth was calculated according to the following formula: inhibition rate%=(blank control well OD490−experimental well OD490)/control well OD490×100%.

Embodiment 3: compound synthesis and pharmacological experiment result

Thirteen target compounds that have not been reported in the literature were synthesized, and the structural formula was confirmed by hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR), carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) and mass spectrum (MS) (Figure 8). The obtained target compounds are easily soluble in tetrahydrofuran and ethyl acetate, soluble in methanol and ethanol, and almost insoluble in water.

3.1 Compound spectrum analysis

Taking the target compound M-1-b (molecular formula C ₃₉ H ₅₆ O ₈ , molecular weight 652.40, see Fig. 9 for the structural formula) as an example, its related spectrum data were analyzed.

3.1.1 Proton nuclear magnetic resonance spectrum ¹ H-NMR:

¹ H-NMR (400MHz, CDCl ₃ ) δ: 5.69 (1H, s, H-12), 4.89-5.05 (3H, m, 3CH), 4.52-4.54 (1H, m, CH), 4.13-4.15 (1H ,m,H-3),3.01-3.08(3H,m,COOCH ₃ ),2.37(1H,s,H-9),2.08(1H,m,H-18),1.86(2H,m,CH ₂ ),1.22-1.70(17H,m,H-2,H-6,H-7,H-15,H-16,H-19,H-21,H-22,H-27),1.63(2H ,m,CH ₂ ), 0.90 (3H,m,-CH ₃ ).

3.1.2 Carbon Nuclear Magnetic Resonance Spectrum ¹³ C-NMR:

¹³ C-NMR (100MHz, CDCl ₃ ) δ: 200.3(C-11), 176.9(C-30), 174.9(8'), 170.4(9'), 169.5(C-13), 128.4(C-12 ), 81.9 (C-3), 61.7 (C-9), 51.8 (C-31).

The chemical shift of the carbonyl group is usually 160-220ppm. There are 4 carbonyl groups in the molecule of the target compound M-1-b, and the chemical shifts are 200.3, 176.9, 174.9, 170.4, and δ200.3 are attributed to the chemical shift of the carbonyl carbon connected to the alkene group , δ170.4 is assigned to the chemical shift of the carbonyl carbon of the carboxylic acid, δ176.9, 174.9 is assigned to the chemical shift of the carbonyl carbon of the carboxylate, and δ169.5, 128.4 is assigned to the chemical shift of the two carbons of the alkene group.

3.1.3 Mass spectrometry ESI-MS:

In the ESI-MS of the target compound M-1-b, m/z M+Na ⁺ (675), it can be known that the molecular weight is 652, which is consistent with the molecular weight of the target compound M-1-b.

In conclusion, by ¹ H-NMR, ¹³ C-NMR, and ESI-MS, it can be confirmed that the synthesized compound is the target compound M-1-b.

The structure determination of other target compounds is similar to this, and the specific ¹ H-NMR, ¹³ C-NMR, and MS spectrum data are shown in Table 2, Table 3, and Table 4.

3.2 The proton nuclear magnetic resonance spectrum ( ¹ H-NMR), carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) and mass spectrometry (MS) data of some synthetic intermediates and target products are shown in Table 2, Table 3, and Table 4, respectively.

Table 2 ¹ H-NMR spectral data of the target product

Table 3 ¹³ C-NMR data of M-3 series compounds

Table 4 Mass spectrometry (MS) data of the target compound

3.3 Target compounds M-1-b, M-1-d, M-2-g, M-1-e, M-1-f act on the proliferation inhibitory effect of liver cancer HepG2 cells

The effects of different concentrations of five compounds on the proliferation of liver cancer HepG2 cells are shown in Table 5 and Table 6; the positive drug and the same drug concentration of the five drugs acted at 24h, 48h, and 2 time points respectively, that is, the inhibition rate increased with time. , with the highest inhibition rate at 48h. Different concentration groups act on Hepg2 cells, and the inhibition rate increases with the increase of concentration, and the inhibition rate is the highest at 14 μg/mL. The inhibitory effect was time and dose dependent.

SPSS13.0 statistical software was used to process all the data, and all data were represented by X, and the comparison of OD values between groups was analyzed by one way-ANOVA method, and P<0.05 indicated that the difference was statistically significant.

Table 5: Proliferation inhibitory effect (OD value) (X) of five compounds at different concentrations on liver cancer HepG2 cells at different times

Table 6: Proliferation inhibition rate (%) (X) of five kinds of drugs with different concentrations acting on liver cancer HepG2 cells for different time

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (6)

1. compound and a steric isomer thereof, described compound is selected from

2. a composition, it comprises the compound of claim 1 and pharmaceutically acceptable carrier.

3. the composition that claim 1-2 is arbitrary is in the purposes preparing Hepatoma therapy medicine.

4. the preparation method of composition in claim 1, is characterized in that: using glycyrrhetinic acid and Enoxolone derivative as carrier, become Lipase absobed with C3 position hydroxyl with Norcantharidin and norcantharidin derivative.

5. according to the preparation method of composition in claim 4, it is characterized in that: glycyrrhetinic acid and Enoxolone derivative are selected from glycyrrhetinic acid, deoxy-glycyrrhetinic acid and 18 α-glycyrrhetinic acid and methyl esters thereof.

6. according to the preparation method of composition in claim 4, it is characterized in that: Norcantharidin and norcantharidin derivative are the Norcantharidin and the mono ethyl ester thereof that retain epoxy bridge.