阅读说明：本技术 达玛烷皂苷元和齐墩果烷皂苷元衍生物及其制备和应用 (Dammarane sapogenin and oleanane sapogenin derivatives, and preparation and application thereof ) 是由 赵余庆 丁萌 王旭德 张雨蒙 徐磊 于 2019-04-18 设计创作，主要内容包括：本发明属于医药技术领域,涉及达玛烷皂苷元和齐墩果烷皂苷元衍生物及其制备方法和应用。本发明通过将植物来源的达玛烷皂苷的酸水解产物或分离后的皂苷元灌胃给予SD大鼠,收集7周的生物样本,并采用乙酸乙酯进行样本物的提取,而后对提取所得浸膏进行分离纯化后,依赖于化学和光谱实验精确鉴定其结构,并探讨其生物合成途径的推导过程。本发明对该衍生物进行抗癌生物活性评价及机制研究,结果表明,本发明制备的达玛烷皂苷元和齐墩果烷皂苷元衍生物具有明显的抗癌作用。可以用于制备治疗癌症的药物。(The invention belongs to the technical field of medicines, and relates to dammarane sapogenin and oleanane sapogenin derivatives, and a preparation method and application thereof. The invention provides a method for identifying the structure of an extract of dammarane saponin by gastric lavage, which comprises the steps of feeding an acid hydrolysis product of the dammarane saponin of a plant source or separated sapogenin into an SD rat, collecting a biological sample for 7 weeks, extracting a sample substance by using ethyl acetate, separating and purifying the extract obtained by extraction, accurately identifying the structure by relying on chemical and spectral experiments, and discussing the derivation process of a biosynthesis route. The invention carries out anticancer biological activity evaluation and mechanism research on the derivatives, and the results show that the dammarane sapogenin and oleanane sapogenin derivatives prepared by the invention have obvious anticancer effect. Can be used for preparing medicine for treating cancer.)

1. Dammarane sapogenin and oleanane sapogenin derivatives having the structure shown in A, B, C, D, E, F or G:

wherein R is H, C₁-C₄Alkyl, hydroxy, C₁-C₄Carboxy, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄An aldehyde group.

2. Dammarane sapogenin and oleanane sapogenin derivatives according to claim 1 having the structure A, B, C, D, E, F or G, selected from:

3. dammarane sapogenin and oleanane sapogenin derivatives according to claim 1 having the structure A, B, C, D, E, F or G,

wherein the content of the first and second substances,

in the formula A, R is H, C₁-C₄Alkyl, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄A carboxyl group;

in the formula B, R is H, C₁-C₄Alkyl, hydroxy, C₁-C₄Aldehyde, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄A carboxyl group;

in the formula C, R is H, C₁-C₄An alkyl group;

in the formula D, R is H, C₁-C₄An alkyl group;

in the formula E, R is H, C₁-C₄An alkyl group;

in the formula F, R is H, C₁-C₄An alkyl group;

in the formula G, R is H, C₁-C₄An alkyl group.

4. Dammarane sapogenin and oleanane sapogenin derivatives according to any one of claims 1 to 3, having the structure A, B, C, D, E, F or G, selected from:

5. a pharmaceutical composition comprising one or more of dammarane sapogenin and oleanane sapogenin derivatives having the structure A, B, C, D, E, F or G according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. The process for the preparation of dammarane sapogenin and oleanane sapogenin derivatives according to claim 2,

step 1: preparing a gastric lavage liquid medicine;

step 2: perfusing a rat with stomach, and preparing biological samples of blood, urine, feces and bile;

and step 3: performing column chromatography separation on each biological sample extract obtained in the step 2 to obtain different dammarane sapogenin and oleanane sapogenin derivatives 1a,2a,4a,7a,1b,3b-7b,1c,5c,6d,1e,7e,5f and 5 g;

and 4, step 4: and (3) carrying out metabolic spectrum and biosynthetic pathway conjecture according to the structures of dammarane sapogenin and oleanane sapogenin obtained in the step (3), and predicting according to the determined metabolic site and metabolic rule obtained by the invention to obtain dammarane sapogenin and oleanane sapogenin derivatives 3a,5a,6a,8a-12a,2b,8b-12b,2c-4c,6c-12c,1d-5d,7d-12d,2e-6e,8e-12e,1f-4f,6f,1g-4g and 6g acceptable in the biosynthetic pathway.

7. The method for preparing dammarane sapogenin and oleanane sapogenin derivatives according to claim 6,

in the step 3, a sectional separation mode is adopted, firstly, coarse separation is carried out, a column is flushed under normal pressure, and petroleum ether: eluting ethyl acetate with 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to obtain mixed fractions 1-10 respectively;

mixed fraction 4 was separated using silica gel column and semi-preparative HPLC-ELSD, silica gel column chromatography eluent petroleum ether: ethyl acetate is 5-3: 1, and a mobile phase of semi-preparative HPLC-ELSD is separated by 85% -90% methanol-water to obtain 5f and 5g of derivatives;

mixed fraction 5 was separated using silica gel column, eluent petroleum ether: 1-2: 1, recrystallizing with ethyl acetate, and separating with semi-preparative HPLC-ELSD (high performance liquid chromatography-evaporative light scattering) to obtain a pair of epimers 1e and 7e, wherein the mobile phase is 80-90% methanol-water;

mixed fraction 7 was separated using silica gel column, eluent petroleum ether: separating ethyl acetate to obtain a crude derivative, recrystallizing the crude derivative with ethyl acetate to obtain a derivative 1a, separating a recrystallization mother liquor by using semi-preparative HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) with a mobile phase of 80-90% methanol-water to obtain a pair of epimers 1a and 7 a;

mixed fraction 8 was separated using silica gel column and gradient eluent petroleum ether: obtaining five fractions of 8-1, 8-2, 8-3, 8-4 and 8-5 by using ethyl acetate which is 1-3: 1 and 1: 1-3, wherein the 8-3 fraction is continuously separated by using semi-preparative HPLC-ELSD, and the mobile phase is 80-90% methanol-water to obtain a derivative 4 b;

mixed fraction 9 was separated using silica gel column, eluent dichloromethane: methanol is 30-50:1, 20-10: 1 and 1-3: 1, five fractions of 9-1, 9-2, 9-3, 9-4 and 9-5 are obtained, the fraction 9-2 is further purified by semi-preparative HPLC-ELSD, and the mobile phase is 75-85% methanol-water, so that the derivatives 2a,4a, 1c and 5c are obtained.

Mixed fraction 10 was separated using silica gel column, eluent dichloromethane: obtaining four fractions of 10-1, 10-2, 10-3 and 10-4 by using methanol of 30-50:1, 20-10: 1 and 1-3: 1, further purifying the fraction 10-1 by using semi-preparative HPLC-ELSD, and obtaining derivatives 6b and 6d by using 75-85% methanol-water as a mobile phase; further purifying fraction 10-3 by semi-preparative HPLC-ELSD with mobile phase of 70% -80% methanol-water to obtain derivative 3 b; fraction 10-4 was separated by open ODS column chromatography with eluents of 10% -90% methanol-water in order to obtain 70% mixed elution fraction, which was further separated, prepared and purified by semi-preparative HPLC-ELSD with mobile phase of 70% -80% methanol-water to obtain derivatives 1b, 5b and 7 d.

8. The method for preparing dammarane sapogenin and oleanane sapogenin derivatives according to claim 6, wherein in the preparation of the gastric lavage liquid medicine in step 1, 20(R) -25-methoxy-protopanoxadiol is used for preparing the gastric lavage liquid medicine, the solvent is a mixture of ethanol, propylene glycol and purified water, and the ratio of ethanol: propylene glycol: the volume ratio of the purified water is 1:30-50: 10-20.

9. Use of dammarane sapogenin and oleanane sapogenin derivatives according to any one of claims 1 to 4 and having the structure of A, B, C, D, E, F or G, or a pharmaceutical composition according to claim 5, in the preparation of an anti-tumour medicament.

10. The use of claim 9, wherein the tumor is selected from the group consisting of ovarian cancer, lung cancer, liver cancer, colon cancer, stomach cancer, prostate cancer, pancreatic cancer, cervical cancer and breast cancer.

The technical field is as follows:

the invention belongs to the technical field of medicines, relates to dammarane sapogenin and oleanane sapogenin derivatives, and a preparation method and application thereof, and particularly relates to a series of dammarane sapogenin and oleanane sapogenin derivatives with new metabolic sites, which are biologically converted in dammarane sapogenin bodies, and a preparation method and application thereof in preparing anti-cancer drugs.

Background art:

dammarane sapogenins can be mainly classified into dammarane type, oxtriptolone type, oleanane type and others through their parent nuclear frameworks. Oleanane-type saponins are pentacyclic triterpene dammarane sapogenins, but in the current research, two dammarane sapogenins of Ro and Ri are found to be oleanane-type mother nuclei. Dammarane type saponin is mainly in tetracyclic triterpene structure. Habituation is classified into diol type (e.g. Ra) according to the number of hydroxyl groups on the parent nucleus₁，Ra₂，Rb₁， Rb₂，Rb₃，Rc，Rd，Rg₃And Rh, etc.) and triols (e.g. Re, Rg₂Rf and Rh) which differ by: the diol type is C-3, C-12, C-20 position each containing a hydroxyl position. The triol type contains a hydroxyl group at each of the C-3, C-6, C-12 and C-20 positions.

Ginseng, American ginseng, pseudo-ginseng and the like are plants in the genus of Panax in the family of Araliaceae, and a large amount of dammarane sapogenins found in the plants of Ginseng, American ginseng, pseudo-ginseng and the like are main functional substances in the plants in the genus by modern scientific research. The dammarane sapogenin derivatives with the same and similar structures are also found in the research of the overground parts (stems and leaves) of the plants. At present, a new anticancer medicine 'Shenyi Capsule' in China is marketed in 2003, and the main component of the new medicine is diol group bisglucoside-dammarane sapogenin Rg found in ginseng₃. This provides important opportunity and basis for the full utilization of Panax plants and the development of medicinal resources. A large number of earlier studies in this group have demonstrated that dammarane sapogenins and their derivatives from ginseng, American ginseng and pseudo-ginseng stems and leaves of the genus Panax have anti-tumor activity. In particularThe series of researches on 20(R) -25-methoxy-protopanaxadiol, 20(R) -25-hydroxy-protopanaxadiol and homologues thereof, which are discovered in the subject group, discover that a new structure-activity relationship generated by the structural change of the side chain of dammarane sapogenin becomes a hot subject of the synthesis research of dammarane type derivative structure modification for the first time.

Dammaranes are abundant in plants of the genus Panax. Modern pharmacological studies show that the dammarane sapogenin has a plurality of biological activities of obviously resisting tumors, regulating immunity, improving life quality and the like, and the low-dose dammarane sapogenin can promote tumor cells to be differentiated towards a normal direction. Therefore, the research and application of the component in tumor prevention and treatment have become one of the research hotspots in the international tumor treatment community. In the classification of dammarane-type sapogenins, they are classified into (20S) -protopanaxadiol and (20S) -protopanaxatriol groups based on their aglycones. The total ginsenoside and monomer compound have important effects in promoting cancer cell apoptosis, promoting differentiation, improving cancer cell sensitivity to chemotherapeutic drugs, improving immunity, and preventing and treating various cancers. A series of studies were conducted on its antitumor activity, this subject group and other subject groups. Researches of Guo yumei and the like find that dammarane sapogenin and low-sugar-chain saponin have obvious anti-tumor effect. In the study of this group, 25-methoxy-protopanaxadiol (25-OCH)₃PPD, AD-1), a potent anticancer natural compound isolated from ginseng acid hydrolysate. It exhibits activity against a variety of cancer cells, such as breast, prostate, lung, stomach and pancreatic cancers. And Rg which has been approved as a cancer therapeutic drug in China₃In contrast, the compound has 10-100 fold cytotoxic activity against the growth of several cancer cell lines. In vivo 25-OCH₃Pharmacokinetic studies of PPD found that it was metabolized to produce 25-OH-PPD in plasma. Up to now, with respect to 25-OCH₃The number of studies on PPD metabolism is limited. Only the metabolism in mammalian liver microsomes was examined, and to gain more information on its metabolism to improve its clinical use, 25-OCH₃The metabolic fate of PPD is understood.

The invention is orally administered to rats 25-OCH₃After PPD, novel dammarane sapogenin and oleanane sapogenin derivatives are found in collected feces, and an oleanane sapogenin is also found to have excellent anti-tumor activity. Therefore, the invention aims to find a lead compound with better activity so as to develop a novel cancer inhibitor.

The invention content is as follows:

the invention aims to provide a dammarane sapogenin in-vivo biotransformation derivative, which comprises dammarane sapogenin and oleanane sapogenin derivatives, wherein the series of derivatives have traceable metabolic rules and characteristics of a biosynthesis pathway, metabolic sites are novel, and a ring-expanded product has high metabolic rate and higher anticancer effect.

The technical scheme of the invention is as follows:

the invention provides dammarane sapogenin and oleanane sapogenin derivatives prepared by in vivo biotransformation, and salts and isomers thereof:

wherein:

r is H, C₁-C₄Alkyl, hydroxy, C₁-C₄Carboxy, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄An aldehyde group;

the derivatives and the salts and isomers thereof can be:

the following dammarane sapogenin and oleanane sapogenin derivatives, and salts and isomers thereof are preferred in the present invention:

in the formula A, R is H, C₁-C₄Alkyl, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄A carboxyl group;

in the formula B, R is H, C₁-C₄Alkyl, hydroxy, C₁-C₄Aldehyde, hydroxy substituted C₁-C₄Alkyl radical, C₁-C₄A carboxyl group;

in the formula C, R is H, C₁-C₄An alkyl group;

in the formula D, R is H, C₁-C₄An alkyl group;

in the formula E, R is H, C₁-C₄An alkyl group;

in the formula F, R is H, C₁-C₄An alkyl group;

in the formula G, R is H, C₁-C₄An alkyl group;

the following dammarane sapogenin and oleanane sapogenin derivatives, and salts and isomers thereof are preferred in the present invention:

the invention uses rat gavage to give mother drugs: 20(R) -25-methoxy-protopanoxadiol, obtaining and determining metabolic sites and metabolic rules, and carrying out the speculation of metabolic spectrum and biosynthetic pathway:

20(R) -25-methoxy-protopanoxadiol

The dammarane sapogenin is total saponin acid hydrolysis product of Panax ginseng C.A.Meyer, Panax quinquefolium L.and Panax notoginseng (Burk.) F.H.Chen of Araliaceae and Gynostemma pentaphyllum of Cucurbitaceae.

The invention provides a preparation method of the dammarane sapogenin and oleanane sapogenin derivatives, which comprises the following steps:

step 1: preparing a gastric lavage liquid medicine;

step 2: perfusing a rat with stomach, and preparing biological samples of blood, urine, feces and bile;

and step 3: performing column chromatography separation on each biological sample extract obtained in the step 2 to obtain different dammarane sapogenin and oleanane sapogenin derivatives 1a,2a,4a,7a,1b,3b-7b,1c,5c,6d,1e,7e,5f and 5 g;

and 4, step 4: predicting a metabolic spectrum and a biosynthetic pathway according to the structures of dammarane sapogenin and oleanane sapogenin obtained in the step 3, and predicting according to the determined metabolic site and metabolic rule obtained by the method to obtain dammarane sapogenin and oleanane sapogenin derivatives 3a,5a,6a,8a-12a,2b,8b-12b,2c-4c,6c-12c,1d-5d,7d-12d,2e-6e,8e-12e,1f-4f,6f,1g-4g and 6g which are acceptable in the biosynthetic pathway;

specifically, the method comprises the following steps:

step 1: placing saponin acid hydrolysis product or separated sapogenin of Panax ginseng C.A.Meyer, Panax quinquefolium L.and Panax notoginseng belonging to Araliaceae and Gynostemma pentaphyllum belonging to Cucurbitaceae in a conical flask, ultrasonically dissolving with ethanol as little as possible, slowly adding propylene glycol while stirring with a glass rod, and dropwise adding distilled water to obtain clear and transparent intragastric perfusion liquid medicine without medicine precipitation;

step 2: and (2) administering the intragastric lavage liquid medicine obtained in the step (1mL/100g) according to the weight of the rat, collecting biological samples (including blood, urine, excrement and bile) for 24 hours, centrifuging, filtering and the like, and then placing the biological samples in a refrigerator for freezing. After the process lasts for 7 weeks, all the obtained biological samples are mixed, and after ultrasonic extraction and reduced pressure concentration, extractum of each biological sample is obtained;

and step 3: performing column chromatography separation on each biological sample extract obtained in the step 2 to obtain different dammarane sapogenin and oleanane sapogenin derivatives 1a,2a,4a,7a,1b,3b-7b,1c,5c,6d,1e,7e,5f and 5 g;

and 4, step 4: predicting a metabolic spectrum and a biosynthetic pathway according to the structures of different dammarane sapogenins and oleanane sapogenin obtained in the step 3, and predicting according to the determined metabolic sites and metabolic rules obtained by the method, so as to obtain dammarane sapogenin and oleanane sapogenin derivatives 3a,5a,6a,8a-12a,2b,8b-12b,2c-4c,6c-12c,1d-5d,7d-12d,2e-6e,8e-12e,1f-4f,6f,1g-4g and 6g which are acceptable in the biosynthetic pathway;

the final products 1a,2a,4a,7a,1b,3b-7b,1C,5C and 6d prepared by the invention are dammarane sapogenins, mainly subjected to metabolic conversion at C-29, 1e and 7e are side chain ring closure dammarane sapogenin derivatives, and 5f and 5g are oleanane sapogenin derivatives subjected to mother nucleus ring expansion biotransformation.

In the step 1, the gastric lavage liquid medicine is prepared by using the saponin acid hydrolysis product or the separated sapogenin, the preparation solvent is the mixture of ethanol, propylene glycol and purified water, and the weight ratio of ethanol: propylene glycol: the volume ratio of the purified water is 1:30-50: 10-20.

In the step 2, when each biological sample is subjected to reduced pressure concentration, a water bath is adopted for keeping the temperature at 40-50 ℃;

in the step 3: the method adopts a sectional separation mode:

firstly, carrying out crude separation, flushing a column at normal pressure, and respectively using petroleum ether: eluting ethyl acetate with 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to obtain mixed fractions 1-10 respectively;

mixed fraction 4 was separated using silica gel column, eluent petroleum ether: ethyl acetate 5-3: 1 and semi-preparative HPLC-ELSD eluent 85% -90% methanol-water were separated to give derivatives 5f and 5 g.

Mixed fraction 5 was separated using silica gel column, eluent petroleum ether: and (3) carrying out ethyl acetate recrystallization and semi-preparative HPLC-ELSD (80-90% methanol-water) separation to obtain a pair of epimers 1e and 7e, so as to realize the achiral column resolution of chiral substances.

Mixed fraction 7 was separated using silica gel column, eluent petroleum ether: separating ethyl acetate 2-3: 1 to obtain a crude derivative, recrystallizing the crude derivative with ethyl acetate to obtain a derivative 1a, and separating a recrystallization mother liquor by using semi-preparative HPLC-ELSD (mobile phase is 80-90% methanol-water) to obtain a pair of epimers 1a and 7a, so that achiral column resolution of chiral substances is realized;

mixed fraction 8 was separated using silica gel column and gradient eluent petroleum ether: ethyl acetate 1-3: 1 and 1: 1-3 gave five fractions of 8-1, 8-2, 8-3, 8-4 and 8-5, of which 8-3 was further separated using semi-preparative HPLC-ELSD (mobile phase 80% -90% methanol-water) to give derivative 4 b.

Mixed fraction 9 was separated using silica gel column, eluent dichloromethane: methanol is 30-50:1, 20-10: 1 and 1-3: 1, five fractions of 9-1, 9-2, 9-3, 9-4 and 9-5 are obtained, and the fraction 9-2 is further purified by semi-preparative HPLC-ELSD (75-85% methanol-water as a mobile phase) to obtain derivatives 2a,4a, 1c and 5 c.

Mixed fraction 10 was separated using silica gel column, eluent dichloromethane: the methanol is 30-50:1, 20-10: 1 and 1-3: 1, four fractions of 10-1, 10-2, 10-3 and 10-4 are obtained, and the fraction 10-1 is further purified by semi-preparative HPLC-ELSD (mobile phase is 70-80% methanol-water) to obtain derivatives 6b and 6 d. Fraction 10-3 was further purified by semi-preparative HPLC-ELSD (mobile phase 70% -80% methanol-water) to give derivative 3 b. Fraction 10-4 was separated by open ODS column chromatography with eluents of 10% -90% methanol-water in order to obtain 70% mixed elution fraction, which was further separated, prepared and purified by semi-preparative HPLC-ELSD (mobile phase of 70% -80% methanol-water) to obtain derivatives 1b, 5b and 7 d.

The compound in step 3, due to the absence of chromophores in the chemical structure, shows no absorption in its Ultraviolet (UV) spectrum. Therefore, the uv detector is not the best tool for detecting notoginsenoside and its trace metabolites. An Evaporative Light Scattering Detector (ELSD) is commonly used to detect compounds that do not have UV absorption, which can be suitable for the determination of notoginsenoside.

The compound obtained in the step 3 is: 1 a: 20(R) -25-methoxy-dammarane-3 β,12 β, 20-triol, 2 a: 20(R) -dammarane-3 β,12 β,20,25, 29-pentaol, 4 a: 20(R) -25-methoxy-3 β,12 β, 20-trihydroxy-dammarane-4-carboxylic acid, 7 a: 20(S) -25-methoxy-dammarane-3 β,12 β, 20-triol, 1 b: 20(R) -dammarane-3 β,12 β,20, 25-tetraol, 3 b: 20(R) -3 β,12 β,20, 25-tetrahydroxy-dammarane-4-aldehyde, 4 b: 20(R) -3 β,12 β,20, 25-tetrahydroxy-dammarane-4-carboxylic acid, 5 b: 20(R) -4-nordammarane-3 β,12 β,20, 25-tetraol, 6 b: 20(R) -dammarane-3 β,4 β,12 β,20, 25-pentaol, 7 b: 20(S) -dammarane-3 β,12 β,20, 25-tetraol, 1 c: 20(R) -12 β,20,25 trihydroxy-dammaran-3-one, 5 c: 20(R) -12 β,20, 25-trihydroxy-4-nordammaran-3-one, 6 d: (20R,24R) -4-nordammarane-3 β,12 β,20, 25-pentaol, 1 e: 3 β,12 β,20(R) -20, 25-epoxydimethane-3, 12-diol, 7 e: 3 β,12 β,20(S) -20, 25-epoxydimethane-3, 12-diol, 5 f: 3 β,21 α,22 β -hydroxy-24-norlean-12-ene, 5 g: 3 beta, 21 alpha-hydroxy-24-norlean-12-en-22-one.

Metabolite biosynthesis analysis was performed based on the structure of the compound described in step 3 and found that the metabolic derivative was changed mainly from C-25-demethylation of 1a to 1 b. Then formed after multiple oxidations (3b → 4b) and decarboxylations (5b) at the C-29 position of 1 b. Since 1a, 7a,1b and 7b are present simultaneously and the 20R isomer predominates, this indicates that dehydration can occur at C-20, resulting in a carbocation equilibrium. In animals, lanosterol is converted to cholesterol, including loss of 3 methyl groups, reduction of side chain double bonds, and Δ^5,6Formation of double bonds, instead of Δ^8,9-a double bond. The two canthamethyl groups of C-4 of ovine sterols are identical to those of 1 a. Based on the metabolic pathway analysis of the metabolites that have been isolated, it was found that the metabolism of 1a is very similar to the synthesis of cholesterol in animals (process of removing horny methyl group on C-4). It is presumed that the site of metabolism of aglycone represented by 1a and 1b occurs mainly at C-29, and from this, a series of metabolic derivatives related to this could be deduced, and the reason why these derivatives were not isolated by preparation may be the generationMetabolic trace or instability.

In the step 4, the dammarane sapogenin and oleanane sapogenin derivative compounds acceptable in the biosynthetic pathway are predicted according to the determined metabolic sites and metabolic rules obtained by the method and are 3a,5a,6a,8a-12a,2b,8b-12b,2c-4c,6c-12c,1d-5d,7d-12d,2e-6e,8e-12e,1f-4f,6f,1g-4g and 6 g;

the invention aims to provide a dammarane sapogenin in-vivo biotransformation derivative, the series of derivatives have a traceable metabolic rule and characteristic of a biosynthesis route, a metabolic site is novel, and an expanded ring product has high metabolic rate and higher anticancer effect.

The invention provides dammarane sapogenin and oleanane sapogenin derivatives, and application of metabolic site change, isomers and active derivatives acceptable in a biosynthesis pathway thereof in preparation of anti-cancer drugs.

Has the advantages that: the invention provides a series of dammarane sapogenin metabolic derivatives, and the partial derivatives have obvious inhibition effect on cancer cells, so that the metabolic derivatives with anticancer effect can be applied to the preparation of anticancer drugs.

Drawings

FIG. 1 is a cell cycle arrest experiment.

A: compound 5f acts on MCF-7 cells. B: compound 3b acted on IOSE144 cells. C: 20(R) -25-methoxy-protopanaxadiol (AD-1) was applied to MCF-7 cells.

FIG. 2 is the mechanism of 5f induction of apoptosis in HO-8901 cells.

The translocation of Bax and the release of mitochondrial cytochrome c (cytc) were detected by western blot assay in HO-8901 cells.

Data are presented as mean ± SD of triplicate experiments performed independently. P <0.05, P <0.01, P < 0.001.

The specific implementation mode is as follows:

metabolic derivatives preparation example 1:

step 1: weighing 400mg of acid hydrolysis product of dammarane saponin or separated sapogenin, placing in a 100 mL conical flask, ultrasonically dissolving with 2mL of ethanol, slowly adding 68mL of propylene glycol while continuously stirring with a glass rod, and finally dropwise adding 32mL of distilled water to obtain clear and transparent gastric lavage liquid medicine without medicine precipitation;

step 2: and (2) administering the intragastric lavage liquid medicine obtained in the step (1mL/100g) according to the weight of the rat, collecting biological samples (including blood, urine, excrement and bile) for 24 hours, centrifuging, filtering and the like, and then placing the biological samples in a refrigerator for freezing. After the process lasts for 7 weeks, all the obtained biological samples are mixed, and after ultrasonic extraction and reduced pressure concentration, extractum of each biological sample is obtained;

and step 3: and (3) performing column chromatography separation on each biological sample extract obtained in the step (2), wherein petroleum ether: eluting ethyl acetate by 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to respectively obtain mixed fractions 1-10, separating the mixed fraction 9 by using a silica gel column, and eluting by using dichloromethane: methanol is 30-50:1, 20-10: 1 and 1-3: 1, five fractions of 9-1, 9-2, 9-3, 9-4 and 9-5 are obtained, and the fraction 9-2 is further purified by semi-preparative HPLC-ELSD (mobile phase is 82% methanol-water) to obtain the novel metabolic derivatives 2a,4a and 5 c.

20(R) -dammarane-3 beta, 12 beta, 20,25, 29-pentaol (2a)

Compound 2a was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.67 (1H,m),0.92(1H,m),1.96(1H,m),1.39(1H,m),3.65(1H,m),0.95(1H,m),1.93 (1H,m),1.84(1H,m),1.61(1H,m),1.07(1H,m),1.52(1H,m),2.13(2H,m),3.95 (1H,td,J_1,2＝5.1,J_1,3＝10),2.05(1H,m),1.51(1H,m),1.27(1H,m),2.03(1H,m), 2.44(1H,m),0.96(3H,s),0.91(3H,s),1.43(3H,s),1.69(2H,m),1.74(1H,m),1.51 (1H,m),1.90(1H,m),1.55(2H,m),1.16(3H,s),1.16(3H,s),4.51(2H,d, J_1,2＝10.86),1.56(3H,s),1.04(3H,s),3.17(3H,s)；¹³C-NMR(pyridine-d₅,400MHz, ppm):_C39.55(C-1),27.05(C-2),80.40(C-3),43.74(C-4),57.26(C-5),18.47(C-6), 31.82(C-7),40.45(C-8),50.95(C-9),37.55(C-10),32.84(C-11),71.20(C-12), 49.67(C-13),52.11(C-14),35.96(C-15),28.96(C-16),51.18(C-17),17.71(C-18), 17.21(C-19),73.60(C-20),23.22(C-21),44.25(C-22),19.55(C-23),41.56(C-24), 74.93(C-25),25.55(C-26),25.53(C-27),64.83(C-28),24.00(C-29),16.16(C-30), 49.33(OCH₃).MS:m/z 531.4020[M+Na]⁺.

20(R) -25-methoxy-3 beta, 12 beta, 20-trihydroxy-dammarane-4-carboxylic acid (4a)

Compound 4a was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.67 (1H,m),0.92(1H,m),1.96(1H,m),1.39(1H,m),3.38(1H,dd,J_1,2＝3.78, J_1,3＝11.70),1.048(1H,m),1.92(1H,m),1.84(1H,m),1.60(1H,m),1.08(1H,m), 1.54(1H,m),2.18(2H,m),3.97(1H,td,J_1,2＝4.92,J_1,3＝10.08),2.06(1H,m),1.54 (1H,m),1.39(1H,m),2.48(1H,m),1.94(1H,m),2.44(1H,m),0.98(3H,s),1.14 (3H,s),1.41(3H,s),1.68(2H,m),2.35(2H,m),1.54(2H,m),1.15(3H,s),1.15(3H, s),1.74(3H,s),1.12(3H,s),3.16(3H,s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C40.28(C-1),27.07(C-2),78.59(C-3),50.06(C-4),57.47(C-5),18.46(C-6),31.80 (C-7),40.37(C-8),50.36(C-9),38.24(C-10),32.89(C-11),71.23(C-12),49.74 (C-13),52.20(C-14),35.75(C-15),29.67(C-16),51.21(C-17),17.67(C-18),14.93 (C-19),73.59(C-20),23.19(C-21),44.23(C-22),21.17(C-23),41.57(C-24),74.92 (C-25),25.55(C-26),25.52(C-27),25.10(C-29),16.12(C-30),49.33(OCH₃).MS: m/z 523.3993[M+H]⁺.

20(R) -12 beta, 20, 25-trihydroxy-4-nordammarane-3-one (5c)

Compound 5c was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.84 (1H,m)；1.11(1H,m),2.39(1H,m)；2.28(1H,m),2.21(1H,td,J_1,2＝6.5,J_1,3＝12.9), 0.97(1H,s),1.38(1H,m)；1.18(1H,m),1.46(1H,m),2.13(1H,m)；1.63(1H,m), 3.92(1H,td,J_1,2＝5.2,J_1,3＝9.9),2.04(1H,m),1.58(1H,m)；1.03(1H,m),2.42(1H, m),1.05(3H,s),0.97(3H,s),1.42(3H,s),1.74(2H,m),2.12(1H,m)；2.05(1H,m), 1.73(2H,m),1.43(3H,s),1.43(3H,s),1.16(3H,s),1.07(3H,d,J_1,2＝6.54),0.89(3H, s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C40.80(C-1),37.96(C-2),212.4(C-3), 44.89(C-4),53.87(C-5),22.75(C-6),34.27(C-7),40.01(C-8),48.41(C-9),37.16 (C-10),32.98(C-11),71.01(C-12),49.72(C-13),52.14(C-14),31.73(C-15),27.03 (C-16),51.19(C-17),16.02(C-18),13.85(C-19),73.76(C-20),23.12(C-21),44.41 (C-22),19.09(C-23),45.96(C-24),70.08(C-25),30.59(C-26),30.30(C-27),12.40 (C-29),17.55(C-30).MS:m/z 485.3601[M+Na]⁺.

Metabolic derivatives preparation example 2:

step 1: weighing 400mg of acid hydrolysis product of dammarane saponin or separated sapogenin, placing in a 100 mL conical flask, ultrasonically dissolving with 2mL of ethanol, slowly adding 68mL of propylene glycol while continuously stirring with a glass rod, and finally dropwise adding 32mL of distilled water to obtain clear and transparent gastric lavage liquid medicine without medicine precipitation;

step 2: and (2) administering the intragastric lavage liquid medicine obtained in the step (1mL/100g) according to the weight of the rat, collecting biological samples (including blood, urine, excrement and bile) for 24 hours, centrifuging, filtering and the like, and then placing the biological samples in a refrigerator for freezing. After the process lasts for 7 weeks, all the obtained biological samples are mixed, and after ultrasonic extraction and reduced pressure concentration, extractum of each biological sample is obtained;

and step 3: and (3) performing column chromatography separation on each biological sample extract obtained in the step (2), wherein petroleum ether: eluting ethyl acetate by 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to respectively obtain mixed fractions 1-10, separating the mixed fraction 8 by using a silica gel column, and using a gradient eluent of petroleum ether: ethyl acetate 1-3: 1 and 1: 1-3 gave five fractions of 8-1, 8-2, 8-3, 8-4 and 8-5, of which 8-3 was further separated using semi-preparative HPLC-ELSD (mobile phase 84% methanol-water) to give derivative 4 b.

20(R) -3 beta, 12 beta, 20, 25-tetrahydroxy-dammarane-4-carboxylic acid (4b)

Compound 4b was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.68 (2H,m),2.66(1H,m),1.41(1H,m),0.92(1H,m),1.24(1H,m),1.67(1H,m),1.06 (1H,m),3.48(1H,m),1.56(1H,m),1.28(1H,m),1.15(1H,m),1.40(1H,m),1.87 (1H,m),1.40(1H,m),1.87(1H,m),0.78(3H,s),0.77(3H,s),0.94(3H,s),1.25(2H, m),1.60(2H,m),1.23(2H,m),1.02(3H,s),1.02(3H,s),1.08(3H,s),0.91(3H,s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C40.46(C-1),25.78(C-2),77.45(C-3), 47.64(C-4),57.03(C-5),17.56(C-6),30.63(C-7),39.61(C-8),48.92(C-9),36.93 (C-10),31.49(C-11),69.87(C-12),48.14(C-13),51.10(C-14),35.06(C-15),29.62 (C-16),49.45(C-17),16.81(C-18),14.64(C-19),72.18(C-20),22.36(C-21),42.82 (C-22),20.25(C-23),44.53(C-24),68.92(C-25),29.45(C-26),29.30(C-27),180.34 (C-28),25.33(C-29),15.25(C-30).MS:m/z 531.5656[M+Na]⁺.

Metabolic derivatives preparation example 3:

step 1: weighing 400mg of acid hydrolysis product of dammarane saponin or separated sapogenin, placing in a 100 mL conical flask, ultrasonically dissolving with 2mL of ethanol, slowly adding 68mL of propylene glycol while continuously stirring with a glass rod, and finally dropwise adding 32mL of distilled water to obtain clear and transparent gastric lavage liquid medicine without medicine precipitation;

step 2: and (2) administering the intragastric lavage liquid medicine obtained in the step (1mL/100g) according to the weight of the rat, collecting biological samples (including blood, urine, excrement and bile) for 24 hours, centrifuging, filtering and the like, and then placing the biological samples in a refrigerator for freezing. After the process lasts for 7 weeks, all the obtained biological samples are mixed, and after ultrasonic extraction and reduced pressure concentration, extractum of each biological sample is obtained;

and step 3: and (3) performing column chromatography separation on each biological sample extract obtained in the step (2), wherein petroleum ether: eluting ethyl acetate by 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to respectively obtain mixed fractions 1-10, separating the mixed fraction 10 by using a silica gel column, and eluting by using dichloromethane: methanol is 30-50:1, 20-10: 1 and 1-3: 1, four fractions of 10-1, 10-2, 10-3 and 10-4 are obtained, and the fraction 10-1 is further purified by semi-preparative HPLC-ELSD (mobile phase is 74% methanol-water) to obtain the new metabolic derivatives 6b and 6 d.

20(R) -dammarane-3 beta, 4 beta, 12 beta, 20, 25-pentaol (6b)

Compound 6b was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.78 (1H,m),0.99(1H,m),2.19(1H,m),1.87(1H,m),3.55(1H,dd,J_1,2＝4.86, J_1,3＝11.58),0.96(1H,s),1.59(1H,m),1.35(1H,m),1.58(1H,m),2.23(1H,m),1.69 (1H,m),3.99(1H,td,J_1,2＝5.04,J_1,3＝10.20),2.47(1H,m),1.63(1H,m),1.09(1H,m), 1.97(1H,m),1.40(1H,m),2.47(1H,m),1.16(3H,s),1.33(3H,s),1.43(3H,s),1.76 (2H,m),2.06(1H,m),1.95(1H,m),1.76(2H,m),1.44(3H,s),1.44(3H,s),1.59 (3H,s),0.99(3H,s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C39.3(C-1),28.2 (C-2),76.0(C-3),74.5(C-4),55.10(C-5),30.6(C-6),35.4(C-7),40.3(C-8),40.1 (C-9),37.7(C-10),32.58(C-11),71.3(C-12),49.70(C-13),52.3(C-14),31.8(C-15), 27.1(C-16),51.19(C-17),16.02(C-18),13.85(C-19),73.76(C-20),23.12(C-21), 44.41(C-22),19.09(C-23),45.96(C-24),70.08(C-25),30.59(C-26),30.30(C-27), 26.1(C-29),17.7(C-30).MS:m/z 503.3653[M+Na]⁺.

(20R,24R) -4-nordammarane-3 beta, 12 beta, 20, 25-pentaol (6d)

Compound 6d was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.79 (1H,m),1.31(1H,m),3.09(1H,m),1.31(1H,m),0.68(1H,s),1.48(1H,m),1.28 (1H,m),1.44(1H,m),2.23(1H,m),1.69(1H,m),3.62(1H,m),1.75(1H,m),1.06 (2H,m),1.85(1H,m),1.23(1H,m),2.08(1H,m),1.01(3H,s),0.87(3H,s),1.16 (3H,s),1.48(2H,m),1.61(2H,m),1.84(2H,m),1.26(3H,s),1.26(3H,s),0.96(3H, d,J_1,2＝5.28),0.91(3H,s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C38.38(C-1), 29.85(C-2),76.69(C-3),38.61(C-4),51.89(C-5),22.84(C-6),34.14(C-7),39.50 (C-8),48.27(C-9),36.61(C-10),31.8(C-11),71.27(C-12),48.61(C-13),51.93 (C-14),31.08(C-15),26.60(C-16),50.20(C-17),15.80(C-18),14.14(C-19),74.49 (C-20),22.15(C-21),43.19(C-22),21.03(C-23),44.36(C-24),71.19(C-25),29.60 (C-26),29.51(C-27),15.22(C-29),17.20(C-30).MS:m/z 481.3888[M+Na]⁺.

Fraction 10-3 was further purified by semi-preparative HPLC-ELSD (mobile phase 76% methanol-water) to give the novel metabolic derivative 3 b.

20(R) -3 beta, 12 beta, 20, 25-tetrahydroxy-dammarane-4-aldehyde (3b)

Compound 3b was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.75(1H, m),0.98(1H,m),2.25(1H,m),2.20(1H,m),3.63(1H,m),1.07(1H,m),1.87(1H, m),1.61(1H,m),1.45(1H,m),1.25(1H,m),3.61(1H,m),1.56(1H,m),1.03(1H, m),3.94(1H,m),2.02(1H,m),2.15(1H,m),1.93(1H,m),1.36(1H,m),2.43(1H, m),0.98(3H,s),0.82(3H,s),1.42(3H,s),1.74(2H,m),2.09(1H,m),1.73(1H,m), 1.74(2H,m),1.42(3H,s),1.50(3H,s),0.93(3H,s)；¹³C-NMR(pyridine-d₅,400 MHz,ppm):_C38.86(C-1),28.96(C-2),76.45(C-3),54.02(C-4),57.71(C-5),19.65 (C-6),35.30(C-7),40.19(C-8),50.05(C-9),37.63(C-10),31.76(C-11),71.08 (C-12),49.66(C-13),52.12(C-14),32.89(C-15),27.02(C-16),51.14(C-17),16.08 (C-18),17.09(C-19),73.75(C-20),23.18(C-21),44.43(C-22),19.10(C-23),45.98 (C-24),70.08(C-25),30.61(C-26),30.03(C-27),207.95(C-28),21.77(C-29),17.63 (C-30).MS:m/z 493.3888[M+H]⁺.

And continuously separating the fraction 10-4 by using an open type ODS chromatographic column, sequentially eluting by 10-90% methanol-water to obtain a 70% mixed elution fraction, and further separating, preparing and purifying by using semi-preparative HPLC-ELSD (mobile phase is 76% methanol-water) to obtain the new metabolic derivative 5 b.

20(R) -4-nordammarane-3 beta, 12 beta, 20, 25-tetraol (5b)

ppm):_H1.71(1H,m),0.92(1H,m),1.96(1H,m),1.39(1H,m),3.28(1H,td),1.52 (1H,m),0.67(1H,m),1.61(1H,m),1.21(1H,m),1.47(1H,m),1.23(1H,m),1.50 (1H,m),2.01(1H,m),1.82(1H,m),3.95(1H,m),2.07(1H,m),1.59(1H,m),1.05 (1H,m),1.97(1H,m),1.38(1H,m),2.48(1H,m),1.05(3H,s),0.85(3H,s),1.44 (3H,s),2.37(1H,m),1.90(1H,m),2.36(1H,m),1.31(1H,m),3.85(1H,m),1.56 (3H,s),1.54(3H,s),1.25(3H,d,J_1,2＝6.4),0.95(3H,s)；¹³C-NMR(pyridine-d₅,400 MHz,ppm):_C39.04(C-1),27.09(C-2),76.18(C-3),39.66(C-4),52.53(C-5),21.75 (C-6),34.73(C-7),40.02(C-8),48.89(C-9),37.06(C-10),32.16(C-11),71.25 (C-12),49.65(C-13),52.25(C-14),31.75(C-15),27.09(C-16),51.50(C-17),16.21 (C-18),14.62(C-19),73.66(C-20),23.12(C-21),41.43(C-22),26.37(C-23),80.43 (C-24),73.27(C-25),26.54(C-26),26.54(C-27),16.21(C-29),17.58(C-30).MS: m/z 487.3758[M+Na]⁺.

Metabolic derivatives preparation example 4:

step 1: weighing 400mg of acid hydrolysis product of dammarane saponin or separated sapogenin, placing in a 100 mL conical flask, ultrasonically dissolving with 2mL of ethanol, slowly adding 68mL of propylene glycol while continuously stirring with a glass rod, and finally dropwise adding 32mL of distilled water to obtain clear and transparent gastric lavage liquid medicine without medicine precipitation;

step 2: and (2) administering the intragastric lavage liquid medicine obtained in the step (1mL/100g) according to the weight of the rat, collecting biological samples (including blood, urine, excrement and bile) for 24 hours, centrifuging, filtering and the like, and then placing the biological samples in a refrigerator for freezing. After the process lasts for 7 weeks, all the obtained biological samples are mixed, and after ultrasonic extraction and reduced pressure concentration, extractum of each biological sample is obtained;

and step 3: and (3) performing column chromatography separation on each biological sample extract obtained in the step (2), wherein petroleum ether: eluting ethyl acetate by 10-20: 1, 3-8: 1, 1-2: 1, 1: 2-5 to respectively obtain mixed fractions 1-10, and eluting the mixed fraction 4 by using a silica gel column as an eluent, wherein the eluent is petroleum ether: ethyl acetate 5-3: 1 and semi-preparative HPLC-ELSD eluent 87.5% methanol-water were separated to give derivatives 5f and 5 g.

3 β,21 α,22 β -hydroxy-24-norlean-12-ene (5f)

Compound 5f was obtained as a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.65(1H, m),1.01(1H,m),3.31(1H,td,J_1,2＝5.16,J_1,3＝10.5),0.73(1H,td,J_1,2＝1.56,J_1,3＝12.3),1.43(1H,m),1.35(1H,m),1.93(1H,m),1.28(1H,m),1.65(1H,m),2.06(2H,m), 5.37(1H,m),1.07(2H,m),2.61(1H,dd,J_1,2＝3.60,J_1,3＝13.68),2.11(1H,m),1.39 (1H,m),3.77(1H,d,J_1,2＝2.70),3.86(1H,d,J_1,2＝3.00),1.27(3H,m),0.9(3H,s), 1.07(3H,s),1.27(3H,s),1.30(3H,s),1.49(3H,s)；¹³C-NMR(pyridine-d₅,400MHz, ppm):_C38.99(C-1),31.96(C-2),76.13(C-3),39.62(C-4),52.18(C-5),21.52(C-6), 31.98(C-7),40.39(C-8),46.21(C-9),36.98(C-10),24.72(C-11),123.15(C-12), 145.06(C-13),42.60(C-14),21.03(C-15),27.89(C-16),36.95(C-17),44.46(C-18), 47.66(C-19),39.62(C-20),80.02(C-21),75.04(C-22),16.34(C-23),14.51(C-25), 17.51(C-26),26.86(C-27),22.73(C-28),32.49(C-29),21.75(C-30).MS:m/z 467.3496[M+Na]⁺.

3 β,21 α -hydroxy-24-norlean-12-en-22-one (5g)

5g of the obtained compound was a white powder.¹H-NMR(pyridine-d₅,400MHz,ppm):_H1.65 (1H,m),1.03(1H,m),2.04(1H,m),1.85(1H,m),3.33(1H,m),1.58(1H,m),0.73 (1H,m),1.44(1H,m),1.26(1H,m),1.65(1H,m),1.28(2H,m),5.31(1H,m),1.69 (1H,m)；0.98(1H,m),2.41(1H,m),4.58(1H,s),0.93(3H,s),0.97(3H,s),2.01(3H, m),1.30(3H,s),0.90(3H,s)；¹³C-NMR(pyridine-d₅,400MHz,ppm):_C38.96(C-1), 31.98(C-2),76.12(C-3),39.61(C-4),52.18(C-5),21.46(C-6),32.56(C-7),40.02 (C-8),46.12(C-9),37.02(C-10),25.91(C-11),125.01(C-12),141.73(C-13),42.52 (C-14),25.66(C-15),28.71(C-16),46.23(C-17),48.17(C-18),40.54(C-19),48.44 (C-20),79.84(C-21),216.73(C-22),16.34(C-23),14.48(C-25),17.42(C-26),24.65 (C-27),21.53(C-28),29.23(C-29),19.26(C-30).MS:m/z 465.3339[M+Na]⁺.