阅读说明：本技术 一种银杏酚酸的酰胺化合物的制备及应用 (Preparation and application of amide compound of ginkgolic acid ) 是由 赵俸艺 徐莉 吴文龙 王未凡 曹福亮 李维林 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种具体的银杏酚酸(C13:0)与具有抗癌活性的脱氢枞胺的改性产物,即一种银杏酚酸的酰胺化合物的制备方法及其在抗癌活性方面的应用。该酰胺化合物的制备方法易操作。结合两种经济林树种中的活性化合物,富有创新性。其对Hela细胞的IC-(50)值为1.83μM,对HepG2细胞的IC-(50)值为3.31μM,对MCF-7细胞的IC-(50)值为9.98μM,对A549细胞的IC-(50)值为5.80μM。具有较好的抗Hela细胞、HepG2细胞、MCF-7细胞和A549细胞活性,且毒性较小,改变了传统概念中“银杏有毒,生食或过量食用会引起过敏反应,甚至导致死亡等现象”,为抗癌药的开发提供了新的思路。该化合物结构新颖,含有长链供电子基团(#C-(13)H-(27)),具有低毒性及独特的抗癌活性,是一类具有优良前景的抗癌药剂,为研究化合物的抗癌活性与结构的关系创造条件,为新药研发奠定基础。(The invention discloses a preparation method of a modified product of ginkgolic acid (C13: 0) and dehydroabietylamine with anticancer activity, namely an amide compound of ginkgolic acid and application of the amide compound in the aspect of anticancer activity. The preparation method of the amide compound is easy to operate. The combination of active compounds in two economic forest species is innovative. Its IC on Hela cells 50 IC value of 1.83. mu.M for HepG2 cells 50 IC value of 3.31. mu.M for MCF-7 cells 50 IC for A549 cells with a value of 9.98. mu.M 50 The value was 5.80. mu.M. Has better activity of resisting Hela cells, HepG2 cells, MCF-7 cells and A549 cells, has less toxicity, and changes the traditional concept that gingko is toxicThe phenomenon that the raw or excessive eating can cause anaphylactic reaction and even death and the like provides a new idea for the development of anti-cancer drugs. The compound has novel structure and contains long-chain electron-donating groups (-C) 13 H 27 ) The compound has low toxicity and unique anticancer activity, is an anticancer medicament with excellent prospect, creates conditions for researching the relation between the anticancer activity and the structure of the compound and lays a foundation for the research and development of new drugs.)

1. An amide compound of a ginkgolic acid, characterized in that: the amide compound of ginkgolic acid is the product of ginkgolic acid C13:0 and dehydroabietylamine, and its structural formula is shown in figure 1.

2. The preparation method of the ginkgolic acid amide compound comprises the following steps: by adopting a DCC-HoBt condensation method, 0.04 mmol of ginkgolic acid C13:0, 0.04 mmol of HoBt are dissolved by 10 mL of ethyl acetate, stirred for 30 minutes at 0 ℃, added with 0.04 mmol of DCC and reacted for 2.5 hours at 0 ℃. Then, 2 mL of an ethyl acetate solution of 0.04 mmol of dehydroabietylamine was added dropwise, the reaction was followed by TLC, and the mixture was stirred in an ice bath for 30 minutes to sufficiently precipitate DCU. Filtering to remove DCU, filtering, performing reduced pressure rotary evaporation, and performing column chromatography on the product with column chromatography V ethyl acetate: v n-hexane =1:3, isolated and purified to give a light brown oil, 0.014 g, yield 60%.

3. The method of claim 2, wherein: the molar ratio of the ginkgolic acid C13:0 to the dehydroabietylamine is 1:1.0-1: 1.2.

4. The use of the ginkgolic acid amide compounds of claim 1 for anticancer activity.

Technical Field

The invention belongs to the field of natural product chemistry, and particularly relates to a preparation method of an amide compound which is a modified product of ginkgolic acid (C13: 0) and dehydroabietylamine, and application of the amide compound in the aspect of anticancer activity.

Background

Many studies in recent years have shown that various ginkgo extracts have significant anti-cancer activity against tumor strains of both human and murine origin. Itokawa and the like research the anticancer activity of long-chain phenolic acid substances in ginkgo episperm, and the results show that the compounds can inhibit mouse sarcoma S₁₈₀And V-79 cell of Chinese rat. [ Itokawa H, Totsuka N, Nakahara K, Takeya K, Lepoittevin J P, Asakawa Y, Anticancer drugs fromGinkgo biloba L.[J]. Chem Pharm Bull, 1987, 35(7): 3016-3020.]Lee and the like separate the ginkgolic acid from the extract of the ginkgo biloba exocarp, and find the extremely strong inhibitory activity of phosphatidylinositol-specific phospholipase, wherein the IC of the ginkgolic acid with the strongest inhibitory activity₅₀2.22 mu mol/L; meanwhile, the ginkgolic acid is proved to be capable of effectively inhibiting the proliferation of a plurality of human-derived cancer cells such as A-549, MCF-7, HCT-15, SKOV-3 and HT-1197, and has lower toxicity to normal colon cells (CCD-18 Co) than corresponding colon cancer cells. [ Lee J S, Cho Y S, Park E J, et al, Phospholipase C γ 1 inhibiting principles from the sarcotesis ofGinlgo biloba L.[J]. J. Nat. Proud, 1998, 61(7): 867-871.]Nichow et al found that ginkgolic acid can inhibit Hela cell proliferation and is time-dependent and dose-dependent, but ginkgolic acid has an optimal dose range for regulating the immunity of tumor-bearing mice, and if the dose is too high, the immune function of the mice can be inhibited. With the increase of the concentration of the ginkgolic acid, the inhibition rate of the ginkgolic acid on the tumor cells is increased, but the toxic and side effects on normal cells are also increased, namely the killing rate of the ginkgolic acid with high concentration on the normal cells and the tumor cells tends to be consistent. [ Nissan, WucollumStudy of antitumor Activity of ginkgolic acids in Ginkgo testa [ J]The university of agriculture, Huazhong, 2006, 25(4): 449-451.]

Studies have shown that ginkgolic acid has the potential to cause allergy, non-immunotoxicity, and induce organism mutation and carcinogenesis. [ Helke H, Reiner J, Egon K, et al, In vitro evaluation of the cytoxic potential of alkylphenols fromGinkgo biloba L.[J]. Toxicology, 2002, 177(2/3): 167-177; Ahlemeyer B, Selke D, Schaper C, et al. Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C[J]. European Journal of Pharmacology, 2001, 430(1): 1-7.]Lepoittevin et al examined the allergy test of guinea pigs and the results indicated that ginkgolic acid was the major allergen. [ Lepoittevin J P, Benezra C, Asakawa Y. Allergic contact dermatologis toGinkgo biloba L.: relationship with urushiol[J]. Archives of Dermatological Research, 1989, 281(4): 227-230.]The phenomenon that the gingko is toxic, the phenomenon that the gingko causes anaphylactic reaction and even death when eaten raw or excessively, and the like, is generally considered to be caused by ginkgolic acid in the gingko at present.

Dehydroabietylamine (DHAA), an important modified product of rosin, is a main component of disproportionated abietylamine, and in recent years, research on the anti-cancer activity of DHAA derivatives has attracted much attention. DHAA can be used as a multi-targeted drug to inhibit three major deregulated signaling pathways in melanoma by disrupting cholesterol transport, resulting in decreased cell proliferation, increased apoptosis and decreased cancer vascularization. Scholars at home and abroad intensively research the biological activity of the DHAA derivatives, and Vicky and other researches find that the dehydroabietic acid and the DHAA compounds have good antiviral activity. [ Roa-lines V C, Brand Y M, Agudelo-Gomez L S, et al, Anti-viral and Anti-dendritic activity of abietane derived collagen analogues synthesized from (+) -dehydroabietylamine [ J]. Eur. J. Med. Chem., 2016, 108: 79-88.]The research of Ling and the like finds that the DHAA derivatives have great potential in the aspect of anticancer, and tests show that the compounds can effectively inhibit the growth of breast cancer cells. [ Ling T, My T, et al. (+) -Dehydroabietylamine derivatives target triple-negative breast cancer[J]. European Journal of Medicinal Chemistry, 2015, 102: 9-13.]Screening a series of anti-cancer activities of DHAA amide derivatives by virtue of Roo Xiaoping and the like, and finding out the IC of the DHAA amide derivatives on liver cancer cells SMMC7721, lung cancer cells A549, glioma cells C6 and breast cancer cells MCF-7₅₀The values were 6.65, 0.75, 0.81 and 10.65. mu.M, respectively. [ Rao X, Huang X, He L, et al, Anticancer Activity and Structure-Activity Relationship of variants with a Dehydroabietyl Skeleton [ J]. Comb. Chem. High T. Scr., 2012, 15(10): 840-844.]

If we can modify the toxic ginkgolic acid into a compound with anticancer activity, a new idea is provided for the development of anticancer drugs. Therefore, the work selects a novel compound with better anticancer activity after the ginkgolic acid is combined with DHAA with the anti-activity, and the cultivation and utilization of economic forests are improved.

Disclosure of Invention

The invention aims to provide a specific modified product of ginkgolic acid (C13: 0) and dehydroabietylamine, a preparation method of an amide compound of ginkgolic acid and application of the amide compound in the aspect of anticancer activity.

The preparation method of the amide compound is easy to operate, the toxicity of the required articles in the experiment is low, and the amide compound is innovative by combining the active compounds in two economic forest species.

Further discloses the application of the amide compound, namely the application in the preparation of anti-cancer drugs.

The amide compound has a novel structure and contains a long-chain electron-donating group (-C)₁₃H₂₇) The compound has low toxicity and unique anticancer activity, is an anticancer medicament with excellent prospect, creates conditions for researching the relation between the anticancer activity and the structure of the compound and lays a foundation for the research and development of new drugs.

A ginkgolic acid amide compound has a structural formula shown in figure 1.

1. The preparation method of the ginkgolic acid amide compound comprises the following steps:

by adopting a DCC-HoBt condensation method, 0.04 mmol of ginkgolic acid (C13: 0) and 0.04 mmol of HoBt are dissolved in 10 mL of ethyl acetate, stirred for 30 minutes at 0 ℃, added with 0.04 mmol of DCC and reacted for 2.5 hours at 0 ℃. Then, 2 mL of an ethyl acetate solution of 0.04 mmol of dehydroabietylamine was added dropwise, the reaction was followed by TLC, and the mixture was stirred in an ice bath for 30 minutes to sufficiently precipitate DCU. The DCU was filtered off and the product was isolated and purified by column chromatography (V ethyl acetate: V n-hexane =1: 3) under reduced pressure to give a light brown oil, 0.014 g, 60% yield.

The molar ratio of the ginkgolic acid (C13: 0) to the dehydroabietylamine is 1: 1.0.

2. Application of ginkgolic acid amide compound in resisting cancer is provided.

MTT thiazole blue colorimetric method is adopted to research the toxic activity of ginkgolic acid amide compound on cervical cancer cells (Hela), liver cancer cells (HepG 2), breast cancer cells (MCF-7), non-small cell lung cancer cells (A549) and normal cells Human Umbilical Vein Endothelial Cells (HUVEC). Taking the cells to be tested in logarithmic growth phase, preparing into 10⁵Inoculating 100 mu L of single cell suspension per mL on a 96-well culture plate, culturing in an incubator with the volume fraction of 5% carbon dioxide, the saturation humidity and the temperature of 37 ℃ for 24 h, and adding 100 mu L of samples to be detected with different concentrations onto the culture plate, wherein each concentration is 6 multiple wells; continuing to culture for 48 h, adding 100 μ L of 1mg/mL MTT staining solution into each well, continuing to culture for 4 h in the incubator, removing supernatant, adding 200 μ L DMSO into each well, shaking for 15 min, measuring OD sample value at 595nm wavelength on the microplate reader, replacing sample with 100 μ L serum-free DMEM culture solution, calculating the cell inhibition rate of ginkgolic acid amide compounds on Hela, HepG2, MCF-7, A549 and HUVEC by the following formula, and finally obtaining IC through origin analysis₅₀。

m=1−n=1−OD_{Sample (A)}/OD_{Blank space}m: the inhibition ratio n: cell viability.

Has the advantages that:

the ginkgolic acid amide compound protected by the invention is IC (Integrated Circuit) on Hela cells₅₀IC value of 1.83. mu.M for HepG2 cells₅₀The value is 3.IC of 31. mu.M on MCF-7 cells₅₀IC for A549 cells with a value of 9.98. mu.M₅₀The value was 5.80. mu.M. Has better activity for resisting Hela cells, HepG2 cells, MCF-7 cells and A549 cells, has lower toxicity, changes the phenomenon that gingko in the traditional concept is toxic, causes anaphylactic reaction and even death when eaten raw or excessively, and provides a new idea for developing anticancer drugs.

The preparation method of the ginkgolic acid amide compound is easy to operate, the toxicity of the required substances in the experiment is low, and the combination of the active substances in two economic forest tree species is innovative.

The ginkgolic acid amide compound has novel structure and contains long-chain electron-donating groups (-C)₁₃H₂₇) The compound has low toxicity and unique anticancer activity, is an anticancer medicament with excellent prospect, creates conditions for researching the relation between the anticancer activity and the structure of the compound and lays a foundation for the research and development of new drugs.

Drawings

FIG. 1 shows the structural formula of ginkgolic acid amide compounds.

FIG. 2 is a nuclear magnetic hydrogen spectrum of ginkgolic acid amide compounds in example 1 of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

The preparation method of the ginkgolic acid amide compound comprises the following steps: by adopting a DCC-HoBt condensation method, 0.04 mmol of ginkgolic acid (C13: 0) and 0.04 mmol of HoBt are dissolved in 10 mL of ethyl acetate, stirred for 30 minutes at 0 ℃, added with 0.04 mmol of DCC and reacted for 2.5 hours at 0 ℃. Then, 2 mL of an ethyl acetate solution of 0.04 mmol of dehydroabietylamine was added dropwise, the reaction was followed by TLC, and the mixture was stirred in an ice bath for 30 minutes to sufficiently precipitate DCU. The DCU was filtered off and the product was isolated and purified by column chromatography (V ethyl acetate: V n-hexane =1: 3) under reduced pressure to give a light brown oil, 0.014 g, 60% yield.

The molar ratio of the ginkgolic acid (C13: 0) to the dehydroabietylamine is 1: 1.0.

The test results were as follows: m.p. 38-39 ℃ IR (neat)νmax 3416, 2924, 2852, 1630, 1534, 1454, 1375, 1280, 1216, 819, 739, 628; 1H NMR(DMSO-d6, 600MHz): δ(ppm)0.90 (3H, s), 1.13-1.15 (3H, s), 1.18 (9H, t, J = 11.4 Hz), 1.23-1.28 (22H, m), 1.30-1.35 (2H, m), 1.38-1.45 (2H, m), 1.50-1.62 (2H, m), 1.67-1.74 (1H, m), 1.92-2.01 (1H, m), 2.23 (1H, d, J = 12.6 Hz), 2.39-2.41 (2H, m), 2.74-2.78 (2H, m), 2.87-2.92 (1H, m), 2.99 (1H, d, J = 7.8 Hz), 3.23 (1H, d, J =13.8 Hz), 6.57 (1H, d, J = 7.8 Hz), 6.62 (1H, d, J = 7.8 Hz), 6.81 (1H, s), 6.91 (1H, d, J = 4.2 Hz), 6.98 (1H, dd, J =3.6, 4.8 Hz), 7.06 (1H, d, J = 8.4 Hz), 7.99 (1H, s), 9.33 (1H, s, −OH); 13C NMR (DMSO-d6, 151MHz): δ (ppm) 23.95, 25.24, 28.74, 28.85, 28.91, 28.92, 29.05, 29.08, 29.41, 30.36, 30.97, 31.32, 32.77, 32.87, 35.39, 36.93. 37.55, 37.88, 43.72, 49.33, 112.69, 119.09, 123.29, 123.78, 126.33, 126.53, 128.41, 134.70, 140.41, 144.64, 147.11, 153.73, 167.69; ESI-MS [M+Na]+ m/z 610.4594 (calcd for C40H61NO2 587.4702). Anal. calcd for C40H61NO2: C, 81.72; H, 10.46; N, 2.38. Found: C, 81.79; H, 10.41; N, 2.41。

MTT thiazole blue colorimetric method is adopted to research the toxic activity of cervical cancer cell (Hela), liver cancer cell (HepG 2), breast cancer cell (MCF-7), non-small cell lung cancer cell (A549) and normal cell Human Umbilical Vein Endothelial Cell (HUVEC). Ginkgolic acid amide compounds, IC on Hela cells₅₀IC value of 1.83. mu.M for HepG2 cells₅₀IC value of 3.31. mu.M for MCF-7 cells₅₀IC for A549 cells with a value of 9.98. mu.M₅₀The value was 5.80. mu.M. Has better activity for resisting Hela cells, HepG2 cells, MCF-7 cells and A549 cells, has lower toxicity, changes the phenomenon that gingko in the traditional concept is toxic, causes anaphylactic reaction and even death when eaten raw or excessively, and provides a new idea for developing anticancer drugs.

Example 2

The preparation method of the ginkgolic acid amide compound comprises the following steps: by adopting a DCC-HoBt condensation method, 0.04 mmol of ginkgolic acid (C13: 0) and 0.04 mmol of HoBt are dissolved in 10 mL of ethyl acetate, stirred for 30 minutes at 0 ℃, added with 0.04 mmol of DCC and reacted for 2.5 hours at 0 ℃. Then, 2 mL of an ethyl acetate solution of 0.044 mmol of dehydroabietylamine was added dropwise thereto, followed by reaction by TLC, and the mixture was stirred in an ice bath for 30 minutes to thereby sufficiently precipitate DCU. DCU was filtered off, and the product was isolated and purified by column chromatography (V ethyl acetate: V n-hexane =1: 3) under reduced pressure to give a light brown oil, 0.015 g, 64% yield.

The molar ratio of the ginkgolic acid (C13: 0) to the dehydroabietylamine is 1: 1.1. The test results showed the same results as in example 1.

Example 3

The preparation method of the ginkgolic acid amide compound comprises the following steps: by adopting a DCC-HoBt condensation method, 0.04 mmol of ginkgolic acid (C13: 0) and 0.04 mmol of HoBt are dissolved in 10 mL of ethyl acetate, stirred for 30 minutes at 0 ℃, added with 0.04 mmol of DCC and reacted for 2.5 hours at 0 ℃. Then, 2 mL of an ethyl acetate solution of 0.048 mmol of dehydroabietylamine was added dropwise thereto, followed by reaction by TLC, and the mixture was stirred in an ice bath for 30 minutes to thereby sufficiently precipitate DCU. The DCU was filtered off and the product was isolated and purified by column chromatography (V ethyl acetate: V n-hexane =1: 3) under reduced pressure to give a light brown oil, 0.014 g, 60% yield.

The molar ratio of the ginkgolic acid (C13: 0) to the dehydroabietylamine is 1: 1.2. The test results showed the same results as in example 1.