A centella asiatica acid derivative, preparation method and application thereof

Abstract

The present application relates to the technical field of asiatic acid, and particularly relates to an asiatic acid derivative and a preparation method. The asiatic acid derivative provided by the present application has R which is an alkyl group formed by removing a hydroxyl group from a saturated or unsaturated monohydric alcohol, and the saturated or unsaturated monohydric alcohol includes one of ethanol, octanol, hexadecanol, stearyl alcohol, 2-hexyl-1-decanol, decyltetradecin, oleyl alcohol, cis,cis-9,12-octadecadienol and bisabolol. The asiatic acid derivative of the present application has higher water solubility and oil solubility, especially oil solubility, so that the asiatic acid derivative has obviously enhanced liposolubility, is easily soluble in natural oil, alkane and other ester solvents, and has a significant inhibitory effect on EDNRA and TRPV1 and obviously enhanced bioactivity. The asiatic acid derivative of the present application has high availability, and the asiatic acid derivative has no safety risk.

Description

Technical Field

[0001] This invention relates to the field of asiatic acid technology, specifically to an asiatic acid derivative, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is a malignant tumor that occurs in the colon or rectum, and it is a common cancer in modern society. Colorectal cancer is generally treated with surgery and chemotherapy, but there are not many drugs available for treating colorectal cancer. Currently, broad-spectrum anticancer drugs such as 5-fluorouracil (5-FU) and irinotecan are used to treat colorectal cancer, often in combination with antibody therapies such as cetuximab or bevacizumab. However, broad-spectrum drugs are deadly to both normal and cancer cells, and drug resistance leads to high recurrence rates. Natural anticancer compounds have great potential in the treatment of various diseases. Some natural compounds extracted from traditional herbs have shown considerable regulatory ability against specific targets; for example, icariin enhances CD8 expression by inhibiting PD-L1 expression. + T-cell infiltration is a drug approved by the State Food and Drug Administration for the treatment of advanced liver cancer.

[0003] Endothelin Receptor Type A (EDNRA) belongs to the G protein-coupled receptor (GPCR) family and is mainly expressed in endothelial cells and smooth muscle cells. It is one of the main receptors for endothelin-1 (ET-1). In colorectal cancer, the EDN1 / EDNRA / β-arrestin axis promotes tumor cell proliferation and invasion by activating STAT3 phosphorylation. Drug development targeting EDNRA mainly focuses on antibody drugs. TRPV1 (transient receptor potential vanillic acid isoform 1) is a non-selective cation channel protein, also known as the capsaicin receptor (VR1). Capsaicin can regulate the proliferation of colorectal cancer.

[0004] Asiatic acid (AA) is a pentacyclic triterpenoid natural compound derived from Centella asiatica. It possesses multi-target and multi-domain pharmacological activities, with core efficacy focused on skin repair and regeneration, promoting wound healing and scar repair, anti-aging and firming, and anti-photoaging. It also exhibits broad-spectrum anti-inflammatory activity. Recent studies have found that asiatic acid has a protective effect on the central nervous system, showing promise for the treatment of neurodegenerative diseases. However, asiatic acid has extremely poor water solubility (due to its pentacyclic triterpenoid structure) and low oral bioavailability (<5%), limiting its efficacy. Currently, there is an urgent need to find a natural anticancer compound targeting EDNRA and TRPV1. Therefore, improving the structure of asiatic acid to enhance its inhibitory effects on EDNRA and TRPV1, thereby improving its anti-colorectal cancer efficacy, is a key technical challenge that needs to be addressed for its application. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defect of poor inhibitory effect of targeting EDNRA and TRPV1 in the prior art, so as to provide a asiatic acid derivative, its preparation method and application, so as to improve its inhibitory effect of targeting EDNRA and TRPV1, thereby improving the effect of anti-colorectal cancer.

[0006] To address the aforementioned technical problems, the following technical solutions are proposed:

[0007] The present invention provides a asiatic acid derivative having a structure as shown in formula (1);

[0008] Equation (1) In formula (1), R is an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol, and the saturated or unsaturated monohydric alcohol includes one of ethanol, octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.

[0009] Preferably, the asiatic acid derivative has at least one of the following structures:

[0010] Equation (2)

[0011] Equation (3)

[0012] Equation (4)

[0013] Equation (5)

[0014] Equation (6)

[0015] Equation (7)

[0016] Equation (8)

[0017] Equation (9)

[0018] Equation (10).

[0019] This invention provides a method for preparing the asiatic acid derivative described in the above technical solution, comprising: Triacetyl asiatic acid ester, organic solvent, water and strong base are reacted to obtain a reaction solution; the reaction solution is concentrated and extracted to obtain asiatic acid derivative; the triacetyl asiatic acid ester has the structure shown in formula (11);

[0020] Equation (11) In formula (11), R is an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol, wherein the saturated or unsaturated monohydric alcohol is selected from one of ethanol, octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.

[0021] Preferably, the organic solvent includes tetrahydrofuran.

[0022] Preferably, the ratio of triacetyl asiatic acid ester to organic solvent is (0.2-0.5):50, where the unit of ratio is mmol / mL; the molar ratio of triacetyl asiatic acid ester to strong base is (0.2-0.5):(1-1.7); and the volume ratio of organic solvent to water is (10-20):1.

[0023] Preferably, the concentration method includes rotary evaporation; the extractant used in the extraction includes ethyl acetate; the auxiliary extraction system used in the extraction includes saturated brine; and the strong base substance includes at least one of LiOH, NaOH, and KOH.

[0024] Preferably, the reaction temperature is 20-25°C, more preferably 25°C; the reaction continues until triacetyl asiatic acid ester is completely consumed.

[0025] This invention provides the application of the asiatic acid derivative described in the above technical solution or the asiatic acid derivative obtained by the preparation method described in the above technical solution in at least one of the following: 1) Preparation of EDNRA inhibitors; 2) Preparation of TRPV1 inhibitors; 3) Prepare a drug for the treatment of colorectal cancer; 4) Prepare skin anti-aging agents.

[0026] The technical solution of this invention has the following advantages: 1. The asiatic acid derivative of the present invention exhibits a significant inhibitory effect on EDNRA and TRPV1 compared to asiatic acid.

[0027] 2. The solubility of the asiatic acid derivative of the present invention is significantly improved and its bioavailability is high.

[0028] In summary, this invention modifies the structure of asiatic acid molecules to obtain asiatic acid derivatives, thereby expanding the comprehensive utilization value of asiatic acid analogs in the fields of anti-cancer and anti-aging, and opening up new avenues for their application in more fields. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a process flow diagram of a specific example of asiatic acid derivative in Embodiment 10 of the present invention; wherein A represents triacetyl asiatic acid ester and B represents the asiatic acid derivative prepared in this invention. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0033] The materials and reagents used in the following examples and comparative examples are as follows: All raw materials and chemical reagents involved in this invention were purchased from Sigma-Aldrich.

[0034] The room temperature in the following examples is 25°C.

[0035] Example 1 This embodiment provides a specific preparation method and operating parameters for a asiatic acid derivative, as follows: 1. Add asiatic acid (488 mg, 1.0 mmol, 1.0 equiv.) to a 100 mL single-necked flask, then add 50 mL of dichloromethane solvent and acetic anhydride (408 mg, 4.0 mmol, 4.0 equiv.), and add DMAP (12 mg, 0.1 mmol, 0.1 equiv.) as a catalyst. Stir at room temperature for 12 h. Then transfer the reaction mixture to a separatory funnel, extract twice with 50 mL of saturated saline solution, and dry the organic phase with anhydrous sodium sulfate. The final product, triacetylasiatic acid, is then purified by silica gel column chromatography.

[0036] 2. Add triacetyl asiatic acid (614 mg, 1.0 mmol, 1.0 equiv) to a 100 mL single-necked flask, then place the flask in an ice-water bath. Add 50 mL of dichloromethane solvent and two drops of DMF as a catalyst to obtain the first reaction solution. Separately, weigh oxalyl chloride (140 mg, 1.2 mmol, 1.2 equiv) into a 25 mL flask and dissolve it in 10 mL of dichloromethane to obtain an oxalyl chloride solution. Slowly add the oxalyl chloride solution dropwise to the first reaction solution. After the addition is complete, allow the mixture to rise naturally to room temperature and stir for 3 hours to obtain the second reaction solution. The second reaction solution was then evaporated to dryness using a rotary evaporator with dichloromethane solvent. Another 30 mL of dichloromethane was added to dissolve the residue, and the solvent was evaporated again. Using 50 mL of dichloromethane solvent, 1.2 mmol (1.2 equiv) of ethanol was added, and the reaction was carried out at room temperature for 12 hours. TLC analysis showed that triacetyl asiaticoyl chloride was completely consumed. The mixture was then transferred to a separatory funnel and extracted twice with 50 mL of saturated saline solution (volume ratio of extracted substance to saturated saline solution 1:1). The organic phase was dried over anhydrous sodium sulfate. The final product, triacetyl asiaticoyl ester A01, was obtained by separation and purification using a silica gel column (200-300 mesh, purchased from Qingdao Ocean Chemical Co., Ltd.). The chemical structure of A01 is shown below:

[0037] A01 A01 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.21 – 5.16 (m, 1H), 5.15 – 5.05 (m, 1H),5.02 (d, J = 10.3 Hz, 1H), 3.99 (q, J = 7.1 Hz, 2H), 3.79 (d, J= 11.8 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.2 Hz, 1H), 2.05 – 2.01 (m, 3H), 1.97 –1.95 (m, 3H), 1.93 – 1.90 (m, 3H), 1.87 (d, J = 6.8 Hz, 2H), 1.78 – 1.18 (m,14H), 1.15 (t, J = 7.1 Hz, 4H), 1.11 – 0.96 (m, 9H), 0.91 – 0.67 (m, 13H); 13 C NMR (100 MHz, CDCl3) δ 176.40, 169.86, 169.43, 169.37, 137.28,123.93, 73.82, 68.91, 64.25, 59.02, 51.79, 46.79, 46.58, 46.46, 42.75, 41.03,40.86, 38.56, 38.03, 37.81, 36.76, 35.55, 31.52, 29.64, 26.85, 23.06, 22.35,22.32, 20.17, 20.08, 19.88, 19.78, 16.86, 16.03, 16.02, 15.95, 13.22, 12.92. Example 2 The preparation method is the same as in Example 1, except that ethanol is replaced with octanol. The final product is triacetyl asiaticoside ester A02, and the chemical structural formula of A02 is shown below:

[0038] A02 A02 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.17 (t, J = 3.7 Hz, 1H), 5.10 (t, J = 10.7 Hz, 1H), 5.02 (d, J = 10.3 Hz, 1H), 3.96 – 3.86 (m, 2H), 3.79 (d,J = 11.8 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.1 Hz, 1H), 2.02 (s, 3H), 1.96 (s, 3H), 1.91 (s, 3H), 1.86 (d, J = 10.4 Hz, 3H), 1.77 – 1.13 (m, 299H), 1.08 (d, J = 11.9Hz, 2H), 1.02 (d, J = 11.6 Hz, 7H), 0.90 – 0.75 (m, 9H), 0.69 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 177.54, 170.90, 170.47, 170.41, 138.37,124.97, 74.85, 69.94, 65.29, 64.33, 52.81, 48.00, 47.61, 47.49, 43.77, 42.06,41.89, 39.58, 39.06, 38.84, 37.79, 36.66, 32.55, 31.81, 30.68, 29.24, 29.19,28.58, 27.87, 26.07, 24.12, 23.38, 22.66, 21.20, 21.11, 20.91, 20.81, 17.90, 17.08, 17.04, 16.99, 14.13, 13.95. Example 3 The preparation method is the same as in Example 1, except that ethanol is replaced with hexadecyl alcohol. The final product is triacetyl asiatic acid ester A03, and the chemical structural formula of A03 is shown below:

[0039] A03 A03 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.20 – 5.15 (m, 1H), 5.10 (t, J = 10.7 Hz, 1H), 5.02 (d, J= 10.3 Hz, 1H), 3.91 (t, J = 8.5 Hz, 2H), 3.78 (d, J = 11.8 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.5 Hz, 1H), 2.02 (s, 3H), 1.96 (s,3H), 1.92 (s, 3H), 1.87 (d, J = 7.0 Hz, 3H), 1.76 – 1.12 (m, 41H), 1.11 – 0.96(m, 9H), 0.91 – 0.75 (m, 13H), 0.69 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.51, 169.89, 169.45, 137.30, 123.92,73.79, 68.89, 64.23, 63.29, 51.75, 46.95, 46.55, 46.42, 42.70, 41.00, 40.83,38.51, 38.01, 37.79, 36.73, 35.61, 31.50, 30.90, 29.63, 28.69, 28.64, 28.59,28.54, 28.35, 28.18, 27.53, 26.82, 25.02, 23.07, 22.33, 21.68, 20.17, 20.09, 19.90, 19.79, 16.84, 16.03, 16.01, 15.95, 13.12, 12.92. Example 4 The preparation method is the same as in Example 1, except that ethanol is replaced with stearyl alcohol. The final product is triacetyl asiatic acid ester A04, and the chemical structural formula of A04 is shown below:

[0040] A04 A04 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.18 (d, J = 3.7 Hz, 1H), 5.10 (t, J= 10.7 Hz, 1H), 5.02 (d, J = 10.3 Hz, 1H), 3.91 (q, J = 6.4 Hz, 2H), 3.78 (d, J = 11.8 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.4 Hz, 1H), 2.02 (s, 3H), 1.96 (s,3H), 1.92 (s, 3H), 1.90 – 1.83 (m, 2H), 1.78 – 1.12 (m, 47H), 1.11 – 0.96 (m,9H), 0.91 – 0.75 (m, 12H), 0.69 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.52, 169.90, 169.45, 169.40, 137.30,123.92, 73.79, 68.90, 64.23, 63.29, 51.75, 46.95, 46.55, 46.42, 42.71, 41.00,40.84, 38.51, 38.01, 37.79, 36.73, 35.61, 31.50, 30.90, 29.63, 28.69, 28.64,28.59, 28.54, 28.35, 28.18, 27.53, 26.82, 25.02, 23.07, 22.33, 21.68, 20.17, 20.09, 19.89, 19.79, 16.85, 16.03, 15.95, 13.12, 12.91. Example 5 The preparation method is the same as in Example 1, except that ethanol is replaced with 2-hexyl-1-decyl alcohol. The final product is triacetyl asiaticoside A05, and the chemical structure of A05 is shown below:

[0041] A05 A05 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.17 (d, J= 3.6 Hz, 1H), 5.09 (t, J = 10.8 Hz, 1H), 5.01 (d, J = 10.3 Hz, 1H), 3.81 (d, J = 5.2 Hz, 2H), 3.78 (d, J = 11.7 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.1 Hz, 1H), 2.02 (s, 3H), 1.96 (s,3H), 1.91 (s, 3H), 1.87 (d, J = 7.5 Hz, 2H), 1.19 (s, 41H), 1.02 (d, J = 10.1 Hz,9H), 0.91 – 0.75 (m, 16H), 0.69 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.61, 169.87, 169.43, 169.38, 137.38,123.95, 73.83, 68.89, 65.61, 64.26, 51.80, 47.17, 46.57, 46.46, 42.73, 41.02,40.86, 38.52, 38.03, 37.88, 36.76, 36.23, 35.74, 31.51, 30.92, 30.87, 30.84,30.47, 30.41, 29.66, 28.99, 28.65, 28.62, 28.33, 26.84, 25.75, 25.69, 23.13, 22.38, 22.33, 21.68, 21.66, 20.15, 20.07, 19.88, 19.78, 16.86, 16.08, 15.97, 13.11, 12.90. Example 6 The preparation method is the same as in Example 1, except that ethanol is replaced with tetradecanol. The final product is triacetyl asiaticoside A06, and the chemical structure of A06 is shown below:

[0042] A06 A06 1 H NMR and 13The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.20 – 5.15 (m, 1H), 5.11 (t, J = 10.8 Hz, 1H), 5.01 (d, J = 10.3 Hz, 1H), 3.81 (d, J = 5.2 Hz, 2H), 3.78 (d, J = 11.7 Hz, 1H), 3.51 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.2 Hz, 1H), 2.02 (s, 3H), 1.95 (s, 3H), 1.91 (s, 3H), 1.87 (d, J = 7.5 Hz, 2H), 1.77 – 1.12 (m, 69H), 1.02 (d, J =9.9 Hz, 10H), 0.92 – 0.75 (m, 24H), 0.75 – 0.71 (m, 1H), 0.69 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.53, 169.77, 169.34, 137.38, 123.97,73.83, 68.86, 65.57, 64.23, 51.81, 47.17, 46.59, 46.46, 42.75, 41.03, 40.87,38.53, 38.05, 37.89, 36.77, 36.23, 35.75, 31.53, 30.92, 30.49, 30.42, 29.69,28.99, 28.73, 28.68, 28.37, 26.86, 25.75, 23.15, 22.38, 22.34, 21.69, 20.16, 20.05, 19.85, 19.76, 16.88, 16.10, 16.00, 15.98, 13.13, 12.90. Example 7 The preparation method is the same as in Example 1, except that ethanol is replaced with oleyl alcohol. The final product is triacetyl asiatic acid ester A07, and the chemical structural formula of A07 is shown below:

[0043] A07 A07 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.33 – 5.15 (m, 2H), 5.09 (d, J = 10.6 Hz, 1H), 5.02 (d, J = 10.4 Hz, 1H), 3.94 (d, J = 32.0 Hz, 1H), 3.79 (d, J = 11.8 Hz, 1H), 3.51 (d, J = 11.9 Hz, 1H), 2.16 (d, J = 4.8 Hz, 1H), 2.04 – 2.01 (m, 3H), 1.97 – 1.95 (m, 3H), 1.93 – 1.91 (m, 3H), 1.89 – 1.14 (m, 45H), 1.05 (q, J =14.6, 10.0 Hz, 9H), 0.93 – 0.74 (m, 15H), 0.69 (s, 2H); 13 C NMR (100 MHz, CDCl3) δ 176.48, 169.84, 169.42, 169.38, 137.33,128.94, 128.91, 128.77, 128.73, 123.96, 73.83, 68.90, 64.25, 63.28, 61.94,59.37, 51.79, 46.97, 46.59, 46.47, 42.74, 41.03, 40.87, 38.55, 38.04, 37.83,36.76, 35.63, 31.77, 30.90, 29.66, 28.75, 28.65, 28.44, 28.31, 28.21, 28.18, 27.56, 26.85, 26.19, 25.04, 24.75, 23.10, 22.35, 21.68, 20.17, 20.06, 20.02, 19.86, 19.77, 16.88, 16.05, 15.96, 13.18. Example 8 The preparation method is the same as in Example 1, except that ethanol is replaced with cis-cis-9,12-octadecadienol. The final product is triacetyl asiatic acid ester A08, and the chemical structure of A08 is shown below:

[0044] A08 A08 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.21 (dt, J = 23.2, 3.6 Hz, 1H), 5.10 (t, J =10.6 Hz, 1H), 5.02 (d, J = 10.3 Hz, 1H), 3.99 (q, J = 7.1 Hz, 1H), 3.79 (d, J =11.6 Hz, 1H), 3.50 (d, J = 11.8 Hz, 1H), 2.17 (d, J = 11.9 Hz, 1H), 2.02 (s, 3H), 1.96 (s, 3H), 1.91 (s, 3H), 1.83 – 0.99 (m, 46H), 0.88 (s, 5H), 0.81 (d, J =15.8 Hz, 16H), 0.72 (t, J = 3.2 Hz, 1H), 0.70 (s, 2H); 13 C NMR (100 MHz, CDCl3) 13 C NMR (101 MHz, Chloroform- d) δ 177.44,170.90, 170.48, 170.41, 138.31, 136.70, 126.51, 124.96, 74.85, 69.94, 69.89,65.29, 60.06, 58.79, 53.98, 52.81, 47.83, 47.56, 47.48, 43.78, 42.06, 42.04,41.89, 39.71, 39.59, 39.05, 38.83, 38.58, 37.78, 36.58, 35.28, 32.55, 32.47,30.67, 30.43, 29.70, 27.87, 27.70, 25.27, 24.09, 23.38, 21.20, 21.11, 20.97, 20.92, 17.89, 17.12, 17.06, 17.04, 16.98, 16.93, 16.89, 14.25, 13.95. Example 9 The preparation method is the same as in Example 1, except that ethanol is replaced with bisabolol. The final product is triacetyl asiatic acid ester A09, and the chemical structural formula of A09 is shown below:

[0045] A09 A09 1 H NMR and 13 The C NMR values ​​are as follows: 11H NMR (400 MHz, CDCl3) δ 5.30 (t, J = 4.2 Hz, 2H), 5.16 – 5.07 (m, 3H), 3.86 (d, J = 11.5 Hz, 1H), 3.58 (d, J = 11.5 Hz, 1H), 2.23 (d, J = 11.0 Hz, 1H), 2.13 (s, 1H), 2.10 (s, 3H), 2.08 (s, 2H), 2.03 (d, J = 1.6 Hz, 6H), 1.92 (d, J = 13.9 Hz, 4H), 1.87 – 1.75 (m, 3H), 1.72 (d, J = 15.6 Hz, 4H), 1.66 (s, 3H), 1.63 (s, 3H), 1.61 (d, J = 9.0 Hz, 10H), 1.36 (s, 3H), 1.35 – 1.32 (m, 2H), 1.31 (s, 3H), 1.27 (s, 3H), 1.21 – 1.16 (m, 1H), 1.12 (s, 3H), 1.08 (m, 1H), 0.91 – 0.83 (m, 12H), 0.80 (d, J = 6.5 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ 176.82, 170.95, 170.28, 170.24, 138.13, 134.19, 132.10, 125.26, 124.50, 120.60, 83.27, 75.20, 70.10, 65.60, 52.87, 48.11, 48.00, 47.90, 43.90, 42.77, 42.20, 42.14, 39.93, 38.98, 38.92, 38.20, 38.12, 36.55, 32.77, 30.94, 30.54, 27.99, 27.24, 25.70, 24.21, 24.10, 23.95, 23.72, 23.67, 23.18, 23.16, 21.13, 21.11, 21.05, 20.55, 18.20, 17.70, 17.09, 17.00, 16.96, 14.10. Example 10 In the A01 (0.5 mmol, 1.0 equiv.) prepared in Example 1, 50 mL of tetrahydrofuran solvent and 5 mL of water were added sequentially as solvents, followed by LiOH (1.7 mmol, 3.1 equiv.). The mixture was stirred at room temperature, and the reaction was stopped when A01 was completely consumed, as detected by TLC. The procedure is as follows: Figure 1 Add 5 mL of 0.1 M hydrochloric acid solution and evaporate most of the tetrahydrofuran using a rotary evaporator. Add 50 mL of ethyl acetate to the residue and extract twice with saturated brine to obtain the organic phase. Dry the organic phase with anhydrous sodium sulfate. Then, separate and purify the final product, asiatic acid derivative B01, using silica gel column chromatography. The chemical structure is as follows:

[0046] B01 B01 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ5.38 (t, J = 3.1 Hz, 1H), 4.53 (dd, J = 10.3,9.4, Hz, 1H), 4.24 – 4.17 (m, 2H), 4.01 (dd, J = 18.7, 7.2 Hz, 2H), 3.74 (dd, J =11.2, 7.7 Hz, 1H), 2.18 (d, J = 11.1 Hz, 1H), 2.01 (m, 3H), 1.84 (m, 1H), 1.77(m, 1H), 1.67 (m, 5H), 1.60 – 1.22 (m, 11H), 1.12 (s, 3H), 1.10 (d, J = 10.4Hz, 1H), 1.06 (d, J = 2.8 Hz, 3H), 1.04 (d, J = 7.1 Hz, 5H), 0.96 – 0.90 (m, 1H), 0.90 – 0.79 (m, 10H); 13C NMR (100 MHz, CDCl3) δ176.82, 138.13, 125.26, 78.20, 68.90, 66.50,61.08, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98, 38.92,38.30, 36.55, 33.20, 30.54, 27.99, 24.21, 23.80, 23.67, 21.13, 18.50, 17.80,17.60, 16.96, 14.40, 14.09. Example 11 Same as Example 10, except that A01 was replaced with A02 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B02, with the following chemical structure:

[0047] B02 B02 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ5.38 (t, J = 3.1 Hz, 1H), 4.53 (d, J = 11.2 Hz, 1H), 4.20 (d, J = 7.8 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.74 (dd, J = 11.2, 7.7Hz, 1H), 2.20 (d, J = 11.2 Hz, 1H), 2.02 (d, J = 12.6 Hz, 3H), 1.81 (d, J = 20.9Hz, 2H), 1.74 – 1.62 (m, 7H), 1.60 – 1.20 (m, 27H), 1.12 (s, 4H), 1.06 (s,3H), 0.97 – 0.91 (m, 1H), 0.91 – 0.78 (m, 9H); 13C NMR (100 MHz, CDCl3) δ 176.82, 138.13, 125.26, 78.20, 68.90,66.50, 64.20, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98,38.92, 38.30, 36.55, 33.20, 31.74, 30.54, 29.41, 29.40, 28.88, 27.99, 27.00,24.21, 23.80, 23.67, 22.72, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.10. Example 12 Same as Example 10, except that A01 was replaced with A03 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B03, with the following chemical structure:

[0048] B03 B03 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.38 (t, J = 3.1 Hz, 1H), 4.53 (d, J = 10.1 Hz, 2H), 4.20 (d, J = 7.8 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.74 (dd, J = 11.2, 7.7Hz, 1H), 2.20 (d, J = 11.2 Hz, 1H), 2.01 (d, J = 11.3 Hz, 4H), 1.87 – 1.75 (m,4H), 1.73 – 1.62 (m, 12H), 1.60 – 1.20 (m, 32H), 1.12 (s, 4H), 1.06 (s, 3H),0.97 – 0.78 (m, 12H); 13C NMR (100 MHz, CDCl3) δ176.82, 138.13, 125.26, 78.20, 68.90, 66.50,64.20, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98, 38.92,38.30, 36.55, 33.20, 32.36, 30.54, 30.45, 29.58, 29.54, 29.53, 29.49, 29.40,28.88, 27.99, 27.00, 24.21, 23.80, 23.67, 22.87, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.14. Example 13 Same as Example 10, except that A01 was replaced with A04 (0.5 mmol, 1.0 equiv.), and then the final product, asiatic acid derivative B04, was obtained by silica gel column separation and purification, with the following chemical structure:

[0049]

[0050] B04 B04 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ5.38 (t, J = 3.1 Hz, 1H), 4.52 (d, J = 6.1 Hz, 1H), 4.20 (t, J = 10.6 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.77 – 3.72 (m, 1H), 2.20 (d, J = 11.2 Hz, 1H), 2.02 (d, J = 10.6 Hz, 3H), 1.87 – 1.77 (m, 2H), 1.72 –1.62 (m, 8H), 1.60 – 1.22 (m, 46H), 1.12 (s, 4H), 1.06 (s, 3H), 0.96 – 0.85(m, 13H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ176.82, 138.13, 125.26, 78.20, 68.90, 66.50,64.20, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98, 38.92,38.30, 36.55, 33.20, 32.00, 30.54, 30.45, 29.58, 29.54, 29.53, 29.49, 29.36,28.88, 27.99, 27.00, 24.21, 23.80, 23.67, 22.60, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.14. Example 14 Same as Example 10, except that A01 was replaced with A05 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B05, with the following chemical structure:

[0051] B05 B05 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ5.38 (t, J = 3.1 Hz, 1H), 4.81 (d, J = 8.1 Hz, 1H), 4.52 (d, J = 6.1 Hz, 1H), 4.24 – 4.17 (m, 2H), 3.74 (dd, J = 11.2, 7.7 Hz, 1H), 2.22 (d, J = 11.2 Hz, 1H), 2.11 – 1.99 (m, 7H), 1.87 – 1.63 (m, 11H), 1.61 (s, 3H), 1.59 – 1.23 (m, 30H), 1.15 (d, J = 6.9 Hz, 1H), 1.12 (s, 3H), 1.06 (s,3H), 0.93 (dd, J= 12.5, 9.2 Hz, 1H), 0.90 – 0.83 (m, 11H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ176.55, 138.13, 125.26, 78.20, 74.33, 68.90,66.50, 52.85, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98, 38.92, 38.30,36.54, 34.06, 33.20, 31.98, 31.71, 30.54, 29.50, 29.47, 29.36, 29.17, 27.99,25.26, 25.24, 24.18, 23.80, 23.67, 22.72, 22.55, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.10. Example 15 Same as Example 10, except that A01 was replaced with A06 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B06, with the following chemical structure:

[0052] B06 B06 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ5.38 (t, J = 3.1 Hz, 1H), 4.81 (t, J = 9.0 Hz,1H), 4.56 – 4.48 (m, 1H), 4.24 – 4.17 (m, 2H), 3.77 – 3.71 (m, 1H), 2.24 –2.20 (m, 1H), 2.13 – 1.99 (m, 6H), 1.88 – 1.78 (m, 2H), 1.75 – 1.62 (m, 6H), 1.61 (s, 3H), 1.57 – 1.23 (m, 47H), 1.15 (d, J= 6.9 Hz, 1H), 1.13 – 1.11 (m,3H), 1.08 – 1.05 (m, 2H), 0.96 – 0.90 (m, 1H), 0.90 – 0.85 (m, 16H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.82, 138.13, 125.26, 78.20, 74.33,68.90, 66.50, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14, 40.20, 38.98,38.92, 38.30, 36.55, 34.06, 33.20, 31.98, 30.54, 29.58, 29.54, 29.53, 29.51,29.49, 29.47, 29.36, 27.99, 25.27, 24.21, 23.80, 23.67, 22.72, 22.68, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.10, 14.09. Example 16 Same as Example 10, except that A01 was replaced with A07 (0.5 mmol, 1.0 equiv.), and then the final product, asiatic acid derivative B07, was obtained by silica gel column separation and purification, with the following chemical structure:

[0053]

[0054] B07 B07 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.38 (t, J = 3.1 Hz, 1H), 5.25 (t, J = 6.5 Hz,2H), 4.56 – 4.49 (m, 1H), 4.23 – 4.17 (m, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.74(dd, J = 11.2, 7.7 Hz, 1H), 2.20 (d, J= 11.1 Hz, 1H), 2.06 – 1.99 (m, 3H), 1.86 – 1.76 (m, 6H), 1.72 – 1.21 (m, 42H), 1.12 (s, 4H), 1.06 (s, 3H), 0.96 – 0.91(m, 1H), 0.91 – 0.85 (m, 10H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.82, 138.13, 130.02, 129.94, 125.26,78.20, 68.90, 66.50, 65.39, 52.87, 48.11, 48.10, 48.00, 47.90, 43.60, 42.14,40.20, 38.98, 38.92, 38.30, 36.55, 33.20, 32.68, 32.60, 31.92, 30.54, 29.86,29.65, 29.62, 29.41, 29.29, 29.26, 29.23, 29.00, 27.99, 27.00, 24.21, 23.80, 23.67, 22.71, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.22.

[0055] Example 17 Same as Example 10, except that A01 was replaced with A08 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B08, with the following chemical structure:

[0056] B08 B08 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.54 – 5.44 (m, 2H), 5.40 – 5.33 (m, 2H),5.26 (d, J = 15.6 Hz, 1H), 4.56 – 4.49 (m, 1H), 4.23 – 4.17 (m, 2H), 4.05 (t, J=6.6 Hz, 2H), 3.77 – 3.72 (m, 1H), 2.20 (d, J = 11.1 Hz, 1H), 2.06 – 1.99 (m,4H), 1.94 (d, J = 6.9 Hz, 2H), 1.86 – 1.76 (m, 2H), 1.73 – 1.59 (m, 9H), 1.58 –1.21 (m, 32H), 1.12 (s, 4H), 1.08 – 1.03 (m, 3H), 0.94 (d, J = 9.2 Hz, 1H),0.92 – 0.85 (m, 8H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.82, 138.13, 131.30, 131.00, 129.80,128.80, 125.26, 78.20, 68.90, 66.50, 64.20, 52.87, 48.11, 48.10, 48.00,47.90, 43.60, 42.14, 40.20, 38.98, 38.92, 38.30, 36.55, 33.20, 32.46, 31.53,31.00, 30.54, 29.35, 29.34, 29.29, 29.23, 28.88, 27.99, 27.00, 24.21, 23.80, 23.67, 22.57, 21.13, 18.50, 17.80, 17.60, 16.96, 14.40, 14.08.

[0057] Example 18 Same as Example 10, except that A01 was replaced with A09 (0.5 mmol, 1.0 equiv.), and then purified by silica gel column chromatography to obtain the final product, asiatic acid derivative B09, with the following chemical structure:

[0058] B09 B09 1 H NMR and 13 The C NMR values ​​are as follows: 1 H NMR (400 MHz, CDCl3) δ 5.38 (t, J= 3.1 Hz, 1H), 5.30 (t, J = 4.3 Hz, 1H), 5.12 (t, J = 6.5 Hz, 1H), 4.56 – 4.49 (m, 1H), 4.23 – 4.17 (m, 2H), 3.74(dd, J = 11.2, 7.7 Hz, 1H), 2.23 (d, J = 11.1 Hz, 1H), 2.17 – 2.11 (m, 1H), 2.10 – 1.75 (m, 9H), 1.74 (s, 3H), 1.74 – 1.67 (m, 4H), 1.66 (s, 3H), 1.63 (s,3H), 1.61 (s, 3H), 1.57 – 1.32 (m, 12H), 1.31 (s, 3H), 1.30 – 1.22 (m, 2H), 1.12 (s, 3H), 1.11 – 1.07 (m, 1H), 1.06 (s, 3H), 0.93 (dd, J = 12.5, 9.2 Hz, 1H), 0.87 (d, J = 6.3 Hz, 10H), 0.80 (d, J = 6.5 Hz, 3H); 13 C NMR (100 MHz, CDCl3) δ 176.82, 138.13, 134.19, 132.10, 125.26,124.50, 120.60, 83.27, 78.20, 68.90, 66.50, 52.87, 48.11, 48.10, 48.00,47.90, 43.60, 42.77, 42.14, 40.20, 38.98, 38.92, 38.30, 38.12, 36.55, 33.20,30.94, 30.54, 27.99, 27.24, 25.70, 24.21, 24.10, 23.95, 23.80, 23.67, 23.18, 23.16, 21.13, 18.50, 17.80, 17.70, 17.60, 16.96, 14.40. Efficacy test (1) Construct SH-SY5Y cells overexpressing EDNRA and TRPV1 SH-SY5Y cells were purchased from BeiNa Biotechnology.

[0059] The vector plasmids and target genes used to construct stable overexpression cell lines were provided by Shanghai Merckson Biotechnology Co., Ltd. The EDNRA gene sequence number is NM_001957.4. The base vector is CMV-MCS-PGK-Puro, and the restriction enzyme sites are Xhol and EcoR1. After restriction enzyme digestion and recombination, the EDNRA gene recombinant vector is obtained.

[0060] The TRPV1 gene sequence is NCBI numbered AY131289.1, the base vector is pCDH-CMV-MCS-EF1a-Puro, and the restriction sites are EcoR1 and BamH1; the TRPV1 gene recombinant vector is obtained after restriction digestion and recombination.

[0061] Following the transfection method described in the Lipofectamine™ 3000 manual, the EDNRA gene recombinant vector and the TRPV1 gene recombinant vector were transfected into SH-SY5Y cells, respectively, to obtain SH-SY5Y-EDNRA overexpressing cell lines and SH-SY5Y-TRPV1 overexpressing cell lines.

[0062] (2) Target inhibition efficacy test of B01-B09 Sample Group 1: SH-SY5Y-EDNRA overexpressing cell lines and SH-SY5Y-TRPV1 overexpressing cell lines were seeded at a density of 35,000 cells / well in black 96-well plates and cultured for 48 hours in complete medium (DMEM / F12 (1:1) + 10% FBS + 0.1% penicillin-streptomycin + 1% NEAA + 1mM sodium pyruvate + 0.4 mM glutamine). After culture, the medium was discarded, and 200 μL of 1×HHBS was added to each well to gently wash the cells once. Then, 75 μL of 1×HHBS and 25 μL of Cal-520 fluorescent probe (Cal-520®AM working solution: 50 μL of 1 mg / ml Cal-520®AM stock solution diluted with 6 mL of 1×HHBS) were added to each well, and the plates were incubated in a CO2 incubator for 60 min. After incubation, the staining solution was removed, and 200 μL of 1×HHBS (containing 20 mM HEPES) was added to each well to gently wash the cells once. Then, 75 μL of 1×HHBS and 25 μL of different concentrations (125.86, 41.95, 13.98, 4.66, 1.55, 0.517, 0.172 mM) of the test sample B01-B09 were added to each well, and the cells were incubated in a CO2 incubator for 10 min. After incubation, 25 μL of the stimulant endothelin-1 (final concentration 50 nM) was automatically added to each well of the incubation solution in a FlexStation 3. The excitation / emission wavelengths were 485 / 525 nm, the detection time was 90 s, and the fluorescence intensity (RFU) was collected.

[0063] Model Group 1: Same as Sample Group 1, except that no test sample is added.

[0064] Sample group 2: Same as sample group 1, except that SH-SY5Y-TRPV1 overexpressing cell line was added, and 25 μL of capsaicin (final concentration 40 nM) was automatically added to each well.

[0065] Model Group 2: Same as Sample Group 2, except that no test sample is added.

[0066] The inhibition rate of each receptor in the model group (the test group with added stimulant) was set to 0%, and the inhibition rate of the test substance on the receptor was calculated using formula (1) based on the fluorescence intensity value: Inhibition rate (%) = (RFU) 模型组 -RFU 样品组 ) / RFU 模型组 ×100%(1) The results were based on the mean half-maximal inhibitory concentration (IC50) of the receptor. 50The results are presented as mean ± standard deviation (SD). One-way ANOVA was used in GraphPad Prism 8 software for significance analysis. The results showed that compounds B03, B04, B07, and B09 exhibited inhibitory effects on EDNRA and TRPV1, all stronger than asiatic acid (see Table 1). Other compounds did not show inhibitory effects on EDNRA and TRPV1. (“ / ” indicates that at the maximum cell-safe concentration, the maximum inhibitory rate of the compound on the receptor is less than 50%).

[0067] Table 1. Inhibitory effects of B01-B09 on EDNRA and TRPV1 (IC50) 50 Unit: mM)

[0068] In summary, this invention modifies the structure of asiatic acid molecules to obtain asiatic acid derivatives with higher biological activity, better lipid solubility, and higher safety, thereby expanding the comprehensive utilization value of asiatic acid and opening up new paths for its application in more fields.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A asiatic acid derivative, characterized in that, The asiatic acid derivative has the structure shown in formula (1); Equation (1) In formula (1), R is an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol, and the saturated or unsaturated monohydric alcohol includes one of ethanol, octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.

2. The asiatic acid derivative according to claim 1, characterized in that, The asiatic acid derivative has at least one of the following structures: Equation (2) Equation (3) Equation (4) Equation (5) Equation (6) Equation (7) Equation (8) Equation (9) Equation (10).

3. The method for preparing the asiatic acid derivative according to claim 1 or 2, characterized in that, include: Triacetyl asiatic acid ester, organic solvent, water, and strong base are reacted to obtain a reaction solution; The reaction solution was concentrated and extracted to obtain asiatic acid derivative; the triacetyl asiatic acid ester has the structure shown in formula (11); Equation (11) In formula (11), R is an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol, wherein the saturated or unsaturated monohydric alcohol is selected from one of ethanol, octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.

4. The preparation method according to claim 3, characterized in that, The organic solvent includes tetrahydrofuran.

5. The preparation method according to claim 3, characterized in that, The ratio of triacetyl asiatic acid ester to organic solvent is (0.2-0.5):50, with the unit being mmol / mL.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the triacetyl asiatic acid ester to the strong base is (0.2-0.5):(1-1.7).

7. The preparation method according to claim 3, characterized in that, The volume ratio of the organic solvent to water is (10-20):

1.

8. The preparation method according to claim 3, characterized in that, The concentration method includes rotary evaporation; the extractant used in the extraction includes ethyl acetate; the auxiliary extraction system used in the extraction includes saturated brine; and the strong base substance includes at least one of LiOH, NaOH, and KOH.

9. The preparation method according to claim 3, characterized in that, The reaction is carried out at a temperature of 20-25°C until triacetyl asiatic acid ester is completely consumed.

10. The use of the asiatic acid derivative according to claim 1 or 2 or the asiatic acid derivative obtained by any one of the preparation methods of claims 3-9 in at least one of the following: 1) Preparation of EDNRA inhibitors; 2) Preparation of TRPV1 inhibitors; 3) Prepare a drug for the treatment of colorectal cancer; 4) Prepare skin anti-aging agents.

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