A centella asiatica acid derivative and a preparation method thereof
By modifying the structure of asiatic acid molecules, asiatic acid derivatives with better water and oil solubility were prepared, which solved the problem of poor inhibitory effects of asiatic acid on EDNRA and TRPV1, and achieved higher anti-tumor activity and anti-aging effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI JAKA BIOTECH CO LTD
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-29
AI Technical Summary
Asiatic acid has poor inhibitory effects on EDNRA and TRPV1, which limits its widespread application in the pharmaceutical field.
By modifying the structure of asiatic acid molecules, asiatic acid derivatives with specific structures were prepared, improving their water solubility and oil solubility, and enhancing their bioavailability.
The asiatic acid derivatives significantly enhanced the inhibitory effects on EDNRA and TRPV1, improved antitumor activity, and expanded their application value in the fields of anticancer and anti-aging.
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Figure CN122103235A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of asiatic acid technology, specifically to an asiatic acid derivative and its preparation method. Background Technology
[0002] Colorectal cancer (CRC) is a malignant tumor that occurs in the colon or rectum, and is related to factors such as age, genetics, diet, and lifestyle. Early stages may not present with obvious symptoms, but rectal bleeding, changes in bowel habits, and abdominal pain are common warning signs.
[0003] 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, 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 can lead to a high recurrence rate.
[0004] 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). EDNRA is significantly highly expressed in colorectal cancer (CRC) tissues, and its expression level is positively correlated with tumor stage and negatively correlated with overall survival, making it a potential biomarker for poor prognosis in CRC. Therefore, therapeutic drugs for CRC can be developed targeting EDNRA.
[0005] TRPV1, short for Transient Receptor Potential Vanilloid 1, is a widely expressed, non-selective cation channel protein belonging to the vanillic acid receptor subfamily of the TRP superfamily. Previous studies have shown that capsaicin (CAP) inhibits CRC by regulating circadian rhythm genes (such as Per1 / 2 / 3, Bmal1, Cry1 / 2, etc.) and the TRPV1 pathway. Currently, there is an urgent need to find a natural anticancer compound that targets EDNRA and TRPV1.
[0006] Asiatic acid, also known as asiatic acid, is a pentacyclic triterpenoid compound with an ursane-type skeleton extracted from Centella asiatica. It possesses anti-aging, anti-wrinkle, and anti-tumor properties. However, despite its diverse biological activities, its weak pharmacological effects, extremely low water solubility, and moderate lipid solubility, coupled with poor inhibitory effects on EDNRA and TRPV1, greatly limit its widespread application in the pharmaceutical field. Summary of the Invention
[0007] Therefore, the technical problem to be solved by the present invention is to overcome the defect that asiatic acid has poor inhibitory effect on EDNRA and TRPV1 in the prior art, and thus provide an asiatic acid derivative and its preparation method to improve the inhibitory effect of asiatic acid derivative on EDNRA and TRPV1, thereby improving its anti-tumor effect.
[0008] To address the aforementioned technical problems, the following technical solutions are proposed:
[0009] The present invention provides a asiatic acid derivative having a structure as shown in formula (1);
[0010] Equation (1) In formula (1), Ac- is acetyl CH3CO-; R is a straight-chain or branched chain with 1 to 24 carbon atoms, or an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol.
[0011] Preferably, the saturated or unsaturated monohydric alcohol includes one of octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.
[0012] Preferably, the asiatic acid derivative has at least one of the following structures;
[0013] Equation (2)
[0014] Equation (3)
[0015] Equation (4)
[0016] Equation (5)
[0017] Equation (6)
[0018] Equation (7)
[0019] Equation (8)
[0020] Equation (9).
[0021] The present invention modifies the molecular structure of asiatic acid, and the molecular structure of asiatic acid is shown in the following formula (10). The water solubility and oil solubility of the prepared asiatic acid derivative are significantly enhanced, especially the oil solubility, resulting in good application effect, high bioavailability, and high safety.
[0022]
[0023] Equation (10) This invention provides a method for preparing the asiatic acid derivative described in the above technical solution, comprising: S1, react asiatic acid, organic solvent, acetic anhydride and catalyst to obtain a reaction solution; extract, dry and purify the reaction solution to obtain triacetylasiatic acid; S2, the triacetyl asiatic acid, organic solvent and N,N-dimethylformamide (DMF) are mixed to obtain a reaction solution; S3, oxalyl chloride solution is added dropwise to the reaction solution to carry out the reaction, and a second reaction solution is obtained; after evaporating the second reaction solution to dryness, an organic solvent and a monohydric alcohol are added to carry out the reaction, and asiatic acid derivative is obtained.
[0024] Preferably, in step S3, the method for preparing the oxalyl chloride solution includes: mixing oxalyl chloride with an organic solvent to obtain an oxalyl chloride solution; the organic solvent includes dichloromethane; and the mass-volume concentration of oxalyl chloride in the oxalyl chloride solution is 5-15 mg / mL, more preferably 14 mg / mL.
[0025] Preferably, in S1, the ratio of asiatic acid to organic solvent is (5-10):1, where the ratio is expressed in mg / mL. The ratio of acetic anhydride to organic solvent is (5-10):1, with the unit being mg / mL; The catalyst comprises 4-dimethylaminopyridine (DMAP); the ratio of the catalyst to the organic solvent is (0.1-0.5):1, with the unit being mg / mL.
[0026] Preferably, in S2, the ratio of triacetyl asiatic acid to organic solvent is (10-15):1, with the unit of ratio being mg / mL; the volume ratio of organic solvent to N,N-dimethylformamide is 50:(0.1-0.2), more preferably 50:0.1.
[0027] Preferably, in S3, the molar ratio of asiatic acid to monohydric alcohol is 1:(1-1.2), more preferably 1:1; the addition ratio of monohydric alcohol to organic solvent is (15-25):1, more preferably 24:1, with the unit of ratio being mmol / L.
[0028] Preferably, in step S3, after reacting with the organic solvent and monohydric alcohol, extraction and drying are further performed; the extraction includes extraction with saturated brine; the number of extractions is 2-3 times, more preferably 2 times; the volume ratio of the extract to the extracted substance solution during extraction is 1:(1-1.2), more preferably 1:1; the drying agent used includes anhydrous sodium sulfate; the purification method includes adsorption chromatography purification.
[0029] Preferably, the adsorption chromatography purification includes silica gel column separation and purification.
[0030] 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) To prepare drugs for the treatment of colorectal cancer; 4) Prepare skin anti-aging agents.
[0031] The technical solution of this invention has the following advantages: 1. Compared with asiatic acid, the asiatic acid derivatives of the present invention showed significant inhibitory effects on EDNRA and TRPV1. In colorectal cancer, the EDN1 / EDNRA / β-arrestin axis promotes tumor cell proliferation and invasion by activating STAT3 phosphorylation. The asiatic acid derivatives A03, A04, A06, and A09 showed good inhibitory effects on EDNRA and TRPV1, thus concluding that the asiatic acid derivatives A03, A04, A06, and A09 have an inhibitory effect on colorectal cancer.
[0032] 2. Compared with low molecular weight alkyl groups, the solubility of the asiatic acid derivatives of the present invention is significantly improved, with good water and lipid solubility, high bioavailability, and no safety risks.
[0033] 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
[0034] 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.
[0035] Figure 1 This is a process flow diagram of a specific example of a asiatic acid derivative in Embodiment 1 of the present invention; Figure 2 This is a process flow diagram of a specific example of a asiatic acid derivative in Embodiment 1 of the present invention. Detailed Implementation
[0036] 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.
[0037] 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.
[0038] All raw materials and chemical reagents involved in this invention were purchased from Sigma-Aldrich.
[0039] The process for preparing triacetylasic acid is as follows: Figure 1 The process for preparing asiatic acid derivatives from triacetylasic acid is described in [link to procedure]. Figure 2 .
[0040] 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. Add 50 mL of dichloromethane solvent (DCM) 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 and extract twice with 50 mL of saturated saline solution. Dry the organic phase with anhydrous sodium sulfate. Purify the final product, triacetylasiatic acid, using silica gel column chromatography.
[0041] 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 (0.1 mL) as a catalyst to obtain the raw material 2 solution. Separately, weigh oxalyl chloride (140 mg, 1.2 mmol, 1.2 equiv) into a 25 mL flask, dissolve it in 10 mL of dichloromethane to obtain an oxalyl chloride solution. Slowly add the oxalyl chloride solution dropwise to the aforementioned raw material 2 solution. After the addition is complete, allow the mixture to rise naturally to room temperature and stir for 3 hours. The reaction mixture was then evaporated to dryness using a rotary evaporator with dichloromethane solvent. 30 mL of dichloromethane was added again to dissolve the residue, and the solvent was evaporated to dryness again. Another 50 mL of dichloromethane solvent was added, followed by ethanol (1.2 mmol, 1.2 equiv). The reaction was carried out at room temperature for 12 h, and TLC analysis showed complete consumption of triacetyl asiaticoyl chloride. The mixture was then transferred to a separatory funnel and extracted twice with 50 mL of saturated saline solution. The organic phase was dried over anhydrous sodium sulfate. The final product, A01, was obtained by silica gel column chromatography.
[0042]
[0043] A01: R group represents ethanol. 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 AO2, and the chemical structure of AO2 is shown below:
[0044] A02: R group is octanol 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 A03, and the chemical structure of A03 is shown below:
[0045] A03: R group is hexadecyl alcohol 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 A04, and the chemical structure of A04 is shown below:
[0046] A04: R group is stearyl alcohol 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 A05, and the chemical structure of A05 is shown below:
[0047] A05: R group is 2-hexyl-1-decyl alcohol. 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 decyltetradecyl alcohol. The final product is A06, and the chemical structure of A06 is shown below:
[0048] A06: R group is decyltetradecyl alcohol. A06 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), 5.11 (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 A07, and the chemical structural formula of A07 is shown below:
[0049] A07: R group represents oleyl alcohol. 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 A08, and the chemical structure of A08 is shown below:
[0050] A08: R group is cis-, cis-9,12-octadecadienol A08 1 H NMR and13 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 A09, and the chemical structure of A09 is shown below:
[0051] A09: R group represents bisabolol. A09 1 H NMR and 13 The C NMR values are as follows: 1 H 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 = 11.0Hz, 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); 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. Application examples 1. Efficacy Test (1) Construct SH-SY5Y cells overexpressing EDNRA and TRPV1 SH-SY5Y cells were purchased from BeiNa Biotechnology.
[0052] 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.
[0053] 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.
[0054] According to the transfection method shown in the Lipofectamine™ 3000 instruction manual, the recombinant vectors containing the above-mentioned EDNRA gene and TRPV1 gene were transfected into SH-SY5Y cells to obtain SH-SY5Y-EDNRA overexpressing cell lines and SH-SY5Y-TRPV1 overexpressing cell lines, respectively.
[0055] (2) Target inhibition efficacy test of A01-A09 Sample Group 1: SH-SY5Y-EDNRA 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 + 0.4mM L-glutamine, purchased from Sigma). Cells were gently washed once with 200 μL of 1×HHBS in each well, followed by the addition of 75 μL of 1×HHBS and 25 μL of Cal-520 fluorescent probe (Cal-520® AM working solution prepared by diluting 50 μL of 1 mg / mL Cal-520® AM stock solution with 6 mL of 1×HHBS). The plates were incubated in a CO2 incubator for 60 min. After removing the staining solution, each well was washed once again with 200 μL of 1×HHBS (containing 20 mM HEPES). Then, each well was filled with 75 μL of 1×HHBS and 25 μL of different concentrations (125.86 mM, 41.95 mM, 13.98 mM, 4.66 mM, 1.55 mM, 0.517 mM, 0.172 mM) of the test sample A01-A09. The wells were incubated in a CO2 incubator for 10 min. Using a FlexStation 3 multi-mode microplate reader, 25 μL of the stimulant endothelin-1 (final concentration 50 nM) was automatically added to each well. The excitation / emission wavelengths were 485 / 525 nm, the detection time was 90 s, and the fluorescence intensity (RFU) was collected.
[0056] Model Group 1: Same as Sample Group 1, except that no test sample is added.
[0057] 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.
[0058] Model Group 2: Same as Sample Group 2, except that no test sample is added.
[0059] The inhibition rate of each receptor in the model group (test group with added stimuli) was set to 0%, and the inhibition rate of test substances A01-A09 on the membrane potential induced by receptor TRPV1 or EDNRA activation was calculated using formula (1): 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 A03, A04, A06, and A09 exhibited inhibitory effects on EDNRA and TRPV1, all stronger than asiatic acid (see Table 1). For example, A03 at a concentration of 0.44 mM could inhibit endothelin-1-induced changes in cell membrane potential by 50%. 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%).
[0060] Table 1. Inhibitory effects of A01-A09 on EDNRA and TRPV1 (IC50) 50 (Unit: mM)
[0061] 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.
[0062] 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), Ac- is acetyl CH3CO-; R is a straight-chain or branched chain with 1 to 24 carbon atoms, or an alkyl group formed by removing the hydroxyl group from a saturated or unsaturated monohydric alcohol.
2. The asiatic acid derivative according to claim 1, characterized in that, The saturated or unsaturated monohydric alcohols include one of octanol, hexadecyl alcohol, stearyl alcohol, 2-hexyl-1-decyl alcohol, decyltetradecyl alcohol, oleyl alcohol, cis-, cis-9,12-octadecadienol and bisabolol.
3. 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).
4. The method for preparing the asiatic acid derivative according to any one of claims 1-3, characterized in that, include: S1, react asiatic acid, organic solvent, acetic anhydride and catalyst to obtain a reaction solution; extract, dry and purify the reaction solution to obtain triacetylasiatic acid; S2, the triacetyl asiatic acid, organic solvent and N,N-dimethylformamide are mixed to obtain a reaction solution; S3, oxalyl chloride solution is added dropwise to the reaction solution to carry out the reaction, and a second reaction solution is obtained; after evaporating the second reaction solution to dryness, an organic solvent and a monohydric alcohol are added to carry out the reaction, and asiatic acid derivative is obtained.
5. The preparation method according to claim 4, characterized in that, In S3, the method for preparing the oxalyl chloride solution includes: mixing oxalyl chloride with an organic solvent to obtain an oxalyl chloride solution; the organic solvent includes dichloromethane; and the mass-volume concentration of oxalyl chloride in the oxalyl chloride solution is 5-15 mg / mL.
6. The preparation method according to claim 4, characterized in that, In S1, the ratio of asiatic acid to organic solvent is (5-10):1, and the unit of ratio is mg / mL; The ratio of acetic anhydride to organic solvent is (5-10):1, with the unit being mg / mL; The catalyst comprises 4-dimethylaminopyridine; the ratio of the catalyst to the organic solvent is (0.1-0.5):1, with the unit being mg / mL.
7. The preparation method according to claim 4, characterized in that, In S2, the ratio of triacetyl asiatic acid to organic solvent is (10-15):1, with the unit of ratio being mg / mL; the volume ratio of organic solvent to N,N-dimethylformamide is 50:(0.1-0.2).
8. The preparation method according to claim 4, characterized in that, In S3, the molar ratio of asiatic acid to monohydric alcohol is 1:(1-1.2); the addition ratio of monohydric alcohol to organic solvent is (15-25):1, with the unit of ratio being mmol / L.
9. The preparation method according to claim 4, characterized in that, In step S3, after reacting with an organic solvent and a monohydric alcohol, extraction and drying are also performed; the extraction includes extraction with saturated brine; the extraction is performed 2-3 times, and the volume ratio of the extract to the extracted substance solution is 1:(1-1.2). The drying agent used in the drying process includes anhydrous sodium sulfate; the purification method includes adsorption chromatography purification.
10. The use of the asiatic acid derivative according to any one of claims 1-3 or the asiatic acid derivative obtained by any one of claims 4-9 in at least one of the following: 1) Preparation of EDNRA inhibitors; 2) Preparation of TRPV1 inhibitors; 3) To prepare drugs for the treatment of colorectal cancer; 4) Prepare skin anti-aging agents.