Cycloastragenol tetrazole derivative as well as preparation method and anti-inflammatory application thereof
The design of new cyclothermal tetraazole derivatives through bioelectronic isotropic and molecular monolithography strategies has solved the toxic side effects of existing anti-inflammatory drugs and achieved high-efficiency and low-toxic anti-inflammatory effects. In particular, the cyclothermal tetraazole derivative A2 has shown significant anti-inflammatory potential.
Patent Information
- Application Number
- CN202510843264.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-08-08
AI Technical Summary
The existing small molecule anti-inflammatory drugs have toxic side effects such as gastrointestinal mucosa damage, liver and kidney toxicity and immunosuppression. Moreover, the research on the anti-inflammatory field of Astragalus alcohol derivatives has not yet been thoroughly studied, so new anti-inflammatory drugs that are efficient and low-toxic are needed.
Using bioelectronic isostat and molecular splicing strategy, with cyclostatin as the leading compound, 31 new tetrazole derivatives were designed and synthesized through two pathways, and substituted phenyltetrazole fragments were introduced and their structure was optimized to enhance anti-inflammatory activity.
The prepared cycloalis tetraazole derivatives exhibited anti-inflammatory activities better than the parent cycloalis cycloalis, especially the cycloalis tetraazole derivative A2, which significantly enhanced the anti-inflammatory potential and has value in in-depth research.
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Figure CN120441640A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicinal chemistry, and particularly relates to a cycloastragenol tetrazole derivative, a preparation method thereof, and anti-inflammatory application thereof. Background Art
[0002] Inflammation is a key host immune defense mechanism in response to pathogen invasion and tissue damage, and its overactivation is closely associated with the development and progression of various chronic diseases. However, currently used small-molecule anti-inflammatory drugs commonly have side effects such as gastrointestinal mucosal damage, hepatotoxicity, and immunosuppression. Therefore, the development of new, highly effective, and low-toxic anti-inflammatory drugs has become an urgent scientific challenge.
[0003] Cycloastragenol (CAG) is a tetracyclic triterpenoid extracted from the dried root of Astragalus mongolica. It exhibits significant anti-inflammatory activity, primarily through multiple mechanisms, including inhibition of NLRP3 inflammasome-mediated cell activation, NF-κB signaling, and the production of proinflammatory cytokines. However, research on its derivatives in the anti-inflammatory field has not been reported. COX-2 (cyclooxygenase-2) is a key enzyme in inflammatory responses, and selective inhibition of COX-2 can effectively reduce inflammation and minimize gastrointestinal side effects.
[0004] As a carboxyl bioisostere, the tetrazolium group has significant application value in the structural modification of lead compounds due to its unique chemical stability, acid-base amphoteric properties, and excellent metabolic stability. Studies have shown that compounds containing tetrazolium groups can specifically bind to the COX-2 active site, and that oxidative modification of the hydroxyl groups of triterpenoids can enhance their interactions with inflammation-related proteins.
[0005] Therefore, there is a need to develop a cycloastragenol tetrazole derivative, its preparation method and anti-inflammatory application in this field. It is of great research value to develop new anti-inflammatory drugs by structural modification using CAG as the lead compound. Summary of the Invention
[0006] The purpose of the present invention is to provide a cycloastragenol tetrazole derivative, a preparation method and anti-inflammatory application thereof. The method is based on bioisostere and molecular assembly strategies, takes cycloastragenol as the lead compound, and designs and synthesizes 31 novel tetrazole derivatives through two approaches. The prepared cycloastragenol tetrazole derivatives all show anti-inflammatory activity superior to that of the parent cycloastragenol. Among them, the cycloastragenol tetrazole derivative A2 shows significant anti-inflammatory potential, which can lay an important theoretical and structural foundation for subsequent research.
[0007] To achieve the above object, the present invention provides a cycloastragenol tetrazole derivative, the structure of which comprises:
[0008]
[0009]
[0010]
[0011] The present invention also provides a method for preparing a cycloastragenol tetrazole derivative, comprising the following steps:
[0012] Step S1, synthesizing an intermediate;
[0013] Step S11, preparing intermediates 1a-1h;
[0014] Step S12, preparing pure intermediates 2a-2h;
[0015] Step S13, preparing intermediates 3a-3h;
[0016] Step S2, synthesizing cycloastragenol tetrazole derivatives;
[0017] Step S21, synthesizing cycloastragenol tetrazole derivatives A1-A7, B1-B7 and C1-C8;
[0018] Step S22: synthesizing cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4.
[0019] Preferably, step S11 is specifically as follows:
[0020] a. Add 10 mmol of various substituted benzonitrile, 12 mmol of sodium azide, 12 mmol of zinc bromide, 80 mL of water and 20 mL of isopropanol to a 250 mL round-bottom flask and reflux for 24 h under vigorous stirring to react;
[0021] b. After the reaction of the raw materials was complete as monitored by TLC, 35 mL of 3 M HCl and 100 mL of ethyl acetate were added and the reaction was continued until the solid matter in the round-bottom flask disappeared;
[0022] c. Extract the aqueous layer with ethyl acetate, combine and concentrate the organic phase to obtain a concentrate;
[0023] The extraction was performed with ethyl acetate three times, using 120 mL of ethyl acetate each time.
[0024] d. Add 25 mL of 2.5 M NaOH solution to the concentrate, stir at room temperature for 30 min to form a suspension, and filter to obtain a filtrate and a filter cake; wash the filter cake with 12 mL of 1 M NaOH solution;
[0025] e. Add 35 mL of 3 M HCl to the filtrate, stir vigorously, and let the suspension stand to precipitate tetrazolium. Filter with suction to obtain a filter cake.
[0026] f. The filter cake was washed with 1 M HCl and dried to obtain intermediate 1a-1h;
[0027] The HCl washing was performed twice, with 20 mL of HCl used each time.
[0028] Preferably, step S12 is specifically as follows:
[0029] a. Add 5 mmol of intermediate 1a-1h, 10 mmol of K2CO3, and 7.5 mmol of ethyl bromoacetate to 15 mL of acetonitrile to form a mixture, and reflux with stirring overnight to react;
[0030] b. Monitor the reaction using TLC. After all the starting materials disappear, quench the reaction with 1 mL of cold water and filter the reaction mixture to obtain a crude product.
[0031] c. Purify the crude product by column chromatography to obtain pure intermediate 2a-2h;
[0032] The eluent in the column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1.
[0033] Preferably, step S13 is specifically as follows:
[0034] 1 mmol of pure intermediate 2a-2h was added to 2 mL of 10% aqueous sodium hydroxide solution and stirred at room temperature until clear. The reaction was monitored by TLC until the starting material disappeared. The pH was adjusted to 4-5 with 1 M HCl. The reaction solution was cooled, filtered, and dried to obtain intermediates 3a-3h.
[0035] Preferably, step S21 is specifically as follows:
[0036] a. Weigh 196 mg of cycloastragenol (0.4 mmol) into a 25 mL eggplant-shaped flask, and add 1.0 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 0.02 mmol of 4-dimethylaminopyridine (DMAP), and 0.56 mmol of intermediates 3a-3h in this order;
[0037] b. Add 10 mL of anhydrous dichloromethane and stir the reaction at room temperature for 8-24 hours;
[0038] c. Monitor the reaction using TLC until the cycloastragenol disappears completely, then add 50 mL of dichloromethane;
[0039] The wavelength of the ultraviolet lamp in the TLC monitoring reaction was 254 nm, and a 10% sulfuric acid aqueous solution was used for color development;
[0040] d. Extract the organic phase with saturated aqueous ammonium chloride solution, then wash the organic layer with 100 mL of saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product;
[0041] The saturated ammonium chloride aqueous solution was extracted three times, using 50 mL of saturated ammonium chloride aqueous solution each time;
[0042] e. The crude product was purified by normal phase silica gel column chromatography to obtain three compounds, which were then purified again by preparative thin layer chromatography to obtain cycloastragenol tetrazole derivatives A1-A7, B1-B7, and C1-C8;
[0043] The eluent in normal phase silica gel column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1; the developing solvent in preparative thin layer chromatography is a combination of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 30:1.
[0044] Preferably, step S22 is specifically as follows:
[0045] a. Weigh 50 mg of some of the cycloastragenol tetrazole derivatives A1-A7, B1-B7, and C1-C8 into a 25 mL eggplant-shaped flask, add 5 mL of dichloromethane, and add a 2-fold molar amount of Dess-Martin periodinane (DMP) in an ice bath. Stir for 4 h, then return to room temperature and continue the reaction for 2 h.
[0046] b. Monitor the reaction using TLC. After the reaction is complete, add 30 mL of water and extract with dichloromethane to obtain an organic layer.
[0047] The TLC monitoring reaction was performed with an ultraviolet lamp at a wavelength of 254 nm and a 10% aqueous sulfuric acid solution for color development. The dichloromethane extraction was performed three times, using 20 mL of dichloromethane each time.
[0048] c. The organic layer was washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product;
[0049] d. The crude product was purified by normal phase silica gel column chromatography to obtain cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4;
[0050] The eluent in the normal phase silica gel column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1.
[0051] The present invention also provides an application of cycloastragenol tetrazole derivatives in anti-inflammatory treatment.
[0052] The present invention adopts the above-mentioned cycloastragenol tetrazole derivative and its preparation method and anti-inflammatory application, and has the following beneficial effects:
[0053] (1) The present invention uses CAG as the lead compound and, through structural modification and optimization, successfully designs and synthesizes 31 novel tetrazole derivatives using two synthetic pathways: directly introducing substituted phenyltetrazole fragments on the C-3 and C-6 hydroxyl groups of cycloastragenol through condensation reaction, or further oxidizing the hydroxyl groups at positions 3, 6, or 16 after condensation of the C-3 or C-6 hydroxyl groups.
[0054] (2) This invention utilizes multiple medicinal chemistry strategies to optimize the structure of CAG, including bioisostere replacement and molecular splicing, to introduce a tetrazolium small molecule fragment with significant anti-inflammatory activity into the CAG structure. This invention improves the water solubility and physicochemical properties of CAG while enhancing its anti-inflammatory activity, thereby fully exploring its potential for application in the anti-inflammatory field.
[0055] (3) In the in vitro anti-inflammatory activity evaluation, the cycloastragenol tetrazole derivative A2 prepared by the present invention performed outstandingly and exhibited significant anti-inflammatory activity, which indicates that compound A2 has important value for in-depth study of its mechanism of action and further exploration of its anti-inflammatory activity in vivo, and is expected to provide a candidate compound for the development of new and highly effective anti-inflammatory drugs.
[0056] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Figure 1 A schematic diagram of the synthetic routes for preparing cycloastragenol tetrazole derivatives A1-A7, B1-B7, C1-C8, D1-D3, E1-E2 and F1-F4 of the cycloastragenol tetrazole derivatives and their preparation method and anti-inflammatory application examples of the present invention;
[0058] Figure 2 The present invention provides a cycloastragenol tetrazole derivative and its preparation method and anti-inflammatory application experimental example 1, in which the cycloastragenol and cycloastragenol tetrazole derivatives are evaluated for cytotoxicity against mouse RAW264.7 cells at a concentration of 5 μM;
[0059] Figure 3 This is an evaluation of the LPS-induced RAW264.7 cell viability of cycloastragenol and some cycloastragenol tetrazole derivatives at a concentration of 5 μM in Experimental Example 2 of the present invention, a cycloastragenol tetrazole derivative and its preparation method and anti-inflammatory application;
[0060] Figure 4This is an evaluation of the cytotoxicity of cycloastragenol and some cycloastragenol tetrazole derivatives on LPS-induced mouse RAW264.7 cells at 10 μM in Experimental Example 2 of a cycloastragenol tetrazole derivative and its preparation method and anti-inflammatory application. DETAILED DESCRIPTION
[0061] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.
[0062] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.
[0063] Example
[0064] like Figure 1 As shown, a method for preparing a cycloastragenol tetrazole derivative comprises the following steps:
[0065] Step S1, synthesizing an intermediate.
[0066] Step S11, preparing intermediates 1a-1h.
[0067] a. Add 10 mmol of various substituted benzonitrile, 12 mmol of sodium azide, 12 mmol of zinc bromide, 80 mL of water and 20 mL of isopropanol to a 250 mL round-bottom flask and reflux for 24 h under vigorous stirring to react.
[0068] b. After the reaction of the raw materials was complete as monitored by TLC, 35 mL of 3 M HCl and 100 mL of ethyl acetate were added and the reaction was continued until the solid matter in the round-bottom flask disappeared.
[0069] c. Extract the aqueous layer with ethyl acetate three times, using 120 mL of ethyl acetate each time. Combine the organic phases and concentrate to obtain a concentrated solution.
[0070] d. Add 25 mL of 2.5 M NaOH solution to the concentrate and stir at room temperature for 30 min to form a suspension. Filter to obtain a filtrate and a filter cake. Wash the filter cake with 12 mL of 1 M NaOH solution.
[0071] e. Add 35 mL of 3 M HCl to the filtrate and stir vigorously. After forming a suspension, let it stand to allow tetrazole to precipitate. Filter with suction to obtain a filter cake.
[0072] f. The filter cake was washed twice with 1 M HCl, using 20 mL of HCl each time, and dried to obtain intermediates 1a-1h.
[0073] Step S12: prepare pure intermediates 2a-2h.
[0074] a. Add 5 mmol of intermediate 1a-1h, 10 mmol of K2CO3, and 7.5 mmol of ethyl bromoacetate to 15 mL of acetonitrile to form a mixture, and reflux with stirring overnight to react.
[0075] b. Monitor the reaction using TLC. After all the starting materials disappear, quench the reaction with 1 mL of cold water and filter the reaction mixture to obtain a crude product.
[0076] c. Purify the crude product by column chromatography to obtain pure intermediate 2a-2h.
[0077] The eluent in the column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1.
[0078] Step S13: Prepare intermediates 3a-3h.
[0079] 1 mmol of pure intermediate 2a-2h was added to 2 mL of 10% aqueous sodium hydroxide solution and stirred at room temperature until clear. The reaction was monitored by TLC until the starting material disappeared. The pH was adjusted to 4-5 with 1 M HCl. The reaction solution was cooled, filtered, and dried to obtain intermediates 3a-3h.
[0080] Step S2, synthesizing cycloastragenol tetrazole derivatives.
[0081] Step S21, synthesizing cycloastragenol tetrazole derivatives A1-A7, B1-B7 and C1-C8.
[0082] a. Weigh 196 mg of cycloastragenol (0.4 mmol) into a 25 mL eggplant-shaped flask. Add 1.0 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 0.02 mmol of 4-dimethylaminopyridine (DMAP), and 0.56 mmol of intermediates 3a-3h in this order.
[0083] b. Add 10 mL of anhydrous dichloromethane and stir the mixture at room temperature for 8-24 hours.
[0084] c. Monitor the reaction using TLC until the raw material cycloastragenol disappears completely, then add 50 mL of dichloromethane.
[0085] The wavelength of the ultraviolet lamp in the TLC monitoring reaction is 254 nm, and a 10% sulfuric acid aqueous solution is used for color development.
[0086] d. Extract the organic phase with saturated aqueous ammonium chloride solution three times, using 50 mL of saturated aqueous ammonium chloride solution each time. Then, wash the organic layer with 100 mL of saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product.
[0087] e. The crude product was purified by normal phase silica gel column chromatography to obtain three compounds, which were then purified again by preparative thin layer chromatography to obtain cycloastragenol tetrazole derivatives A1-A7, B1-B7 and C1-C8.
[0088] The eluent in normal phase silica gel column chromatography was a combination of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:1. The developing solvent in preparative thin layer chromatography was a combination of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 30:1.
[0089] Step S22: synthesizing cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4.
[0090] a. Weigh 50 mg of some of the cycloastragenol tetrazole derivatives A1-A7, B1-B7, and C1-C8 into a 25 mL eggplant-shaped flask, add 5 mL of dichloromethane, and add a 2-fold molar amount of Dess-Martin periodinane (DMP) in an ice bath. Stir for 4 h, then return to room temperature and continue the reaction for 2 h.
[0091] b. Monitor the reaction using TLC. After the reaction is complete, add 30 mL of water and extract with dichloromethane to obtain an organic layer.
[0092] The reaction was monitored by TLC using an ultraviolet lamp at a wavelength of 254 nm and a 10% aqueous sulfuric acid solution for color development. The extraction was performed with dichloromethane three times, using 20 mL of dichloromethane each time.
[0093] c. The organic layer was washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product.
[0094] d. The crude product was purified by normal phase silica gel column chromatography to obtain cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4.
[0095] The eluent in the normal phase silica gel column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1.
[0096] The performance of the cycloastragenol tetrazole derivatives A1-A7, B1-B7, C1-C8, D1-D3, E1-E2 and F1-F4 prepared in this example was tested.
[0097] The CCK-8 (Cell Counting Kit-8) cell proliferation and toxicity assay kit used in the experimental examples was purchased from APEBIO (Shanghai, China). Its active ingredient is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate phenyl)-2H-tetrazolium monosodium salt (WST-8), which indirectly reflects cell viability by detecting mitochondrial dehydrogenase activity in living cells. RAW264.7 cells were provided by the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (IMBS, CAMS / PUMC, Beijing, China). A microplate reader was purchased from Thermo Scientific (Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). iNOS and COX-2 antibodies were purchased from CST (CST, USA). Heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin (Pen), and 100 μg / mL streptomycin (Strep) were purchased from Gibco (Grand Island, NY, USA). RIPA lysis buffer (containing PMSF and phosphatase inhibitors), Griess reagent, and BCA protein quantification kit were all purchased from Beyotime, China.
[0098] The RAW264.7 cell cryopreservation tube was quickly placed in a 37°C water bath and gently shaken until completely thawed at -80°C. After disinfection with 70% ethanol, it was transferred to a clean bench. The cell suspension was added to preheated DMEM medium containing 10% FBS and 1% Pen-Strep, added to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended with fresh medium and inoculated into culture dishes. After labeling, they were placed in a 37°C, 5% CO2 incubator for adherent culture. When the cell density reached 80-90%, the cells were passaged, the old medium was discarded, 2 mL of DMEM medium was added, the suspension was collected by pipetting, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with fresh medium and aliquoted into new culture dishes, labeled, and placed in an incubator for culture.
[0099] Experimental Example 1
[0100] The cytotoxicity of the cycloastragenol tetrazole derivatives A1-A7, B1-B7, C1-C8, D1-D3, E1-E2 and F1-F4 prepared in the examples was determined.
[0101] (1) Experimental process:
[0102] The cytotoxicity and CC50 values of the prepared tetrazole derivatives of cycloastragenol A1-A7, B1-B7, C1-C8, D1-D3, E1-E2, and F1-F4, as well as cycloastragenol (CAG), on RAW264.7 cells were determined using CCK-8 assay. The cytotoxicity of cycloastragenol and its derivatives on RAW264.7 cells at 5 μM was determined.
[0103] (2) Experimental results:
[0104] like Figure 2 As shown, the cytotoxicity of cycloastragenol and the cycloastragenol tetrazole derivatives A1-A7, B1-B7, C1-C8, D1-D3, E1-E2, and F1-F4 prepared in the examples was evaluated against mouse RAW264.7 cells at a concentration of 5 μM. Data are presented as mean ± standard deviation (n=6). Compared with the blank group, ** p<0.01, **** p<0.0001.
[0105] Among the lead compound cycloastragenol and 31 cycloastragenol tetrazole derivatives tested, cycloastragenol tetrazole derivatives B3, C4, and C6-C7 exhibited significant cytotoxicity at a concentration of 5 μM (p < 0.01 or p < 0.0001), while the remaining 27 cycloastragenol tetrazole derivatives showed no significant cytotoxic effects at 5 μM. To ensure the reliability of the experimental results, the concentration of cycloastragenol tetrazole derivatives was controlled below 5 μM in subsequent experiments to avoid potential false positive interference.
[0106] As shown in Table 1, the cytotoxicity of cycloastragenol and the tetrazole derivatives of cycloastragenol A1-A7, B1-B7, C1-C8, D1-D3, E1-E2 and F1-F4 prepared in the examples to RAW264.7 cells at a concentration of 5 μM and their CC 50 The data are expressed as mean ± standard deviation (n = 6). Compared with the blank group, ** p<0.01, **** p<0.0001.
[0107] Compared with cycloastragenol, the toxic CC50 values of cycloastragenol tetrazole derivatives decreased to varying degrees, indicating that their cytotoxicity was increased.
[0108] Table 1 Cytotoxicity of cycloastragenol and its derivatives on RAW264.7 cells at 5 μM concentration and their CC 50 value
[0109]
[0110]
[0111] (3) Experimental conclusions:
[0112] Among them, 27 tetrazole derivatives of cycloastragenol showed no obvious toxicity at a concentration of 5 μM.
[0113] Experimental Example 2
[0114] The cycloastragenol tetrazole derivatives A1-A7, B1-B7, C1-C8, D1-D3, E1-E2 and F1-F4 prepared in the example were subjected to EC inhibition of NO. 50 Value determination.
[0115] (1) Experimental process:
[0116] RAW264.7 cells were cultured at a rate of 1 × 10 4 Cells were seeded in 96-well plates at a density of 10 μM, 5 μM, 2.5 μM, and 1.25 μM of cycloastragenol tetrazolium derivatives for 1 hour, followed by LPS (1 μg / mL) stimulation for 23 hours. Cell supernatants were collected and NO production was determined using the Griess reagent method. Data were then analyzed using GraphPad 8 software, and the EC values of the compounds were calculated using a nonlinear fitting formula. 50 The cell viability of 27 tetrazole derivatives of cycloastragenol was determined at a concentration of 5 μM.
[0117] (2) Experimental results:
[0118] like Figure 3 As shown in the figure, some cycloastragenol tetrazole derivatives were selected to evaluate the viability of RAW264.7 cells induced by LPS at a concentration of 5μM. Compared with the LPS group, **** p<0.0001, *** p<0.001, ** p<0.01; compared with the blank group, #### Compared with the blank group, the NO concentration in the LPS group increased significantly (p<0.0001), indicating that the inflammatory model was successfully established. At a concentration of 5 μM, except for the cycloastragenol tetrazole derivatives A3, B2, B4, and F1-F4, the remaining cycloastragenol tetrazole derivatives showed significant anti-inflammatory activity compared with the LPS group (p<0.01, p<0.001, p<0.0001), and the inhibitory activity of most cycloastragenol tetrazole derivatives was superior to that of cycloastragenol.
[0119] like Figure 4 As shown in the figure, the cycloastragenol tetrazole derivatives with significant differences (p<0.0001) were selected for toxicity determination at a concentration of 10 μM and compared with the blank group. ** p<0.01,**** p<0.0001. The results showed that the tetrazole derivative A2 of cycloastragenol had the best activity, and its EC 50 =9.36μM, showing good research potential,
[0120] (3) Experimental conclusions:
[0121] As shown in Table 2, the EC of tetrazole derivatives of cycloastragenol with no obvious toxicity against NO was tested. 50 The values were determined and the data were expressed as mean ± standard deviation (n=6). 50 The results of the determination of the EC values show that the cycloastragenol tetrazole derivative A2 prepared in the example exhibits excellent anti-inflammatory activity. 50 =9.36μM, which is significantly better than cycloastragenol and shows good research potential.
[0122] Table 2 EC of some cycloastragenol tetrazole derivatives against NO 50 value
[0123]
[0124]
[0125] Therefore, the present invention adopts the above-mentioned cycloastragenol tetrazole derivative, its preparation method and anti-inflammatory application. The method is based on bioisostere and molecular assembly strategy, takes cycloastragenol as the lead compound, and designs and synthesizes 31 new tetrazole derivatives through two approaches. The prepared cycloastragenol tetrazole derivatives all show anti-inflammatory activity superior to that of the parent cycloastragenol. Among them, the cycloastragenol tetrazole derivative A2 shows significant anti-inflammatory potential, which can lay an important theoretical and structural foundation for subsequent research.
[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A cycloastragenol tetrazole derivative, characterized in that: The structure of the cycloastragenol tetrazole derivatives includes:
2. A method for preparing the cycloastragenol tetrazole derivatives according to claim 1, characterized in that: The following steps are involved: Step S1, synthesizing an intermediate; Step S11, preparing intermediates 1a-1h; Step S12, preparing pure intermediates 2a-2h; Step S13, preparing intermediates 3a-3h; Step S2, synthesizing cycloastragenol tetrazole derivatives; Step S21, synthesizing cycloastragenol tetrazole derivatives A1-A7, B1-B7 and C1-C8; Step S22: synthesizing cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4.
3. The method for preparing a cycloastragenol tetrazole derivative according to claim 2, wherein: Step S11 is specifically as follows: a. Add 10 mmol of various substituted benzonitrile, 12 mmol of sodium azide, 12 mmol of zinc bromide, 80 mL of water and 20 mL of isopropanol to a 250 mL round-bottom flask and reflux for 24 h under vigorous stirring to react; b. After the reaction of the raw materials was complete as monitored by TLC, 35 mL of 3 M HCl and 100 mL of ethyl acetate were added and the reaction was continued until the solid matter in the round-bottom flask disappeared; c. Extract the aqueous layer with ethyl acetate, combine and concentrate the organic phase to obtain a concentrate; The extraction was performed with ethyl acetate three times, using 120 mL of ethyl acetate each time. d. Add 25 mL of 2.5 M NaOH solution to the concentrate, stir at room temperature for 30 min to form a suspension, and filter to obtain a filtrate and a filter cake; wash the filter cake with 12 mL of 1 M NaOH solution; e. Add 35 mL of 3 M HCl to the filtrate, stir vigorously, and let the suspension stand to precipitate tetrazolium. Filter with suction to obtain a filter cake. f. The filter cake was washed with 1 M HCl and dried to obtain intermediate 1a-1h; The HCl washing was performed twice, with 20 mL of HCl used each time.
4. The method for preparing a cycloastragenol tetrazole derivative according to claim 2, wherein: Step S12 is specifically as follows: a. Add 5 mmol of intermediate 1a-1h, 10 mmol of K2CO3, and 7.5 mmol of ethyl bromoacetate to 15 mL of acetonitrile to form a mixture, and reflux with stirring overnight to react; b. Monitor the reaction using TLC. After all the starting materials disappear, quench the reaction with 1 mL of cold water and filter the reaction mixture to obtain a crude product. c. Purify the crude product by column chromatography to obtain pure intermediate 2a-2h; The eluent in the column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:
1.
5. The method for preparing a cycloastragenol tetrazole derivative according to claim 2, wherein: Step S13 is specifically as follows: 1 mmol of pure intermediate 2a-2h was added to 2 mL of 10% aqueous sodium hydroxide solution and stirred at room temperature until clear. The reaction was monitored by TLC until the starting material disappeared. The pH was adjusted to 4-5 with 1 M HCl. The reaction solution was cooled, filtered, and dried to obtain intermediates 3a-3h.
6. The method for preparing a cycloastragenol tetrazole derivative according to claim 2, wherein: Step S21 is specifically as follows: a. Weigh 196 mg of cycloastragenol (0.4 mmol) into a 25 mL eggplant-shaped flask, and add 1.0 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.02 mmol of 4-dimethylaminopyridine, and 0.56 mmol of intermediates 3a-3h in that order. b. Add 10 mL of anhydrous dichloromethane and stir the reaction at room temperature for 8-24 hours; c. Monitor the reaction using TLC until the cycloastragenol disappears completely, then add 50 mL of dichloromethane; The wavelength of the ultraviolet lamp in the TLC monitoring reaction was 254 nm, and a 10% sulfuric acid aqueous solution was used for color development; d. Extract the organic phase with saturated aqueous ammonium chloride solution, then wash the organic layer with 100 mL of saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product; The saturated ammonium chloride aqueous solution was extracted three times, using 50 mL of saturated ammonium chloride aqueous solution each time; e. The crude product was purified by normal phase silica gel column chromatography to obtain three compounds, which were then purified again by preparative thin layer chromatography to obtain cycloastragenol tetrazole derivatives A1-A7, B1-B7, and C1-C8; The eluent in normal phase silica gel column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:1; the developing solvent in preparative thin layer chromatography is a combination of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 30:
1.
7. The method for preparing a cycloastragenol tetrazole derivative according to claim 2, wherein: Step S22 is specifically as follows: a. Weigh 50 mg of some of the cycloastragenol tetrazole derivatives A1-A7, B1-B7, and C1-C8 into a 25 mL eggplant-shaped flask, add 5 mL of dichloromethane, and add a 2-fold molar amount of Dess-Martin periodinane in an ice bath. Stir for 4 h, then return to room temperature and continue the reaction for 2 h. b. Monitor the reaction using TLC. After the reaction is complete, add 30 mL of water and extract with dichloromethane to obtain an organic layer. The TLC monitoring reaction was performed with an ultraviolet lamp at a wavelength of 254 nm and a 10% aqueous sulfuric acid solution for color development. The dichloromethane extraction was performed three times, using 20 mL of dichloromethane each time. c. The organic layer was washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product; d. The crude product was purified by normal phase silica gel column chromatography to obtain cycloastragenol tetrazole derivatives D1-D3, E1-E2 and F1-F4; The eluent in the normal phase silica gel column chromatography is a combination of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 2:
1.
8. Use of the cycloastragenol tetrazole derivative according to claim 1 in anti-inflammatory treatment.