A genipin derivative, a synthetic method and use thereof

Abstract

This invention relates to the field of cellular drug screening technology, specifically to a genipin derivative, its synthesis method, and its uses. The genipin derivative is an N-substituted genipin lactam derivative or a cyclopentenepyridine derivative; the N-substituted genipin lactam derivative is a compound represented by general formula (I), and the cyclopentenepyridine derivative is a compound represented by general formula (II); wherein R is selected from any one of hydrogen, alkyl, phenyl, and aralkyl. This invention uses genipin as a lead compound, modifies the core structure, and performs cellular activity screening to identify lead compounds with good activity and low toxicity, providing a foundation for subsequent research on the anti-inflammatory effects of genipin.

Description

Technical Field

[0001] This invention relates to the field of cellular drug screening technology, specifically to a genipin derivative, its synthesis method, and its uses. Background Technology

[0002] In the clinical treatment of inflammatory diseases, anti-inflammatory drugs are mainly divided into three categories: nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (glucocorticoids), and novel targeted anti-inflammatory drugs. NSAIDs work by inhibiting cyclooxygenase (COX-1 / COX-2) activity to reduce prostaglandin synthesis, but they have significant gastrointestinal side effects, cardiovascular risks, and nephrotoxicity, limiting their effectiveness in treating chronic inflammation. Steroidal anti-inflammatory drugs have strong anti-inflammatory effects, but long-term use can lead to serious systemic side effects such as osteoporosis, hyperglycemia, and weakened immune function, limiting their clinical application. Novel targeted anti-inflammatory drugs are highly specific and have fewer side effects, but they are expensive, prone to drug resistance, and have low response rates in some patients. Furthermore, most are biological agents, requiring stringent storage and transportation conditions. In addition, some of the existing anti-inflammatory small molecule compounds obtained through cell activity screening have problems such as low activity, poor selectivity, and undesirable pharmacokinetic properties (e.g., low bioavailability and rapid metabolism), which make it difficult to meet clinical needs. Therefore, there is an urgent need in this field to develop novel small molecule lead compounds with strong anti-inflammatory activity, high selectivity, few side effects, and excellent pharmacokinetic properties.

[0003] Iridoids are natural monoterpenoids widely found in plants. Their multi-target anti-inflammatory mechanisms have made them a hot topic in natural drug development. They can inhibit the release of pro-inflammatory factors such as TNF-α and IL-6 by regulating key inflammatory pathways such as NF-κB and MAPK. Some derivatives can also form a synergistic "anti-inflammatory-antioxidant" effect, showing potential therapeutic value in various inflammatory diseases. However, they suffer from high extraction costs, insufficient activity of some natural products, rapid in vivo metabolism, and low bioavailability. Furthermore, there are few clinical translational studies, limiting their widespread application as anti-inflammatory drugs. Therefore, there is an urgent need to develop highly efficient and drug-like derivatives through structural modification.

[0004] Genipin, a representative iridoid compound derived from gardenia fruit, possesses distinct anti-inflammatory, antioxidant, and tissue-protective activities. Its anti-inflammatory mechanism is related to the regulation of NF-κB and AMPK signaling pathways, inhibiting inducible NO synthase expression and NO synthesis, reducing the release of inflammatory factors, and exhibiting protective effects on various tissues. It has attracted significant attention in the treatment of inflammation-related diseases. While some genipin derivatives have been reported in the current technology, systematic cellular activity screening and toxicity evaluation of genipin derivatives are lacking. This makes it difficult to clarify their application potential in anti-inflammation and to overcome the technical challenges of existing anti-inflammatory drugs being either "effective but toxic" or "non-toxic but ineffective." Summary of the Invention

[0005] This invention provides a genipin derivative, its synthesis method, and its uses. The derivative is based on genipin as a structural template, and the core structure of genipin is modified by chemical modification. By conducting cell activity and toxicity experiments on the derivative, lead compounds with good activity and low toxicity are screened out, which improves the theoretical basis for subsequent research on the anti-inflammatory properties of genipin.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genipin derivative, or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof, wherein the genipin derivative is an N-substituted genipin lactam derivative or a cyclopentenepyridine derivative; wherein the N-substituted genipin lactam derivative is a compound represented by general formula (I), and the cyclopentenepyridine derivative is a compound represented by general formula (II).

[0007] IⅡ R is selected from any one of hydrogen, alkyl, phenyl, and aralkyl.

[0008] Preferably, R is selected from any one of H, Me, Et, Ph, and Bn.

[0009] Preferably, the N-substituted genipin lactam derivative is any one of the following compounds: .

[0010] Preferably, the cyclopentenepyridine derivative is any one of the following compounds: .

[0011] Secondly, the present invention provides a method for synthesizing the above-mentioned genipin derivatives, wherein the method for synthesizing N-substituted genipin lactam derivatives includes the following steps: (1) Genipin is selectively silanized with a hydroxyl protecting agent under the action of a catalyst, so that a protecting group is introduced onto the hydroxyl group of the genipin hydroxymethyl group to obtain intermediate a; (2) An oxidizing agent is added to intermediate a to react and the hydroxyl group in intermediate a is oxidized to carbonyl group to obtain intermediate b; (3) Intermediate b is reacted with an organic amine to form an N-substituted lactam core, and then the protecting group is removed under the action of an organic acid to obtain an N-substituted genipin lactam derivative.

[0012] Preferably, the method for synthesizing the N-substituted genipin lactam derivative includes the following steps: (1) Dissolve the genipin catalyst in a solvent, add a hydroxyl protecting agent to react, and extract, wash, dry, concentrate under reduced pressure and separate by column chromatography to obtain intermediate a; (2) Dissolve intermediate a in a solvent, add an oxidant to react, and concentrate the product under reduced pressure and separate it by column chromatography to obtain intermediate b; (3) Dissolve intermediate b in solvent, add organic amine to react, extract the product after reaction and evaporate the solvent, then add organic acid to carry out deprotection reaction, and extract the product after reaction, collect organic phase, dry, filter, distill under reduced pressure and separate by column chromatography.

[0013] Preferably, the hydroxyl protecting agent in step (1) is dimethyl tert-butylsilicon chloride.

[0014] Preferably, the molar ratio of genipin to hydroxyl protecting agent in step (1) is 1:(1~2).

[0015] Preferably, the molar ratio of genipin and hydroxyl protecting agent in step (1) is 1:1.2.

[0016] Preferably, the catalyst in step (1) is 4-dimethylaminopyridine.

[0017] Preferably, the oxidant in step (2) is pyrrole dichromate (PDC).

[0018] Preferably, the molar ratio of intermediate a to oxidant in step (2) is 1:(1~2).

[0019] Preferably, the molar ratio of intermediate a to oxidant in step (2) is 1:1.3.

[0020] Preferably, the organic amine in step (3) is selected from any one of ammonium acetate, methylamine, aniline, and benzylamine.

[0021] Preferably, the molar ratio of intermediate b to organic amine in step (3) is 1:(1~2).

[0022] Preferably, the molar ratio of intermediate b to organic amine in step (3) is 1:1.2.

[0023] Preferably, the organic acid in step (3) is trifluoroacetic acid.

[0024] Preferably, the column chromatography in steps (1) to (3) uses a mixed solution of n-hexane and ethyl acetate.

[0025] The method for synthesizing the cyclopentenepyridine derivative includes the following steps: S1. React genipin with an oxidizing agent to oxidize the hydroxymethyl group of genipin to an aldehyde group, and obtain intermediate c; S2. Intermediate c is reacted with an amine compound to form a cyclopentenepyridine derivative.

[0026] Preferably, the method for synthesizing the cyclopentenepyridine derivative includes the following steps: S1, genipin and oxidant are dissolved in solvent and reacted under a protective atmosphere. The resulting reactants are filtered, the filtrate is extracted, the organic phases are combined, washed, dried, filtered, desolventized, and purified by column chromatography to obtain intermediate c. S2. Add intermediate c and amine compounds to a solvent, heat to react, remove the solvent after the reaction is complete, and purify by column chromatography to obtain cyclopentenepyridine derivatives.

[0027] Preferably, the oxidant of S1 is 2-iodobenzoic acid.

[0028] Preferably, the molar ratio of genipin to oxidant in S1 is 1:(1~2).

[0029] Preferably, the molar ratio of genipin to oxidant in S1 is 1:1.1.

[0030] Preferably, the column chromatography in S1 is a mixed solution of n-hexane and ethyl acetate.

[0031] Preferably, the amine compound in S2 is selected from any one of ammonia, ethylamine, aniline, and benzylamine.

[0032] Preferably, the molar ratio of intermediate c to amine compound in S2 is 1:(1~2).

[0033] Preferably, the molar ratio of intermediate c to amine compound in S2 is 1:1.07.

[0034] Preferably, the heating reaction conditions for S2 are: heating to 100~120℃ and reacting for 4~6 hours.

[0035] Preferably, the column chromatography in S2 is a mixed solution of dichloromethane and methanol.

[0036] The synthetic routes for the derivatives of genipin in this application—N-substituted genipin lactam derivatives (compound 1) and cyclopentenepyridine derivatives (compound 2)—are as follows: .

[0037] Thirdly, the present invention provides the use of the genipin derivative or the genipin derivative prepared by the synthetic method in the preparation of a drug for protecting normal human lung epithelial cells.

[0038] Preferably, the drug acts on normal human lung epithelial cells of beas-2b.

[0039] Fourthly, the present invention provides the application of the genipin derivative or the genipin derivative prepared by the synthetic method in the preparation of anti-inflammatory drugs.

[0040] Preferably, the dosage form of the drug is selected from any one of tablets, capsules, injections, suspensions, and creams.

[0041] The beneficial effects of this invention are: This application presents genipin derivatives that, through targeted structural modification of genipin, retain its inherent anti-inflammatory and antioxidant activities. Combined with excellent cellular activity and good cell compatibility, these derivatives can be widely used in the preparation of anti-inflammatory drugs and drugs related to the protection of normal human lung epithelial cells. Furthermore, the derivatives exhibit excellent cellular activity and utilize an innovative screening method. For the first time, a CCK-8 cell assay is employed to screen anti-inflammatory lead compounds using cellular activity as the core indicator, overcoming the limitations of existing screening methods. Specifically, the N-substituted genipin lactam derivative and the cyclopentenopyridine derivative demonstrated good cellular activity in BEAS-2B normal human lung epithelial cells, exhibiting low cytotoxicity and good compatibility. This lays a solid foundation for the subsequent development of low-toxicity, high-efficiency anti-inflammatory drugs and effectively addresses the technical shortcomings of existing small-molecule anti-inflammatory compounds, such as low activity and poor selectivity.

[0042] The preparation method of the derivatives in this application features mild reaction conditions and simple operation, requiring no harsh conditions such as high temperature, high pressure, or special catalysts. The reaction can be successfully completed in a mild environment, effectively reducing operational difficulty and production energy consumption, and facilitating laboratory scale-up and industrial mass production. The synthetic process route is scientifically sound, with stable and reproducible yields of intermediates and target products, consistently yielding high-purity products. Furthermore, the reagents used are readily available and low-cost, and the post-processing steps are simple and controllable, effectively reducing raw material waste and product loss. It balances practicality and economy, fully meeting the high-purity requirements of drug synthesis. Attached Figure Description

[0043] Figure 1 This is the 1H NMR spectrum of compound 1a.

[0044] Figure 2 This is the 1H NMR spectrum of compound 2a. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0046] Example 1: N-substituted genipin lactam derivatives and their synthesis Synthesis of intermediate a: Under nitrogen protection, 0.339 g (1.5 mmol) of genipin and 0.5 ml of dry DMF were added to a 10 ml reaction flask, followed by 0.366 g (3 mmol) of 4-dimethylaminopyridine (DMAP), and stirred to dissolve. 0.27 g (1.8 mmol) of dimethyl tert-butylsilicon chloride (TBSCl) was added in portions under ice bath conditions. The reaction was carried out at a constant temperature for 3 h after removing the ice bath. Thin-layer chromatography showed complete reaction. The reaction solution was quenched in ice water, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and separated by n-hexane-ethyl acetate (v / v 15:1) column chromatography to give a white solid intermediate a (0.453 g), yield 90%.

[0047] The chemical structural formula of intermediate a is as follows: .

[0048] The structural characterization data of intermediate a are as follows: 1 H NMR (600 M Hz, CDCl3) δ: 7.49 (s, 1H, =CH), 5.78 (s, 1H, =CH), 4.74(d, J = 6.6 Hz, 1H, CH), 4.31 (s, 2H, CH2), 3.68 (s, 3H, COOCH3), 3.15 (q, J= 8.1 Hz, 2H, CH2), 2.86-2.77 (m, 1H, CH), 2.42 (t, J = 7.5 Hz, 1H, OH), 2.06-1.95 (m, 1H, CH), 0.88 (s, 9H, SiC(CH3)3), 0.07 (s, 6H, Si(CH3)2); 13 C NMR(151 MHz, CDCl3) δ: 168.07, 152.80, 142.14, 129.19, 110.47, 96.40, 62.23,51.24, 48.22, 38.89, 36.73, 25.83, 25.71, -5.45, -5.48.

[0049] Synthesis of intermediate b: Under nitrogen protection, 260 mg (0.77 mmol) of intermediate a was added to a reaction flask, 3 mL of dichloromethane was added to dissolve the sample, and then an appropriate amount of 4A molecular sieve was added. 280 mg (1 mmol) of pyrrole dichromate (PDC) was added under stirring at room temperature, and the mixture was stirred at room temperature for 3 h. After the reaction was confirmed to be complete by thin-layer chromatography, the product was concentrated under reduced pressure and separated by hexane:ethyl acetate (30:1) column chromatography to obtain white compound intermediate b (0.208 g), with a yield of 80%.

[0050] The chemical structural formula of intermediate b is as follows: .

[0051] The structural characterization data of intermediate b are as follows: 1 H NMR (600 MHz, CDCl3) δ: 7.47 (s, 1H, =CH), 5.85 (s, 1H, =CH), 4.514.38 (m, 2H, CH2), 3.77 (s, 3H, COOCH3), 3.64 (d, J = 10.2 Hz, 1H, CH2a),3.56-3.43 (m, 1H, CH2b), 2.94-2.86 (m, 1H, CH), 2.26-2.14 (m, 1H, CH), 0.89(s, 9H, SiC(CH3)3), 0.07 (s, 6H, Si(CH3)2); 13 C NMR (151 MHz, CDCl3) δ: 166.48,166.09, 148.42, 140.91, 127.55, 113.27, 61.44, 51.90, 45.86, 39.22, 36.14,25.89, 25.63, -5.37, -5.42.

[0052] Synthesis of compounds 1a-1d: 50 mg (0.15 mmol) of intermediate b was added to a pressure-resistant tube, followed by 2 ml of dry pyridine to dissolve the sample. Then, 0.18 mmol of organic amines were added (11 mg of ammonium acetate was used to prepare 1a; 5.5 mg of methylamine was used to prepare 1b; 16.5 mg of aniline was used to prepare 1c; and 19 mg of aniline was used to prepare 1d). The temperature was raised to 110 °C, and the reaction was stirred for 2 h. After the reaction was complete, dilute hydrochloric acid was added, and the mixture was extracted with dichloromethane. The solvent was then evaporated. 2 ml of tetrahydrofuran solution was then added dropwise to the pressure-resistant tube, followed by 0.2 ml of trifluoroacetic acid. The temperature was raised to 60 °C, and the reaction was stirred for 3 h. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction mixture to remove trifluoroacetic acid, and the mixture was extracted three times with dichloromethane (3 × 50 ml). The organic phases were combined, dried over anhydrous magnesium sulfate to remove water, filtered, and distilled under reduced pressure. Finally, the mixture was purified by column chromatography using a mixture of n-hexane and ethyl acetate to obtain compounds 1a-1b.

[0053] The chemical structural formula of compound 1a is as follows: .

[0054] The structural characterization data of compound 1a are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.36 (s, 1H), 7.18 (d, J = 5.1 Hz, 1H), 5.83(s, 1H), 4.35 (d, J = 13.2 Hz, 1H), 4.29 (d, J = 13.1 Hz, 1H), 3.74 (s, 3H), 3.64 (d, J = 10.2 Hz, 1H), 3.54 (q, J = 9.7 Hz, 1H), 2.90 (dd, J = 16.8, 8.7 Hz,1H), 2.25 – 2.20 (m, 1H). 13 C NMR (151 MHz, CDCl3) δ 172.57, 167.17, 140.76, 133.17, 129.37, 110.91, 60.88, 51.74, 49.29, 40.14, 37.23.

[0055] The 1H NMR spectrum of compound 1a is shown below. Figure 1 .

[0056] The chemical structural formula of compound 1b is as follows: .

[0057] The structural characterization data of compound 1b are as follows: 1 H NMR (600 MHz, CDCl3) δ 7.13 (s, 1H), 5.75 (s, 1H), 4.28 (d, J = 13.0Hz, 1H), 4.23 (d, J = 12.5 Hz, 1H), 3.69 (s, 3H), 3.60 (d, J = 11.1 Hz, 1H), 3.47 (dt, J = 11.0, 8.7 Hz, 1H), 3.11 (s, 3H), 2.82 (dddt, J = 16.6, 8.7, 3.0,1.4 Hz, 1H), 2.16 (ddq, J = 16.6, 8.8, 2.0 Hz, 1H). 13 C NMR (151 MHz, CDCl3) δ170.86, 166.87, 141.35, 138.42, 129.08, 111.11, 61.04, 51.74, 50.12, 40.25,37.51, 35.62.

[0058] The chemical structural formula of compound 1c is as follows: .

[0059] The structural characterization data of compound 1c are as follows: 1 H NMR (600 MHz, CDCl3) δ 7.44 (t, J = 7.7 Hz, 2H), 7.38 (s, 1H), 7.38– 7.35 (m, 1H), 7.25 – 7.23 (m, 2H), 5.87 (s, 1H), 4.38 (d, J = 14.4 Hz, 1H), 4.32 (d, J = 13.4 Hz, 1H), 3.89 (dt, J = 11.0, 2.0 Hz, 1H), 3.75 (s, 3H), 3.65(dt, J = 11.0, 8.6 Hz, 1H), 2.98 (dd, J= 16.7, 8.6 Hz, 1H), 2.37 (ddq, J =16.7, 8.4, 2.0 Hz, 1H). 13 C NMR (151 MHz, CDCl3) δ 170.50, 166.88, 141.38,139.98, 138.09, 129.58, 129.40, 128.45, 126.55, 111.58, 61.09, 51.82, 50.70, 40.32, 37.54.

[0060] The chemical structural formula of compound 1d is as follows: .

[0061] The structural characterization data of compound 1d are as follows: 1 H NMR (600 MHz, CDCl3) δ 7.32 (t, J = 7.3 Hz, 2H), 7.27 (d, J = 14.6Hz, 1H), 7.21 (s, 2H), 7.19 (s, 1H), 5.82 (s, 1H), 5.27 (s, 1H), 4.78 (d, J =15.0 Hz, 1H), 4.70 (d, J = 15.0 Hz, 1H), 4.32 (q, J = 14.0 Hz, 2H), 3.70 (s,3H), 3.53 (dt, J = 11.0, 8.7 Hz, 1H), 2.90 – 2.85 (m, 1H), 2.25 – 2.20 (m,1H), 1.97 (s, 1H). 13 C NMR (151 MHz, CDCl3) δ 170.66, 166.77, 141.36, 137.12,136.07, 129.35, 129.04, 128.14, 127.66, 111.80, 61.11, 53.58, 51.78, 50.73,50.34, 40.14, 37.5.

[0062] Example 2 Cyclopentenepyridine Derivatives and Their Synthesis Synthesis of intermediate C: 500 mg (2.2 mmol) of genipin, 680 mg (2.4 mmol) of 2-iodobenzoic acid (IBX), and 2 ml of dimethyl sulfoxide were added to a round-bottom flask. Under argon protection, the mixture was stirred at room temperature for 5 hours, and then 100 ml of water was added. The mixture was filtered, and the filtrate was extracted three times with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous magnesium sulfate, filtered, and dissolved. The solution was then removed by column chromatography with a hexane to ethyl acetate ratio of 8:1 to obtain intermediate C.

[0063] Chemical structural formula of intermediate C: .

[0064] The structural characterization data of intermediate c are as follows: 1 H NMR (600 MHz, CDCl3) δ 9.65 (dq, J = 2.1, 1.0 Hz, 1H), 7.48 – 7.44(m, 1H), 6.20 (tt, J = 5.5, 1.6 Hz, 1H), 5.41 (dddd, J = 5.9, 5.2, 3.9, 1.8 Hz,1H), 5.07 (d, J = 4.9 Hz, 1H), 3.72 (s, 2H), 3.38 – 3.29 (m, 1H), 3.12 – 3.04(m, 1H), 2.68 – 2.59 (m, 1H), 2.48 (dtt, J = 14.3, 5.7, 1.1 Hz, 1H). 13 C NMR (151 MHz, CDCl3) δ 192.20, 167.39, 157.57, 152.76, 144.00, 109.32, 96.66, 51.67, 49.69, 41.17, 35.06.

[0065] Synthesis of compounds 2a-2d: 100 mg (0.44 mmol) of intermediate c and 0.47 mmol of amine compounds (2a was prepared from 9 μL of ammonia, 2b from 21 mg of ethylamine, 2c from 44 mg of aniline, and 2d from 50 mg of benzoylamine) were added to a pressure-resistant tube. Then 3 mL of n-butanol was added, the temperature was raised to 110 °C, and the reaction was carried out for 5 h. The n-butanol was dried under vacuum, and the compounds 2a-2d were purified by column chromatography using a mixture of dichloromethane and methanol.

[0066] Chemical structural formula of compound 2a: .

[0067] The structural characterization data of compound 2a are as follows: 1 H NMR (600 MHz, DMSO- D 6) δ 13.35 (s, 1H), 9.77 (s, 1H), 9.04 (s, 1H), 8.35 (s, 1H), 7.83 (dd, J = 3.7, 1.5 Hz, 1H), 6.95 (d, J = 3.7 Hz, 1H), 3.90 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 189.72, 165.95, 136.52, 134.98, 132.21, 131.70, 131.37, 128.09, 125.10, 109.44, 52.04.

[0068] The 1H NMR spectrum of compound 2a is shown below. Figure 2 .

[0069] Chemical structural formula of compound 2b: .

[0070] The structural characterization data of compound 2b are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.17 (q, J = 1.1 Hz, 1H), 8.10 (q, J = 1.1 Hz, 1H), 7.83 (dd, J = 6.7, 1.2 Hz, 1H), 7.78 (d, J = 6.9 Hz, 1H), 4.07 (qt, J =8.2, 0.9 Hz, 2H), 3.76 (s, 3H), 1.24 (t, J = 8.3 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ 188.62, 167.00, 136.87, 135.10, 133.83, 131.80, 129.52, 128.57, 124.29, 110.91, 52.10, 50.96, 14.58.

[0071] Chemical structural formula of compound 2c: .

[0072] The structural characterization data of compound 2c are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.70 – 8.67 (m, 2H), 7.85 (dd, J = 6.6, 1.3Hz, 1H), 7.69 (d, J = 6.6 Hz, 1H), 7.32 – 7.23 (m, 3H), 7.20 – 7.14 (m, 1H), 3.76 (s, 2H). 13 C NMR (151 MHz, CDCl3) δ 188.91, 166.81, 139.33, 136.77,134.14, 130.98, 130.39, 129.58, 128.70, 127.33, 123.79, 123.31, 121.27,113.28, 52.11.

[0073] Chemical structural formula of compound 2d: .

[0074] The structural characterization data of compound 2d are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.24 (q, J = 1.0 Hz, 1H), 8.05 (q, J = 1.1 Hz, 1H), 7.83 (dd, J = 6.7, 1.2 Hz, 1H), 7.78 (d, J = 6.9 Hz, 1H), 7.31 (s, 1H),7.33 – 7.24 (m, 2H), 5.17 (t, J = 0.9 Hz, 2H), 3.76 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 188.62, 167.05, 136.20, 135.14, 134.76, 133.90, 133.75, 129.77,128.70, 128.31, 127.44, 127.31, 124.27, 110.54, 59.42, 52.10.

[0075] The synthesis yields of the N-substituted genipin lactam derivative and cyclopentenepyridine derivative in this embodiment are shown in Table 1.

[0076] Table 1

[0077] Effect of Compound 1 on the Activity of BEAS-2B Normal Human Lung Epithelial Cells Lipopolysaccharide (LPS) can activate the NF-κB and MAPK pathways through TLR4 / MD2 / CD14 receptors, inducing a large release of pro-inflammatory factors and mimicking an in vitro inflammation model. This study used epithelial BEAS-2B cells and conducted in vitro inflammatory activity experiments using the CCK-8 assay. By measuring the anti-inflammatory activity and toxicity of lead compounds, lead compounds with good activity and low toxicity were screened, and the activity of modified lead compound derivatives was further tested using the CCK-8 assay.

[0078] 1. Cell viability assay methods (1) Cell culture Cells were cultured in DEME medium containing 10% serum and 1% penicillin / streptomycin. Cells were grown in an incubator at 37°C and 5% CO2.

[0079] (2) Concentration preparation of the test drug Prepare a 50 mM stock solution of the test compound using DMSO, store at -80°C, and dilute to the required concentration with complete culture medium before use. Set up molar concentration groups of the test compound at 10 μM, 1 μM, and 0.1 μM.

[0080] (3) CCK-8 method Take logarithmically grown BEAS-2B cells and use 5 × 10⁻⁶ cells. 4 Cells were seeded in 96-well plates and cultured at 37°C for 24 hours. Experimental groups were established: control group (cells + complete culture medium), model group (cells + complete culture medium + LPS), and drug-treated group (cells + complete culture medium + LPS + test compound). After culturing at 37°C for 24 hours, 10 μL of CCK-8 reagent was added to each group, and the cells were incubated at 37°C in the dark for 1.5 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated based on the OD value.

[0081] With the blank group as 100% survival rate, calculate the cell survival rate of each group: Cell viability = average OD value of experimental group / average OD value of blank group.

[0082] (4) Experimental results The OD values ​​of each experimental group are shown in Table 2, and the calculated cell viability is shown in Table 3.

[0083] Table 2. OD values ​​of each experimental group

[0084] Table 3. Cell viability of each experimental group

[0085] As shown in Tables 2 and 3, compared with the blank group, the LPS-induced BEAS-2B cell survival rate in the model group was significantly reduced, indicating that the inflammation model was successfully constructed. After intervention with the compounds described in this invention for 1a-1d and 2a-2d, the cell survival rate in each treatment group was significantly higher than that in the model group. Among them, the cell survival rate of most compounds was most significantly improved at a concentration of 10 μM, and the cell survival rate showed a certain dose-response relationship with the compound concentration.

[0086] Meanwhile, the cell survival rates of each compound administration group were similar to those of the blank control group, indicating that within the experimental concentration range, the N-substituted genipin lactam derivative and cyclopentenidine derivative of the present invention have no significant toxicity to normal human lung epithelial cells of BEAS-2B, good cell compatibility, and can effectively improve LPS-induced cell damage and enhance cell viability under inflammatory conditions, thus possessing the potential to serve as lead compounds for lung epithelial cell protection and anti-inflammatory effects.

[0087] 2. Cytotoxicity assay methods (1) Cell culture Cells were cultured in DEME medium containing 10% serum and 1% penicillin / streptomycin. Cells were grown in an incubator at 37°C and 5% CO2.

[0088] (2) Concentration preparation of the test drug Prepare a 50 mM stock solution of the test compound using DMSO, store at -80°C, and dilute to the required concentration with complete culture medium before use. Set up molar concentration groups of the test compound at 100 μM, 10 μM, and 1 μM.

[0089] (3) CCK-8 method Take logarithmically grown BEAS-2B cells and use 5 × 10⁻⁶ cells. 4 Cells were seeded in 96-well plates and cultured at 37°C for 24 hours. Experimental groups were established: a control group (cells + complete culture medium) and a drug-treated group (cells + complete culture medium + test compound). After culturing at 37°C for 24 hours, 10 μL of CCK-8 reagent was added to each group, and the cells were incubated at 37°C in the dark for 1.5 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated based on the OD value.

[0090] (4) Experimental results: The cytotoxicity test results of the blank group and the drug-treated group are shown in Table 4.

[0091] Table 4. Cell viability data in the control group and the drug-treated group

[0092] As shown in Table 4, with the normal cell group having a cell survival rate of 100%, the cell survival rate of BEAS-2B cells remained at a high level after treatment with the compound of the present invention for 1a-1d and 2a-2d within the concentration range of 1-100 μM. This indicates that the derivative of the present invention has low toxicity to normal human lung epithelial cells of BEAS-2B, high safety, and good cell compatibility within the effective concentration range.

[0093] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A genipin derivative, or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof, characterized in that, The genipin derivative is an N-substituted genipin lactam derivative or a cyclopentene pyridine derivative; the N-substituted genipin lactam derivative is a compound represented by general formula (I), and the cyclopentene pyridine derivative is a compound represented by general formula (II); IⅡ R is selected from any one of hydrogen, alkyl, phenyl, and aralkyl.

2. The genipin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, or prodrug, characterized in that, The N-substituted genipin lactam derivative is any one of the following compounds: 。 3. The genipin derivative according to claim 1, or its pharmaceutically acceptable salt, stereoisomer, or prodrug, characterized in that, The cyclopentenepyridine derivative is any one of the following compounds: 。 4. A method for synthesizing the genipin derivative according to claim 1, characterized in that, The method for synthesizing the N-substituted genipin lactam derivative includes the following steps: (1) Genipin is selectively silanized with a hydroxyl protecting agent under the action of a catalyst, so that a protecting group is introduced onto the hydroxyl group of the genipin hydroxymethyl group to obtain intermediate a; (2) An oxidizing agent is added to intermediate a to react and the hydroxyl group in intermediate a is oxidized to carbonyl group to obtain intermediate b; (3) Intermediate b is reacted with an organic amine to form an N-substituted lactam core, and then the protecting group is removed under the action of an organic acid to obtain an N-substituted genipin lactam derivative.

5. The synthesis method according to claim 4, characterized in that, The organic amine in step (3) is selected from any one of ammonium acetate, methylamine, aniline, and benzylamine.

6. A method for synthesizing the genipin derivative according to claim 1, characterized in that, The method for synthesizing the cyclopentenepyridine derivative includes the following steps: S1. React genipin with an oxidizing agent to oxidize the hydroxymethyl group of genipin to an aldehyde group, and obtain intermediate c; S2. Intermediate c is reacted with an amine compound to form a cyclopentenepyridine derivative.

7. The synthesis method according to claim 6, characterized in that, The oxidizing agent of S1 is 2-iodobenzoic acid.

8. The synthesis method according to claim 6, characterized in that, The amine compound in S2 is selected from any one of ammonia, ethylamine, aniline, and benzylamine.

9. The use of a genipin derivative according to any one of claims 1 to 3 or a genipin derivative prepared by the synthetic method according to any one of claims 4 to 8 in the preparation of a drug for protecting normal human lung epithelial cells.

10. The use of a genipin derivative according to any one of claims 1 to 3 or a genipin derivative prepared by the synthetic method according to any one of claims 4 to 8 in the preparation of an anti-inflammatory drug.

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