Genipin derivatives and their preparation methods and applications

Abstract

The present invention belongs to the field of organic synthesis technology and specifically relates to genipin derivatives, preparation methods, and applications thereof. The genipin derivatives provided by the present invention are structurally modified using genipin as a lead compound, attaching an unsaturated enol or alkynol group, and replacing the H atom on the C-1 hydroxyl group of genipin with 3-butene-1-ol, 4-pentene-1-ol, 5-hexene-1-ol, or 3-butyn-1-ol. The resulting four genipin derivatives containing unsaturated groups have excellent anti-inflammatory activity and are more suitable for preparing drugs for treating central nervous system inflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to a genipin derivative and a preparation method and application thereof. Background Art

[0002] Stroke, an acute vascular disease, is primarily caused by external force or other factors, leading to the sudden rupture or blockage of cerebral blood vessels, which prevents normal blood flow to the brain and subsequently causes a series of brain tissue damage. According to relevant data, ischemic stroke accounts for approximately 70% to 80% of all stroke patients. As one of the major diseases that seriously endanger life safety, ischemic stroke (CIS) has high incidence, recurrence, mortality, and disability rates. Cerebral ischemia-reperfusion injury (CIRI) is localized brain damage caused by blood reperfusion after ischemic stroke. It typically occurs during treatment for ischemic disease and can trigger and aggravate brain tissue damage. Ischemic stroke is a serious and common condition worldwide, resulting in at least 6.671 million deaths or disabilities each year.

[0003] Currently, the main treatments for ischemic stroke are intravenous thrombolysis and endovascular mechanical thrombectomy, but both are time-sensitive, and only a very small number of patients can receive timely and effective treatment. For the majority of patients who exceed the treatment window, restoring blood flow to the brain tissue surrounding the infarct and promoting neurological recovery are the key points and challenges of ischemic stroke treatment and the key to improving the prognosis of ischemic stroke patients.

[0004] Related research has shown that cerebral ischemia-reperfusion injury (CIRI) is a complex and interconnected cascade of events encompassing multiple components, including oxidative stress, apoptosis, and inflammation. In Traditional Chinese Medicine (TCM), CIRI is classified as a type of stroke. Stagnation leads to heat, and this heat and toxicity invade the brain, leading to stroke. Therefore, clearing heat and detoxifying methods are effective treatments for stroke. Gardeniae Fructus, the dried, mature fruit of the Rubiaceae plant Gardenia jasminoides Ellis, is yellow or orange-red in color. It was first mentioned in the Shennong Bencao Jing (Classic of Materia Medica), and according to this ancient text, has been highly regarded for its unique medicinal properties since ancient times. Its benefits include purging heat and relieving restlessness, clearing heat and promoting diuresis, and cooling the blood and detoxifying. Numerous studies, both domestically and internationally, have confirmed its significant antipyretic and anti-inflammatory properties, as well as its ability to lower blood pressure, regulate lipids, and lower blood sugar. It also has clear protective effects on the liver, blood vessels, and nerves. According to research reports, genipin also has similar neuroprotective effects and other anti-central nervous system disease effects as gardenia glycosides. However, due to the high polarity of the hydroxyl group of genipin C-1, it has low lipid solubility, is not easily transported by cell membranes, and is not easy to cross the blood-brain barrier to exert central nervous system drug effects. It cannot exert a good anti-inflammatory effect, which limits the application of genipin. Summary of the Invention

[0005] In view of this, the present invention aims to provide genipin derivatives, preparation methods, and applications thereof. The present invention uses 3-butene-1-ol, 4-pentene-1-ol, 5-hexene-1-ol, or 3-butyn-1-ol to replace the hydrogen atom on the C-1 hydroxyl group of genipin, resulting in genipin derivatives with excellent anti-inflammatory activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a genipin derivative, the structural formula of the genipin derivative is shown in Formula I:

[0008]

[0009] In formula I, R is H2C=CH-CH2-CH2-, H2C=CH-CH2-CH2-CH2-, H2C=CH-CH2-CH2-CH2-CH2-, or CH≡C-CH2-CH2-.

[0010] The present invention also provides a method for preparing the genipin derivative described in the above technical solution, comprising the following steps:

[0011] mixing genipin, alcohol, an organic solvent and a catalyst to carry out a substitution reaction to obtain a genipin derivative;

[0012] The alcohol includes an enol or an alkynol, the enol includes 3-butene-1-ol, 4-pentene-1-ol or 5-hexene-1-ol, and the alkynol includes 3-butyn-1-ol.

[0013] Preferably, when the alcohol is an enol, the catalyst is p-toluenesulfonic acid.

[0014] Preferably, the molar ratio of genipin to p-toluenesulfonic acid is 1:1.1 to 1:5.

[0015] Preferably, when the alcohol is an alkynol, the catalyst is boron trifluoride etherate.

[0016] Preferably, the molar ratio of genipin to boron trifluoride etherate is 1:1.1 to 1:5.

[0017] Preferably, the temperature of the substitution reaction is -20 to 0°C, and the time is 4 to 5 hours.

[0018] Preferably, the organic solvent includes dichloromethane or dichloroethane, and the usage ratio of genipin to the organic solvent is 1 mmol: (5-20) mL.

[0019] Preferably, the substitution reaction further includes quenching the reaction, extraction, drying and purification, wherein the purification is silica gel column purification, the eluent of the silica gel column purification is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the mixture is 3:1 to 8:1.

[0020] The present invention also provides the use of the genipin derivative described in the above technical solution or the genipin derivative obtained by the preparation method described in the above technical solution in the preparation of a drug for treating central nervous system inflammation.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a genipin derivative, the structural formula of the genipin derivative is shown in Formula I:

[0023]

[0024] In formula I, R is H2C=CH-CH2-CH2-, H2C=CH-CH2-CH2-CH2-, H2C=CH-CH2-CH2-CH2-CH2-, or CH≡C-CH2-CH2-.

[0025] The present invention uses genipin as a lead compound for structural modification, connects an enol or alkynol unsaturated group, and uses 3-butene-1-ol, 4-pentene-1-ol, 5-hexene-1-ol or 3-butyn-1-ol to replace the H atom on the C-1 hydroxyl group of genipin. The obtained four genipin derivatives containing unsaturated groups have good anti-inflammatory activity and are more suitable for preparing drugs for treating central nervous system inflammation.

[0026] The present invention explores the effects of these unsaturated group-containing genipin derivatives on the NO content in LPS-activated BV-2 cells, laying a solid theoretical foundation for the subsequent development and synthesis of new anti-neuroinflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 is the hydrogen spectrum of 3-butene-1-ol genipin;

[0029] Figure 2 is the hydrogen spectrum of 4-penten-1-ol genipin;

[0030] Figure 3 is the hydrogen spectrum of 5-hexen-1-ol genipin;

[0031] Figure 4 is the hydrogen spectrum of 3-butyn-1-ol genipin;

[0032] Figure 5 is the NO standard curve;

[0033] Figure 6 is the cell survival rate at different concentrations of genipin;

[0034] Figure 7 is the cell survival rate of different concentrations of genipin derivatives, where a is 1-O-(3'-ene-butyl) genipin, b is 1-O-(4'-ene-pentyl) genipin, c is 1-O-(5'-ene-hexyl) genipin, and d is 1-O-(3'-alkyn-butyl) genipin;

[0035] Figure 8 The effect of genipin on NO level;

[0036] Figure 9The figure shows the effects of genipin derivatives on the NO level in the LPS-induced BV-2 cell inflammation model, where a is 1-O-(3'-ene-butyl) genipin, b is 1-O-(4'-ene-pentyl) genipin, c is 1-O-(5'-ene-hexyl) genipin, and d is 1-O-(3'-alkyn-butyl) genipin. DETAILED DESCRIPTION

[0037] The present invention provides a genipin derivative, the structural formula of the genipin derivative is shown in Formula I:

[0038]

[0039] In formula I, R is H2C=CH-CH2-CH2-, H2C=CH-CH2-CH2-CH2-, H2C=CH-CH2-CH2-CH2-CH2-, or CH≡C-CH2-CH2-.

[0040] The genipin derivatives of the present invention have improved lipid solubility, are more easily transported by cell membranes, easily cross the blood-brain barrier, and exert central nervous system drug effects. The genipin derivatives have good anti-inflammatory activity.

[0041] The present invention also provides a method for preparing the genipin derivative described in the above technical solution, comprising the following steps:

[0042] Genipin, alcohol, organic solvent and catalyst are mixed to carry out substitution reaction to obtain a genipin derivative; the alcohol includes an enol or an alkynol, the enol includes 3-butene-1-ol, 4-pentene-1-ol or 5-hexene-1-ol, and the alkynol includes 3-butyn-1-ol.

[0043] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0044] In the present invention, when the alcohol is preferably an enol, the catalyst is preferably p-toluenesulfonic acid, and the molar ratio of genipin to p-toluenesulfonic acid is preferably 1:1.1 to 1:5, more preferably 1:2 to 3; the substitution reaction is preferably S N 1 substitution reaction, the S N The temperature of the substitution reaction is preferably -20 to 0°C, and the time is preferably 4 to 5 hours.

[0045] In the present invention, the molar ratio of genipin to enol is preferably 1:1 to 1:3, more preferably 1:2.

[0046] In the present invention, when the alcohol is preferably an alkynol, the catalyst is preferably boron trifluoride ethyl ether, and the molar ratio of genipin to boron trifluoride ethyl ether is preferably 1:1.1 to 1:5, more preferably 1:2 to 3; the substitution reaction is preferably SN 2 substitution reaction, the S N The temperature of the 2-substitution reaction is preferably -20 to 0°C, and the time is preferably 4 to 5 hours.

[0047] In the present invention, it is preferred that genipin, alcohol and organic solvent are first mixed in an ice bath and then the catalyst is added to lower the system temperature and reduce the generation of by-products.

[0048] In the present invention, the molar ratio of genipin to alkynol is preferably 1:1 to 1:3, more preferably 1:2.

[0049] In the present invention, the organic solvent preferably includes dichloromethane or dichloroethane. The ratio of genipin to the organic solvent is preferably 1 mmol:(5-20) mL, more preferably 2.21 mmol:15 mL. The dichloroethane is preferably 1,2-dichloroethane. The organic solvent of the present invention has low toxicity.

[0050] In the present invention, the substitution reaction preferably further includes quenching reaction, extraction, drying and purification.

[0051] In the present invention, the method for quenching the reaction is preferably: mixing the system obtained by the substitution reaction with water. The extraction reagent is preferably dichloromethane, and the number of extractions is preferably 3. The drying is preferably drying with a desiccant, and the desiccant is preferably anhydrous sodium sulfate.

[0052] In the present invention, the purification is preferably performed by silica gel column purification, the silica gel particle size of the silica gel column is preferably 200-300 mesh, the eluent for the silica gel column purification is preferably a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the mixture is preferably 3:1-8:1, more preferably 5:1.

[0053] The present invention also provides the use of the genipin derivatives described in the above technical solution or the genipin derivatives obtained by the above preparation method in the preparation of drugs for treating central nervous system inflammation.

[0054] Based on the nucleus molecular structure of genipin, the present invention synthesizes a series of genipin derivatives containing unsaturated groups and conducts preliminary research on their anti-inflammatory activities, providing a reference for finding drugs for preventing and treating brain diseases such as neuroinflammation.

[0055] To further illustrate the present invention, the genipin derivatives provided by the present invention, their preparation methods and applications are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] In the embodiments or application examples of the present invention, the BV-2 cells used are a mouse-derived microglial cell line purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0057] Example 1 Synthesis of Genipin Derivatives Containing Alkenyl Unsaturated Groups:

[0058] The reaction formula for synthesizing genipin derivatives containing ethylenically unsaturated groups from genipin and enol is shown below:

[0059]

[0060] In a 50 mL round-bottom flask, 0.50 g (2.21 mmol) of 98% genipin by mass, an enol (3-butene-1-ol, 4-pentene-1-ol or 5-hexene-1-ol, all 4.42 mmol) and 15 mL of dichloromethane were added as the reaction solvent, and p-toluenesulfonic acid (6.63 mmol) was added dropwise to the genipin-enol reaction system; after reacting for 4 hours, the reaction system was poured into distilled water to quench and terminate the reaction.

[0061] The product was then extracted with 150 mL of dichloromethane (3 times, 50 mL each time). The organic phase in the lower layer of the separatory funnel was collected and washed with saturated sodium chloride solution (3 times) until the solution was clear. 5 g of anhydrous sodium sulfate was added for drying. Petroleum ether and ethyl acetate were used as eluents and mixed in a volume ratio of 5:1. 200-300 mesh silica gel was dissolved in petroleum ether and then loaded onto a column to avoid bubbles in the silica gel column. The product was separated from impurities by silica gel column chromatography to obtain a colorless or light yellow oily liquid, which was the purified genipin derivative containing an ethylenically unsaturated group.

[0062] Example 2 Synthesis of Genipin Derivatives Containing Alkyne Unsaturated Groups:

[0063] The reaction formula for synthesizing genipin derivatives containing alkynyl unsaturated groups from genipin and alkynol is as follows:

[0064]

[0065] In a 50 mL round-bottom flask, 0.50 g (2.21 mmol) of 98% genipin, 3-butyn-1 alcohol (4.42 mmol) and 15 mL of dichloromethane were added in sequence as a reaction solvent. After reacting in an ice bath for five minutes in the reaction system of genipin and alkynol, boron trifluoride etherate (6.63 mmol) was added dropwise with a syringe. After reacting for 4 hours, the reaction system was poured into distilled water to quench and terminate the reaction.

[0066] The mixture was then extracted with 150 mL of dichloromethane (3 times, 50 mL each time). The organic phase in the lower layer of the separatory funnel was collected and washed with saturated sodium chloride solution (3 times) until the solution was clear. 5 g of anhydrous sodium sulfate was added for drying. Petroleum ether and ethyl acetate were used as eluents, mixed in a volume ratio of 5:1. 200-300 mesh silica gel was dissolved in petroleum ether and then loaded onto a column to avoid bubbles in the silica gel column. The product was separated from impurities by silica gel column chromatography to obtain a colorless oily liquid, which was the purified genipin derivative containing an alkynyl unsaturated group.

[0067] Figure 1 is the hydrogen spectrum of 3-butene-1-ol genipin, Figure 2 is the hydrogen spectrum of 4-penten-1-ol genipin, Figure 3 is the hydrogen spectrum of 5-hexen-1-ol genipin, Figure 4 The hydrogen spectrum of 3-butyn-1-ol genipin was analyzed by hydrogen spectrum analysis of the compounds obtained in Example 1 and Example 2 as follows:

[0068] 1) GP21-O-(3'-ene-butyl) genipin, colorless oily liquid: 1 H NMR(CDCl3,600MHz),δ:7.48(s,1H),7.41(s,1H),5.72-5.80(m,2H),5.01-5.11(m,2H),4 .51(d,J=8.4Hz,1H),4.22(t,J=7.9Hz,2H),4.13(s,1H),4.01(q,J=7.4Hz,1H),3.69(d,J =4.2Hz,1H),3.57(q,J=7.8Hz,1H),3.15(q,J=8.4Hz,1H),2.82-2.87(m,1H),2.57(t,J=2 .0Hz,1H),2.52(s,1H),2.37(q,J=6.6Hz,1H),2.28(d,J=6.6Hz,1H),2.03(q,J=8.4Hz,1H)

[0069] 2) GP31-O-(4'-ene-pentyl)genipin, yellow oily liquid: 1H NMR (CDCl3, 600MHz), δ: 7.44 (s, 1H), 7.37 (s, 1H), 5.70-5.76 (m, 2H), 5.04 (d, J = 3.0Hz, 1H), 4 .89-4.98(m,2H),4.47(d,J=8.4Hz,1H),4.16(t,J=9.0Hz,2H),3.90(q,J=5.4Hz,1H),3.65(d, J=4.2Hz,1H),3.50(q,J=4.8Hz,1H),3.11(t,J=8.7Hz,1H),2.79-2.83(m,1H),2.53(t,J=8.1 Hz,1H),2.06(q,J=7.2Hz,1H),1.98-2.02(m,2H),1.68(q,J=7.2Hz,2H),1.58(t,J=7.2Hz,1H)

[0070] 3) GP41-O-(5'-ene-hexyl) genipin, yellow oily liquid: 1 H NMR (CDCl3, 600MHz), δ: 7.52 (s, 1H), 7.45 (s, 1H), 5.76-5.84 (m, 1H), 4.94-5.10 (m, 2H), 4.54 (d, J = 8.4H z,1H),4.25(d,J=13.8Hz,2H),3.98(d,J=9.6Hz,1H),3.82(d,J=9.0Hz,2H),3.73(d,J=6.0Hz,1H),3.57( d,J=9.0Hz,1H),3.49(s,1H),3.19(t,J=7.8Hz,1H),3.09(s,1H),2.60(t,J=8.1Hz,1H),2.46(s,1H),2.0 5-2.10(m,2H),1.66(t,J=7.8Hz,1H),1.58(t,J=7.5Hz,1H),1.47(q,J=7.8Hz,1H),1.41(t,J=7.8Hz,1H)

[0071] 4) GP51-O-(3'-Alkyne-butyl)genipin, colorless oily liquid: 1H NMR (CDCl3, 600MHz), δ: 7.50 (s, 1H), 5.83 (s, 1H), 4.61 (d, J = 8.4Hz, 1H), 4.31 (q, J = 13.0Hz, 2H), 4.04-4.08 (m, 1H), 3, 69-3. 73(m,3H),3.19(q,J=7.8Hz,1H),2.86-2.90(m,1H),2.64(m,1H),2.52-2.55(m,2H),2.43(d,J=2.4Hz,2H),2.04-2.09(m,2H)

[0072] Through organic synthesis, the structure of genipin was modified with the parent structure: unsaturated groups enol and alkynol were replaced by substitution reaction of enol in p-toluenesulfonic acid and alkynol in boron trifluoride ether to synthesize four genipin derivatives: GP2, GP3, GP4, GP5, with yields of 66.2%, 56.7%, 48.7% and 49.2%, respectively.

[0073] Comparative Example 1

[0074] The catalyst in Example 1 was replaced with boron trifluoride etherate, and the remaining steps were the same as in Example 1. The reaction of enol and genipin catalyzed by boron trifluoride etherate is as follows:

[0075]

[0076] The experimental results show that the yield of the target product is extremely low and it is difficult to obtain a pure product.

[0077] Comparative Example 2

[0078] The 3-butene-1-ol in Example 1 was replaced with 1-butene-3-ol, and the remaining steps were the same as in Example 1. The reaction formula of 1-butene-3-ol and genipin is as follows:

[0079]

[0080] The experimental results showed that the reaction between 1-butene-3-ol and genipin was not ideal, with many by-products and very little of the required main product, making it impossible to obtain the target product.

[0081] Application Example 1

[0082] 1. Biological activity evaluation method

[0083] LPS, an endotoxin produced by Gram-negative bacteria, is a widely used model for inducing neuroinflammation and neurodegeneration, specifically lipopolysaccharide-induced inflammation. LPS administration is widely used to investigate various inflammatory diseases and test the efficacy of targeted inflammatory drugs.

[0084] BV-2 cells are microglia in the mouse nervous system. Microglia play a key immune-related role in the central nervous system (CNS). They are deeply involved in the initiation and subsequent progression of various CNS-related diseases, playing a particularly crucial role in secondary injury caused by inflammation and trauma. Under physiological conditions, microglia maintain a quiescent state, ensuring stable and normal function, monitoring and monitoring the status of the CNS. After CIS, microglia in the brain become activated and release a variety of cytotoxic molecules and factors, exacerbating brain cell damage. Therefore, using LPS-induced BV-2 cells allows for a more concise and clear investigation of the effects of unsaturated genipin derivatives on their effects and to determine whether the synthesized derivatives can ameliorate the LPS-induced inflammatory response in BV-2 cells.

[0085] NO, produced from L-arginine by nitric oxide synthase (NOS), is widely involved in various physiological and pathological processes. During cerebral ischemia, NO produced by endothelial nitric oxide synthase (eNOS) relaxes vascular smooth muscle, helping to maintain blood flow. LPS-induced BV-2 inflammatory cells produce NO, which can be used as a preliminary indicator for evaluating anti-inflammatory activity.

[0086] 1. BV-2 cell culture

[0087] (1) Cell recovery

[0088] Remove 1 mL of BV-2 cell sample from the freezer and place it securely in a constant temperature water bath set at 37°C for strict thawing. After the thawing process is successfully completed, culture the BV-2 cells in a special DMEM culture medium containing 10% FBS (fetal bovine serum) and 1% double antibody (penicillin / streptomycin mixture) to ensure a suitable growth environment for the cells. To ensure that the cells can grow normally, place the culture dish in a 37°C constant temperature incubator filled with 5% CO2 gas. During the culture process, the culture medium needs to be replaced every two days to ensure the stability of the cell growth environment. Once the BV-2 cells grow to confluence in the culture medium and the cell state is confirmed to be good after observation, the next step of cell passaging can be carried out.

[0089] (2) Cell passage

[0090] To ensure the accuracy of the experiment and cell viability, the following steps are performed: First, the culture medium in the culture flask must be completely removed; then, the cells are gently rinsed twice with 2 mL of phosphate-buffered saline (PBS) to remove residual culture medium components; then, trypsin is added to the cells. To ensure effective digestion of the cells, they need to be placed in the trypsin solution for 1 minute. Subsequently, to terminate the trypsin digestion, 3 mL of fresh culture medium is added. Next, the cell suspension is transferred to a centrifuge tube and centrifuged at 1200 rpm for 3 minutes to allow the cells to precipitate. Finally, the centrifuged cells are transferred to new culture medium to complete the entire treatment process. Then, culture them under appropriate conditions.

[0091] (3) Cell plating

[0092] In this step, growing BV-2 cells are first centrifuged to separate the supernatant from the pellet. The pellet is then retained and resuspended in fresh culture medium to ensure cell viability and purity.

[0093] Then, 100 μL of cell suspension was accurately injected into the 96-well cell plate. At the same time, the cell density in each well was strictly controlled to ensure that the number of cells in each well reached exactly 2×10 4 Each set of experiments requires 4 replicate wells to improve the reliability and stability of the experimental results.

[0094] In order to compare the growth and response of cells under different conditions, a control group and a model group (LPS group) were set up. The control group cells were cultured under conventional culture conditions, while the model group cells were exposed to LPS stimulation to simulate specific physiological or pathological environments.

[0095] Finally, all well plates were placed in a CO2 incubator for 24 hours. During this process, the cells will grow and differentiate in a suitable temperature, humidity, and gas environment for subsequent observation and analysis.

[0096] 2. Determination of the inhibitory rate of different drug concentrations on BV-2 cells

[0097] BV-2 cells were grown at 2 × 10 4The cell density was accurately seeded into a 96-well plate, which was then properly placed in an incubator for a standardized 24-hour culture. After the initial culture was completed, drugs at different concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μmol / L were added to each well, and the well plate was placed in the incubator again for a further 24-hour incubation. Subsequently, 10 μL of CCK-8 reagent was added to each well and incubated for another 2 hours under the same conditions. Finally, the absorbance value of each well was measured at a wavelength of 450 nm using a microplate reader. To ensure the accuracy and reliability of the results, 4 replicate wells were set up for each group of experiments.

[0098] 3. Determination of NO Levels in BV-2 Cells

[0099] The cells were placed in an incubator and cultured for 24 hours to ensure that the cells were in good growth condition. After that, the cells were taken out and pre-treated with drugs at different concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μmol / L. The pre-treatment lasted for 1 hour to ensure that the drugs could fully act on the cells. After the pre-treatment, 2 μg / mL of LPS was added to the cells. After the addition of LPS, the cells were incubated for another 24 hours to observe the changes in the cells after stimulation. In order to further understand the metabolic changes in the cells after stimulation, a nitrate reduction test was performed. A new 96-well cell plate was taken out and 50 μL of cell supernatant was added to it. Then, an equal volume of Griess reagent was added to each well and evenly mixed by shaking. Finally, the absorbance of each well was measured at a wavelength of 550 nm, and the concentration of NO was accurately calculated by referring to the standard curve of NaNO2. Table 1 is the NO standard curve table. Figure 5 is the NO standard curve.

[0100] Table 1 NO standard curve

[0101]

[0102]

[0103] Through experimental measurements, the absolute absorbance data corresponding to NO standard solutions at different concentrations were obtained, and correlation analysis was performed. The analysis results showed that the correlation coefficient R was as high as 0.9978, indicating that there is an excellent correlation between the measured absolute absorbance and the concentration of the NO standard solution.

[0104] 2. Biological activity evaluation results

[0105] 1. Effects of genipin and its derivatives on BV-2 cell proliferation

[0106] Figure 6 is the cell survival rate at different concentrations of genipin, Figure 7 Cell viability of genipin derivatives at different concentrations, where a is 1-O-(3'-ene-butyl)genipin, b is 1-O-(4'-ene-pentyl)genipin, c is 1-O-(5'-ene-hexyl)genipin, and d is 1-O-(3'-alkyn-butyl)genipin; *P<0.05, **P<0.01.

[0107] 2. Effects of genipin derivatives on NO levels in the LPS-induced BV-2 inflammation model

[0108] Figure 8 The effect of genipin on NO levels, Figure 9 The figures show the effects of genipin derivatives on the NO level in the LPS-induced BV-2 cell inflammation model, where a is 1-O-(3'-ene-butyl)genipin, b is 1-O-(4'-ene-pentyl)genipin, c is 1-O-(5'-ene-hexyl)genipin, and d is 1-O-(3'-alkyn-butyl)genipin; *P<0.05, **P<0.01.

[0109] The effects of genipin and its derivatives on LPS-induced BV-2 cell survival were examined using the CCK-8 assay. The data showed that genipin had no effect on BV-2 cell proliferation at concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μmol / L, indicating no significant cytotoxicity. The products 1-O-(3'-ene-butyl)genipin, 1-O-(5'-ene-hexyl)genipin, and 1-O-(3'-alkyn-butyl)genipin did affect BV-2 cell proliferation at 100 μmol / L, demonstrating higher cytotoxicity than genipin. The product 1-O-(4'-ene-pentyl)genipin had no effect on cell proliferation at 200 μmol / L, demonstrating lower cytotoxicity and a safer safety profile than genipin.

[0110] Genipin was tested at concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μmol / L in the LPS-induced BV-2 inflammation model. Results showed that genipin at concentrations of 12.5, 25, 50, and 100 μmol / L significantly affected NO levels compared with the model group (P < 0.01). 1-O-(3'-ene-butyl)genipin also affected NO levels in the LPS-induced BV-2 inflammation model at a concentration of 50 μmol / L (P < 0.05), with a relative inhibition rate of 74.27 ± 11.65%. 1-O-(4'-ene-pentyl)genipin also affected NO levels in the LPS-induced BV-2 inflammation model at a concentration of 50 μmol / L (P < 0.01), with a relative inhibition rate of 68.34 ± 10.26%, demonstrating significant differences at this concentration. 1-O-(5'-ene-hexyl)genipin had an effect on NO levels in the LPS-induced BV-2 inflammation model at concentrations of 12.5, 25, and 50 μmol / L (P < 0.01), with relative inhibition rates of 79.37±3.86%, 73.79±5.80%, and 54.83±1.93%, respectively. 1-O-(3'-alkyn-butyl)genipin had an effect on NO levels in the LPS-induced BV-2 inflammation model at concentrations of 12.5, 25, and 50 μmol / L (P < 0.01), with relative inhibition rates of 84.33±4.38%, 77.37±2.66%, and 69.24±5.60%, respectively. 1-O-(5'-ene-hexyl)genipin showed a clear dose-dependency, while 1-O-(3'-alkyn-butyl)genipin showed no significant dose-dependency. Testing the LPS-induced BV-2 inflammation model with different concentrations of the saturated genipin derivative 1-O-methylgenipin revealed that the relative inhibitory rates at concentrations of 12.5, 25, and 50 μmol / L were 80.14±6.62%, 77.94±7.64%, and 75.73±4.68%, respectively, indicating a lower inhibitory effect than 1-O-(5'-ene-hexyl)genipin.

[0111] The present invention synthesized four genipin derivatives containing unsaturated groups by structurally modifying the genipin nucleus. They all share the basic iridoid skeleton, a hydroxyl group at the C-10 position, and an esterified C-11 position. However, they differ in the unsaturated group attached to the C-1 position, resulting in different anti-inflammatory activities against LPS-induced BV-2 cells. Analysis of preliminary cell-based experimental results revealed that 1-O-(5'-ene-hexyl)genipin exhibited minimal cytotoxicity and the best anti-inflammatory activity, while 1-O-(3'-ene-butyl)genipin exhibited the worst anti-inflammatory activity, achieving both no cytotoxicity and anti-inflammatory activity only at a drug concentration of 50 μmol / L. All four synthesized genipin derivatives exhibited good anti-inflammatory activity at a drug concentration of 50 μmol / L, paving the way for further in-depth research.

[0112] Based on the core structure of genipin, the present invention selects two suitable catalysts for S N 1 and S N 2 substitution reaction, ultimately synthesizing four genipin derivatives containing unsaturated groups. Preliminary experiments have shown that it can reduce NO levels in inflammatory cells without affecting the survival rate of BV-2 cells. These findings may pave the way for future research on anti-inflammatory effects in brain cells.

[0113] Neuroinflammation is a common pathophysiological phenomenon in the central nervous system, which is involved in almost all neurological and psychiatric diseases and has a significant impact on the course and prognosis of the disease. Mediation or alleviation of over-activated neuroinflammation is an important strategy to improve the prognosis of central nervous system diseases. BV-2 cells are the main source of inflammatory factors, and the neuroinflammation caused by their over-activation is the key to many nervous system diseases. The unsaturated group-containing genipin derivatives synthesized by the present invention have been found in preliminary activity evaluation to have a certain anti-inflammatory effect on LPS-induced BV-2 cells, laying the foundation for the future development of new genipin derivatives with less toxicity and better anti-inflammatory activity.

[0114] Although the above embodiments provide a detailed description of the present invention, they are only part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A genipin derivative, characterized in that The structural formula is shown in Formula I: In formula I, R is H2C=CH-CH2-CH2-, H2C=CH-CH2-CH2-CH2-, H2C=CH-CH2-CH2-CH2-CH2-, or CH≡C-CH2-CH2-.

2. The method for preparing the genipin derivative according to claim 1, characterized in that: The following steps are involved: Mixing genipin, an alcohol, an organic solvent, and a catalyst to carry out a substitution reaction to obtain a genipin derivative; the alcohol includes an enol or an alkynol, the enol includes 3-butene-1-ol, 4-pentene-1-ol, or 5-hexene-1-ol, and the alkynol includes 3-butyn-1-ol; When the alcohol is an enol, the catalyst is p-toluenesulfonic acid; When the alcohol is an alkynol, the catalyst is boron trifluoride etherate.

3. The preparation method according to claim 2, characterized in that The molar ratio of genipin to p-toluenesulfonic acid is 1:1.1 to 1:

5.

4. The preparation method according to claim 2, characterized in that The molar ratio of genipin to boron trifluoride ether is 1:1.1 to 1:

5.

5. The preparation method according to any one of claims 2 to 4, characterized in that The temperature of the substitution reaction is -20 to 0° C., and the time is 4 to 5 hours.

6. The preparation method according to claim 2, characterized in that The organic solvent includes dichloromethane or dichloroethane, and the usage ratio of genipin to the organic solvent is 1 mmol: (5-20) mL.

7. The preparation method according to claim 2, characterized in that The substitution reaction further includes quenching the reaction, extraction, drying and purification. The purification is silica gel column purification. The eluent of the silica gel column purification is a mixture of petroleum ether and ethyl acetate. The volume ratio of petroleum ether to ethyl acetate in the mixture is 3:1 to 8:

1.

8. Use of the genipin derivative according to claim 1 or the genipin derivative obtained by the preparation method according to any one of claims 2 to 7 in the preparation of a medicament for treating central nervous system inflammation.

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