A 1-S-alkyl genipin derivative and its preparation method and application

Abstract

The present invention belongs to the technical field of organic synthesis, and specifically relates to a 1-S-alkyl genipin derivative, a preparation method thereof, and an application thereof. The present invention provides a 1-S-alkyl genipin derivative, the structure of which is shown in Formula I. The 1-S-alkyl genipin derivative provided by the present invention has good stability and activity. The preparation method provided by the present invention has simple steps, is easy to operate, has high feasibility, and has good prospects for industrial application. The present invention also provides the use of the 1-S-alkyl genipin derivative described in the above scheme or the 1-S-alkyl genipin derivative obtained by the preparation method described in the above scheme in the preparation of anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases. The 1-S-alkyl genipin derivative provided by the present invention has good stability and activity and can be used to prepare anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and in particular relates to a 1-S-alkyl genipin derivative and a preparation method and application thereof. Background Art

[0002] Glial cells, abbreviated as glial cells, are another major type of cells in nervous tissue besides neurons. They have the functions of connecting and supporting various neural components, distributing nutrients, and participating in repair and phagocytosis.

[0003] Microglia, a type of glial cell characterized by multisynaptic and plastic properties, are resident immune effector cells within the central nervous system (CNS). They play a crucial role in CNS physiological processes and are implicated in the pathogenesis of a range of neurodegenerative diseases. Microglial activation and neuroinflammation are key features of neuropathology. In acute CNS diseases such as stroke, cerebral hypoxia, and traumatic brain injury, microglial phenotypes change and inflammatory mediators are released. Chronic microglial-mediated inflammation is implicated in a variety of chronic neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. During chronic inflammation, microglia are chronically activated and subsequently release a range of inflammatory mediators, leading to oxidative stress. Therefore, inhibiting microglial inflammatory responses is a key step in preventing and treating CNS degenerative diseases.

[0004] In the above-mentioned microglial cells, genipin showed good anti-central inflammatory activity, but its stability and activity could not fully meet the needs and needed to be further improved. Summary of the Invention

[0005] The object of the present invention is to provide a 1-S-alkyl genipin derivative and a preparation method and application thereof. The 1-S-alkyl genipin derivative provided by the present invention has good stability and activity.

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

[0007] The present invention provides a 1-S-alkyl genipin derivative, the structure of which is shown in Formula I:

[0008]

[0009] In Formula I, R is

[0010] The present invention also provides a method for preparing the 1-S-alkyl genipin derivative described in the above scheme, comprising the following steps:

[0011] Genipin, mercaptan, catalyst and polar organic solvent are mixed and S N1 substitution reaction to obtain 1-S-alkyl genipin derivatives;

[0012] The mercaptan includes one or more of ethanethiol, propyl mercaptan, 2-propyl mercaptan, 1-butyl mercaptan, 2-butyl mercaptan and isobutyl mercaptan.

[0013] Preferably, the mixing is: premixing genipin, mercaptan and a polar organic solvent to obtain a premixed liquid, and then dropping the catalyst into the premixed liquid.

[0014] Preferably, the molar ratio of genipin to thiol is 1:1-3.

[0015] Preferably, the catalyst comprises one or more of boron trifluoride etherate, sulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid and trimethyl silicone grease.

[0016] Preferably, the molar ratio of genipin to catalyst is 1:2-5.

[0017] Preferably, the polar organic solvent includes one or more of acetonitrile, chloroform and dichloromethane.

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

[0019] Preferably, the S N 1. The temperature of the substitution reaction is -20 to 20°C, and the insulation time is 6 to 12 hours.

[0020] The present invention also provides the use of the 1-S-alkyl genipin derivatives described in the above scheme or the 1-S-alkyl genipin derivatives obtained by the preparation method described in the above scheme in the preparation of anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases.

[0021] The present invention provides a 1-S-alkyl genipin derivative. The 1-S-alkyl genipin derivative provided by the present invention has good stability and activity because genipin has an unstable hemiacetal structure. After replacing the O in the structure with S, the hemiacetal structure is converted into a more stable acetal structure. The derivative can be used for anti-tumor, anti-inflammatory, and antibacterial purposes.

[0022] The present invention also provides a method for preparing the 1-S-alkyl genipin derivatives described in the above scheme. The preparation method provided by the present invention has simple steps, convenient operation, high feasibility, and good industrial application prospects.

[0023] The present invention also provides the use of the 1-S-alkyl genipin derivatives described in the above schemes or the 1-S-alkyl genipin derivatives obtained by the preparation methods described in the above schemes in the preparation of anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases. The 1-S-alkyl genipin derivatives provided by the present invention have good stability and activity and can be used to prepare anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 This is the hydrogen spectrum of 1-S-ethylgenipin prepared in Example 1 of the present invention;

[0026] Figure 2 This is the hydrogen spectrum of 1-S-propylgenipin prepared in Example 2 of the present invention;

[0027] Figure 3 This is the hydrogen spectrum of 1-S-isopropylgenipin prepared in Example 3 of the present invention;

[0028] Figure 4 This is the hydrogen spectrum of 1-S-butylgenipin prepared in Example 4 of the present invention;

[0029] Figure 5 This is the hydrogen spectrum of 1-S-sec-butylgenipin prepared in Example 5 of the present invention;

[0030] Figure 6 This is the hydrogen spectrum of 1-S-isobutylgenipin prepared in Example 6 of the present invention;

[0031] Figure 7 It is the standard curve diagram of NaNO2 in the present invention;

[0032] Figure 8 This is a graph showing the effect of 1-S-ethylgenipin prepared in Example 1 on the proliferation of BV-2 cells;

[0033] Figure 9 This is a graph showing the effect of 1-S-propylgenipin prepared in Example 2 on the proliferation of BV-2 cells;

[0034] Figure 10 This is a graph showing the effect of 1-S-isopropylgenipin prepared in Example 3 on the proliferation of BV-2 cells;

[0035] Figure 11This is a graph showing the effect of 1-S-butylgenipin prepared in Example 4 on the proliferation of BV-2 cells;

[0036] Figure 12 This is a graph showing the effect of 1-S-sec-butylgenipin prepared in Example 5 on the proliferation of BV-2 cells;

[0037] Figure 13 This is a graph showing the effect of 1-S-isobutylgenipin prepared in Example 6 on the proliferation of BV-2 cells;

[0038] Figure 14 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-ethylgenipin prepared in Example 1;

[0039] Figure 15 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-propylgenipin prepared in Example 2;

[0040] Figure 16 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-isopropylgenipin prepared in Example 3;

[0041] Figure 17 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-butylgenipin prepared in Example 4;

[0042] Figure 18 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-sec-butylgenipin prepared in Example 5;

[0043] Figure 19 This is a graph showing the results of measuring the NO level in BV-2 cells induced by LPS using 1-S-isobutylgenipin prepared in Example 6;

[0044] Figure 20 The present invention provides a synthetic route for the 1-S-alkyl genipin derivatives. DETAILED DESCRIPTION

[0045] The present invention provides a 1-S-alkyl genipin derivative, the structure of which is shown in Formula I:

[0046]

[0047] In Formula I, R is

[0048] The 1-S-alkyl genipin derivative provided by the present invention has good stability and activity and can be used for preparing anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases.

[0049] The present invention also provides a method for preparing the 1-S-alkyl genipin derivative described in the above scheme, comprising the following steps:

[0050] Genipin, mercaptan, catalyst and polar organic solvent are mixed (referred to as the first mixing) and subjected to S N 1 substitution reaction to obtain a 1-S-alkyl genipin derivative; the thiol includes one or more of ethanethiol, propylthiol, 2-propylthiol, 1-butylthiol, 2-butylthiol and isobutylthiol.

[0051] In the present invention, the polar organic solvent preferably includes one or more of acetonitrile, chloroform and dichloromethane; the catalyst preferably includes one or more of boron trifluoride ethyl ether, sulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid and trimethyl silicone grease, more preferably boron trifluoride ethyl ether.

[0052] The present invention has found that the yield of the product substituents increases from butyl to ethyl. As the number of carbon atoms in the side chain of the thiol decreases, the degree of reaction increases, the number of products obtained increases, and the yield also increases. This is because when the number of carbon atoms in the side chain of the reactant increases, the steric hindrance increases, the contact between the compounds becomes difficult, the reaction degree decreases, and thus the yield decreases.

[0053] In the present invention, the amount ratio of the genipin to the polar organic solvent is preferably 1 mmol: (5-20) mL, more preferably 1 mmol: 15 mL; the amount ratio of the genipin to the catalyst is preferably 1: 2-5, more preferably 1: 3-4; the amount ratio of the genipin to the thiol is preferably 1: 1-3, more preferably 1: 2.

[0054] In the present invention, the first mixing is preferably: premixing genipin, mercaptan and a polar organic solvent to obtain a premixed liquid, and then dropping a catalyst into the premixed liquid.

[0055] In the present invention, the premixing is preferably carried out in an ice-salt bath; the premixing is preferably carried out by stirring; the stirring speed is preferably 30 to 60 rpm, more preferably 40 to 50 rpm, and the stirring time is preferably 10 to 40 min, more preferably 30 min.

[0056] In the present invention, the dropping speed is preferably 1 to 3 drops / second, more preferably 1 to 2 drops / second.

[0057] In the present invention, the S N The temperature of the substitution reaction is preferably -20 to 20°C, more preferably 0 to 20°C, and even more preferably 5 to 15°C. The holding time is preferably 6 to 12 hours, more preferably 7 to 10 hours, and even more preferably 8 to 9 hours. By controlling the reaction conditions within the above range, a higher yield can be obtained.

[0058] In the present invention, the S N 1. After the substitution reaction, the obtained reaction system is preferably extracted, and the organic phase obtained by the extraction is sequentially washed, dried, the organic solvent is removed, and purified.

[0059] In the present invention, the extraction is preferably to N The system obtained by the substitution reaction is mixed with water and an extractant, followed by extraction; the extractant is preferably dichloromethane or ethyl acetate. In the present invention, the ratio of genipin to the extractant is preferably 1 mmol:50-150 mL, more preferably 1 mmol:100 mL. In the present invention, the extraction is preferably performed 2-5 times, more preferably 3 times.

[0060] In the present invention, the washing is preferably performed to neutrality; the washing agent is preferably a saturated sodium chloride aqueous solution.

[0061] In the present invention, the drying is preferably carried out using a desiccant; the desiccant is preferably anhydrous sodium sulfate or anhydrous magnesium sulfate; the dosage ratio of genipin to the desiccant is preferably 1 mmol:(2-3) g, more preferably 1 mmol:2 g.

[0062] In the present invention, the removal of the organic solvent is preferably performed by concentration; and the concentration is preferably performed by reduced pressure concentration.

[0063] In the present invention, the purification is preferably silica gel column chromatography; the silica gel column chromatography is preferably eluted by n-hexane-ethyl acetate system and dichloromethane-ethyl acetate elution; the volume ratio of n-hexane to ethyl acetate in the n-hexane-ethyl acetate system is preferably 5:1 to 1:1, more preferably 3:1; the volume ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate system is preferably 100:1 to 20:1, more preferably 50:1.

[0064] In the present invention, after the purification, it is preferred that the product is subjected to a first solvent removal, redissolution and a second solvent removal in sequence, the redissolution and the second solvent removal are repeated for more than 5 times, and then a third solvent removal is performed.

[0065] In the present invention, the first desolventizing agent is preferably rotary evaporation; the rotary evaporation device is preferably a rotary evaporator; the temperature of the rotary evaporation is preferably not more than 45°C, more preferably 42-45°C, and the insulation time is preferably not less than 10 minutes, more preferably 10 minutes.

[0066] In the present invention, the reagent used for redissolution is preferably n-hexane; the amount ratio of the reagent used for redissolution to the solid obtained by the first solvent removal is preferably 1 mmol: 5 to 10 mL, more preferably 1 mmol: 10 mL.

[0067] In the present invention, the second desolventizing method is preferably rotary evaporation; the equipment for rotary evaporation is preferably a rotary evaporator; the temperature of the rotary evaporation is preferably not more than 45°C, more preferably 42-45°C, and the insulation time is preferably not less than 10 minutes, more preferably 10 minutes.

[0068] In the present invention, the redissolution and the second solvent removal are repeated 5 times or more, preferably 5 times.

[0069] In the present invention, the third desolventizing method is preferably rotary evaporation; the equipment for rotary evaporation is preferably a rotary evaporator; the temperature of the rotary evaporation is preferably not more than 45°C, more preferably 42-45°C, and the insulation time is preferably not less than 3h, more preferably 3h.

[0070] In the present invention, the 1-S-alkyl genipin derivative is preferably stored in an environment below -20°C.

[0071] The preparation method of 1-S-alkyl genipin derivatives provided by the present invention has a synthetic route as follows: Figure 20 As shown: Genipin reacts with mercaptan in a catalyst and a polar organic solvent. N 1 substitution reaction to obtain 1-S-alkyl genipin derivatives has simple reaction steps and high feasibility.

[0072] The present invention also provides the use of the 1-S-alkyl genipin derivatives described in the above scheme or the 1-S-alkyl genipin derivatives obtained by the preparation method described in the above scheme in the preparation of anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases.

[0073] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] First, weigh 0.5 g (2.2 mmol) of genipin and add it to a round-bottom flask. Then weigh 4.4 mmol of ethanethiol and add it to the round-bottom flask. Then weigh 15 mL of dichloromethane and add it to the round-bottom flask. After completing the above feeding steps, stir in an ice-salt bath for 30 minutes. Then slowly add the catalyst boron trifluoride etherate (6.6 mmol) dropwise to the round-bottom flask and perform S at 10 ° C. N1 substitution reaction; after 8 hours of reaction, the reaction solution was quickly poured into 50 mL of room temperature water, and the above solution was slowly shaken, and then extracted three times with 150 mL of dichloromethane. The organic phase of the lower layer was collected, and the organic phases obtained by the three extractions were combined and washed with a saturated sodium chloride aqueous solution until neutral. The neutral organic phase was dried over 5.0 g of anhydrous Na2SO4, and then concentrated under reduced pressure to remove the organic solvent. The mixture was then chromatographed on a silica gel column (the eluent for silica gel column chromatography was [V (n-hexane): V (ethyl acetate) = 3:1], [V (dichloromethane): V (ethyl acetate) = 50:1]) to obtain an eluate. The obtained eluate was first rotary evaporated to dryness by evaporation at 45°C, and then mixed with 20 mL of n-hexane. The second rotary evaporation was performed at 45°C to evaporate dryness of n-hexane. The second rotary evaporation was repeated 5 times, and the time for each second rotary evaporation was 10 min. After rotary evaporation, a 1-S-alkyl genipin derivative was obtained, which was designated as GP1.

[0076] GP1, 1-S-ethylgenipin, colorless oily liquid, yield 89.3%, hydrogen spectrum as Figure 1 As shown, the hydrogen spectrum information is: 1 H NMR (CDCl 3, 600MHz)δ:7.54(s,1H),5.86(s,1H),4.98(d,J=7.8Hz,1H),4.36(s,2H),3.74(d,J=8.4Hz,3H),3.20(d,J=7 .8Hz,1H),2.85(d,J=7.8Hz,2H),2.75-2.81(m,2H),2.15-2.19(m,1H),1.79(s,1H),1.32(t,J=7.2Hz,3H).

[0077] Example 2

[0078] The only difference from Example 1 is that ethyl mercaptan is replaced by propyl mercaptan, and the product is recorded as GP2.

[0079] GP2, 1-S-propyl genipin, colorless oily liquid, yield 88.6%, hydrogen spectrum as Figure 2 As shown, the hydrogen spectrum information is: 1 H NMR (CDCl 3,600MHz)δ:7.54(s,1H),7.46(d,J=1.8Hz,1H),5.86(s,1H),5.76(d,J=5.4Hz,1H),4.95(d,J=7.8Hz,1H),4.30- 4.38(m,2H),4.17(d,J=12.6Hz,1H)3.75(s,1H),3.73(s,3H),3.25(t,J=6.6Hz,1H),3.18-3.22(m,1H),3.08-3 .12(m,1H),2.85-2.91(m,1H),2.82-2.83(m,1H),2.67-2.78(m,2H),2.62-2.66(m,1H),2.48-2.52(m,1H),2.3 5-2.40(m,1H),2.15-2.20(m,1H),1.82(s,1H),1.63-1.72(s,2H),1.00(t,J=7.2Hz,3H),0.97(t,J=7.2Hz,1H).

[0080] Example 3

[0081] The only difference from Example 1 is that ethyl mercaptan is replaced by 2-propanethiol, and the product is recorded as GP3.

[0082] GP3, 1-S-isopropylgenipin, colorless oily liquid, yield 87.9%, hydrogen spectrum as Figure 3 As shown, the hydrogen spectrum information is: 1 H NMR (CDCl 3, 600MHz)δ:7.54(s,1H),5.85(s,1H),5.02(d,J=7.8Hz,1H),4.31-4.37(m,2H),3.73(s,3H),3.22-3.2 7(m,1H),3.17-3.21(m,1H),2.82-2.86(m,2H),2.16-2.20(m,1H),0.07(s,1H),1.33(d,J=6.6Hz,6H).

[0083] Example 4

[0084] The only difference from Example 1 is that ethyl mercaptan is replaced by 1-butyl mercaptan, and the product is recorded as GP4.

[0085] GP4, 1-S-butyl genipin, colorless oily liquid, yield 87.0%, hydrogen spectrum as Figure 4 As shown, the hydrogen spectrum information is: 1 H NMR (CDCl 3,600MHz)δ:7.54(s,1H),5.86(s,1H),4.94(d,J=7.8Hz,1H),4.35(s,2H),3.73(s,3H),3.18-3.20(m,1H),2.82-2.87 (m,2H),2.72-2.78(m,2H),2.15-2.19(m,1H),1.81(s,1H),1.60-1.65(m,3H),1.40-1.45(m,2H),0.91-0.94(m,3H).

[0086] Example 5

[0087] The only difference from Example 1 is that ethyl mercaptan is replaced by 2-butyl mercaptan, and the product is recorded as GP5.

[0088] GP5, 1-S-sec-butyl genipin, colorless oily liquid, yield 85.9%, hydrogen spectrum as Figure 5 As shown, the hydrogen spectrum information is: 1 HNMR (CDCl 3, 600MHz)δ:7.53(s,1H),5.85(s,1H),5.00(t,J=7.2Hz,1H),4.34(d,J=7.2Hz,2H),3.75(s,1H),3.74(s,3H),3.19(d,J=6.6 Hz,1H),3.02-3.05(m,1H),2.15-2.18(m,2H),1.65-1.69(m,1H),1.56-1.59(m,1H),1.30-1.34(m,3H),0.95-1.00(m,3H).

[0089] Example 6

[0090] The only difference from Example 1 is that ethyl mercaptan is replaced by isobutyl mercaptan, and the product is recorded as GP6.

[0091] GP6, 1-S-isobutylgenipin, colorless oily liquid, yield 83.7%, hydrogen spectrum as Figure 6 As shown, the hydrogen spectrum information is: 1 HNMR (CDCl 3,600MHz)δ:7.54(s,1H),7.45(d,J=1.2Hz,1H),5.85(s,1H),5.71-5.72(m,1H),4.92(d,J=7.8Hz,1H),4.29-4.39(m,2H),3.74(d,J= 1.8Hz,1H),3.73(s,3H),3.25(t,J=7.2Hz,1H),3.19(q,J=7.2Hz,1H),3.09(q,J=8.4Hz,1H),2.88-2.91(m,1H),2.85-2.87(m,1H), 2.82(d,J=7.8Hz,1H),2.66-2.67(m,1H),2.62-2.66(m,1H),2.6-2.61(m,1H)2.53-2.56(m,1H),2.40-2.43(m,1H),2.36-2.39(m,1 H),2.14-2.18(s,1H),1.94(s,1H),1.84-1.89(m,1H),1.74(s,1H),1.01(d,J=1.2Hz,3H),0.99(d,J=1.8Hz,3H),0.95-0.97(m,2H).

[0092] Test Example 1

[0093] The effects of the 1-S-alkyl genipin derivatives prepared in Examples 1 to 6 on BV-2 cell proliferation and NO levels in a BV-2 cell inflammation model were tested:

[0094] 1. Preliminary preparation:

[0095] (1) Cell culture

[0096] Before conducting experiments with BV-2 cells, remove the cells from the freezer and thaw them in a 37°C water bath. Culture the thawed BV-2 cells in DMEM medium supplemented with 10% FBS (fetal bovine serum). Place the medium in a 37°C incubator filled with 5% CO₂. While maintaining these conditions, change the medium every two days until the BV-2 cells are growing well and filling the medium. Once the cells are in good condition, they can be passaged.

[0097] (2) Cell passage

[0098] The culture medium in the culture flask was removed, and the cells were rinsed twice with 2 mL of PBS (phosphate buffered saline). An appropriate amount of trypsin was added to the rinsed cells to promote cell digestion. The digestion process lasted for 1 minute. 3 mL of culture medium was added to stop the digestion. The digested cells were transferred to a centrifuge tube and centrifuged at 1200 rpm for 3 minutes to separate the cell pellet. The centrifuged cells were transferred to a new culture medium for culture.

[0099] (3) Establishment of LPS-induced BV-2 cell inflammation model

[0100] During the growth phase of BV-2 cells, centrifugation was performed to effectively separate the supernatant and cell pellet. The cell pellet separated by centrifugation was resuspended in new culture medium, and 100 μL of cell suspension was added to the 96-well cell plate, ensuring that the cell density in each well reached 2×10 4 All treated cell plates were placed in a CO2 incubator for 24 h. Microglial BV-2 cells were treated with different concentrations of LPS to analyze and explore cell viability and NO levels. The LPS concentration was determined based on the above analysis.

[0101] 2. Effects of 1-S-alkyl genipin derivatives on BV-2 cell proliferation

[0102] 1-S-alkylated genipin was prepared into a 100 mmol / L stock solution using DMSO, and the stock solution was diluted with DMEM high glucose medium to 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μmol / L 1-S-alkylated genipin solutions for later use;

[0103] BV-2 cells were plated at 2 × 10 4 The cells were seeded at a density of 100 μL in a 96-well plate and cultured in an incubator for 24 h. The above-mentioned different concentrations of 1-S-alkylated genipin (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μmol / L) were added to the cell plate, and 8 replicate wells were set up in each group. The plate was placed in an incubator and incubated for 24 h. 10 μL of CCK-8 reagent was added to each well and incubated for another 2 h. The absorbance of each well was detected at 450 nm to analyze the effects of different compounds at different concentrations on cell proliferation. The results are shown in FIG. Figures 8 to 13 shown.

[0104] 3. Determination of NO levels in BV-2 cells induced by LPS by 1-S-alkyl genipin derivatives

[0105] After 24 hours of incubation in the incubator, the cells were removed and pretreated with 1-S-alkylated genipin at different concentrations (as long as it does not affect the cell viability) for 1 hour. 2 μg / mL LPS was added to the pretreated cells and incubated for 24 hours. A new 96-well cell plate was taken out, and 50 μL of cell supernatant was added thereto. Then 50 μL of Griess reagent was added thereto, and then the plate was thoroughly shaken and mixed. The absorbance was measured at 550 nm, and the NO concentration was calculated using the NaNO2 standard curve.

[0106] The preparation of the standard curve of NaNO2 is as follows: using a series of concentrations of NaNO2 solution, measuring the OD value, calculating the absolute OD value, and then establishing a standard curve with the absolute OD value as the ordinate and the concentration of the NaNO2 solution as the abscissa. Table 1 shows the data of the NaNO2 solution, the measured OD value and the absolute OD value. The standard curve of NaNO2 is as follows Figure 7 The test results are shown in Figures 14-19 shown.

[0107] Table 1 Data of NaNO2 solution, determination of OD value and absolute OD value

[0108] Standard concentration (μmol / L) Determination of OD value Absolute OD value 50 0.254 0.207 25 0.150 0.103 12.5 0.097 0.050 6.25 0.073 0.026 3.125 0.059 0.012 1.5625 0.052 0.005 0.78125 0.050 0.003 0.390625 0.049 0.002 0 0.047 0.000

[0109] according to Figure 7 It can be seen that R=0.9997, indicating that the two are well correlated.

[0110] according to Figures 8 to 13 It can be seen that the GP1-6 series of compounds had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78-6.25 μmol / L. Among them, 1-S-ethylgenipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μmol / L. At 100 μmol / L, it was able to significantly inhibit the release of the inflammatory factor NO in BV-2 cells induced by LPS, indicating that it has significant anti-inflammatory activity and has potential for the prevention and treatment of brain neuroinflammatory diseases.

[0111] 1-S-n-propyl genipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μmol / L. Evaluation of the compound's anti-inflammatory activity at concentrations of 12.5, 25, and 50 μmol / L revealed that the compound significantly inhibited LPS-induced NO release in BV-2 cells at 50 μmol / L, demonstrating its significant anti-inflammatory activity and potential role in the prevention and treatment of brain neuroinflammatory diseases.

[0112] 1-S-isopropylgenipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μmol / L. The anti-inflammatory activity of the compound was evaluated at concentrations of 12.5, 25, and 50 μmol / L, showing that at 50 μmol / L, it significantly inhibited the release of the inflammatory factor NO in BV-2 cells induced by LPS, indicating that it has significant anti-inflammatory activity and has potential for the prevention and treatment of brain neuroinflammatory diseases.

[0113] 1-S-n-Butylgenipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, and 6.25 μmol / L. The compound's anti-inflammatory activity was evaluated at three concentrations: 1.56, 3.12, and 6.25 μmol / L. The compound significantly inhibited LPS-induced NO release in BV-2 cells at 6.25 μmol / L, demonstrating significant anti-inflammatory activity and potential for the prevention and treatment of brain neuroinflammatory diseases.

[0114] 1-S-sec-butylgenipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, 6.25, and 12.5 μmol / L. Evaluation of the compound's anti-inflammatory activity at concentrations of 1.56, 3.12, and 6.25 μmol / L revealed that the compound significantly inhibited LPS-induced NO release in BV-2 cells in a dose-dependent manner, demonstrating its significant anti-inflammatory activity and potential role in the prevention and treatment of brain neuroinflammatory diseases.

[0115] 1-S-isobutylgenipin had no significant effect on the proliferation of BV-2 cells at concentrations of 0.78, 1.56, 3.12, 6.25, and 12.5 μmol / L. The anti-inflammatory activity of the compound was evaluated at concentrations of 1.56, 3.12, and 6.25 μmol / L. The results showed that at 3.12 and 6.25 μmol / L, the compound significantly inhibited the release of the inflammatory factor NO in BV-2 cells induced by LPS in a dose-dependent manner, indicating that it has significant anti-inflammatory activity and has potential for the prevention and treatment of brain neuroinflammatory diseases.

[0116] The above results were analyzed using one-way ANOVA with Graphadprism 7.0 software. P < 0.05 was considered statistically significant. Statistically, the effects of 1-S-alkylgenipin derivatives on BV-2 cell proliferation compared with the control group were *P < 0.05, **P < 0.01. Compared with the model group, the effects of 1-S-alkylgenipin derivatives on LPS-induced NO levels in BV-2 cells were *P < 0.05, **P < 0.01.

[0117] In summary, 1-S-alkyl genipin derivatives have very weak toxicity to BV-2 cells and have a relatively small effect on their proliferation. Significant cytotoxicity often occurs only at high concentrations. The inhibitory effect of such compounds on NO often increases with the increase of the substituent group. For example, 1-S-ethyl genipin can significantly inhibit the release of inflammatory factor NO from BV-2 cells induced by LPS at 100 μmol / L. Two 1-S-propyl genipin derivatives (GP2-GP3) can significantly inhibit the release of inflammatory factor NO from BV-2 cells induced by LPS at 50 μmol / L. Three 1-S-butyl genipin derivatives (GP4-GP6) can all significantly inhibit the release of inflammatory factor NO from BV-2 cells induced by LPS at 6.25 μmol / L.

[0118] In addition, the larger the side chain of the substituent, the stronger its activity. For example, at 50 μmol / L, the inhibitory effect of 1-S-isopropyl genipin on NO is stronger than that of 1-S-n-propyl genipin; at 6.25 μmol / L, the inhibitory activity of 1-S-n-butyl genipin on NO is weaker than that of 1-S-sec-butyl genipin and 1-S-isobutyl genipin, the latter two of which have significant NO inhibitory activity; 1-S-sec-butyl genipin has a significant NO inhibitory effect at 1.56 μmol / L, while 1-S-isobutyl genipin and 1-S-n-butyl genipin do not.

[0119] according to Figures 14-19 It can be seen that there is a significant difference between the control group and the model group in the present invention, indicating that the cell inflammation model was successfully established.

[0120] It can be seen from the above examples that the 1-S-alkyl genipin derivatives provided by the present invention have low cytotoxicity and good anti-inflammatory effects.

[0121] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A 1-S-alkyl genipin derivative, characterized in that The structure is shown in Formula I: Formula I; In formula I, R is 、 、 、 、 ,or .

2. The method for preparing the 1-S-alkyl genipin derivative according to claim 1, characterized in that: The following steps are involved: Genipin, mercaptan, catalyst and polar organic solvent are mixed and S N 1 substitution reaction to obtain 1-S-alkyl genipin derivatives; The mercaptan includes one or more of ethanethiol, propyl mercaptan, 2-propyl mercaptan, 1-butyl mercaptan, 2-butyl mercaptan and isobutyl mercaptan.

3. The preparation method according to claim 2, characterized in that The mixing comprises: premixing genipin, mercaptan and a polar organic solvent to obtain a premixed liquid, and then dropwise adding a catalyst into the premixed liquid.

4. The preparation method according to claim 2 or 3, characterized in that The molar ratio of genipin to thiol is 1:1-3.

5. The preparation method according to claim 2, characterized in that The catalyst comprises one or more of boron trifluoride etherate, sulfonic acid and trifluoroacetic acid.

6. The preparation method according to claim 2 or 5, characterized in that The molar ratio of the genipin to the catalyst is 1:2-5.

7. The preparation method according to claim 2, characterized in that The polar organic solvent includes one or more of acetonitrile, chloroform and dichloromethane.

8. The preparation method according to claim 2 or 7, characterized in that The dosage ratio of genipin to polar organic solvent is 1 mmol:(5~20)mL.

9. The preparation method according to claim 2, characterized in that The S N 1 The temperature of the substitution reaction is -20~20 ℃, and the insulation time is 6~12 h.

10. The preparation method according to claim 5, characterized in that The sulfonic acid is p-toluenesulfonic acid.

11. Use of the 1-S-alkyl genipin derivative according to claim 1 or the 1-S-alkyl genipin derivative obtained by the preparation method according to any one of claims 2 to 10 in the preparation of anti-inflammatory drugs and drugs for preventing and treating central nervous system diseases.

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