Geniposide derivatives and genipin derivatives and preparation methods and applications thereof

Abstract

The invention discloses geniposide derivatives and genipin derivatives and preparation methods and applications thereof, belonging to the field of pharmaceutical chemistry. The invention synthesizes prenylated and geranylated derivatives IA-GNPS and GE-GNPS respectively by esterification modification of the carboxyl group of geniposide acid with an unsaturated olefin side chain, and further obtains two genipin derivatives IA-GNP and GE-GNP that can target UCP2 protein and have potential anti-AD therapeutic effects by beta-glucosidase hydrolysis. The preparation method combines chemical synthesis and biocatalysis, has a simple preparation process, retains the hemiacetal pharmacophore, and effectively improves the drugability of genipin.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and particularly relates to geniposide derivatives and geniposide derivatives, and preparation methods and applications thereof. Background Art

[0002] Studies on Alzheimer's disease (AD) have shown that mitochondrial dysfunction is closely related to the development of the disease, so improving mitochondrial function may be a new idea for treating AD. There is a lot of evidence that thermal homeostasis disorders associated with normal aging may lead to various metabolic changes associated with the development of AD. Effectively controlling the increase in brain temperature in the elderly and improving the brain's heat dissipation and cooling mechanism are of great significance for the treatment of AD. Brain temperature is crucial for understanding the pathophysiological process of neurodegenerative diseases and may provide a new perspective for the diagnosis and treatment of the disease.

[0003] Target identification is very important for developing new drugs and understanding their side effects. Clarifying the binding strength of drugs to targets and the therapeutic mechanism can provide guidance for drug evaluation, and determining the drug-target interaction pattern is crucial for developing new drugs. Based on current research on anti-AD drugs, it has been found that mitochondrial uncoupling protein 2 (UCP2) can protect hippocampal neurons from Aβ-induced oxidative stress and toxic damage, and the expression of UCP2 and UCP4 in brain samples of AD patients is abnormal. The effects of UCP2 are not limited to neurons, but can also affect microglia and astrocytes. Appropriate intervention with UCP2 can prevent hippocampal atrophy, neuronal dendrite retraction, metabolic changes, and spatial memory impairment in AD mouse models.

[0004] Yang et al. [HOU YC, et al. Food Chem Toxicol, 2008, 46 (8): 2764-9.] systematically studied and found that the C-3 position of GNP (genipin) has a hemiacetal structure, which is easy to destroy under physiological conditions and produce a dialdehyde structure that is easy to react with amino or sulfhydryl groups. Structural modification of GNPs and GNPS can resist endoplasmic reticulum stress, Aβ toxicity damage, inflammation, oxidative stress and mitochondrial damage and repair damaged neurons. On this basis, Suzuki et al. [WANG R, et al. Chemmedchem, 2014, 9 (7): 1397-401.] transformed the hemiacetal at the C-3 position of GNP into an acetal structure and synthesized a series of derivatives. The study found that these derivatives can have similar activity on the neurite growth of PC12 cells as GNPs, but the chemical structure is more stable than GNPs. Wang et al. [WANG R et al. Int J Mol Sci, 2016, 17(1): 117.] reduced the double bonds at C-6 and C-7 of GNPs, and the resulting compounds CHR20 / 21 showed stronger neuroprotective effects than the parent compounds CHRa / b. Chen et al. [WANG R, et al. Neuroscience, 2015, 286(1): 242-250.] synthesized a series of N-substituted GA derivatives using GNPs as raw materials, and investigated the neuroprotective activity of the derivatives. In the PC12 cell injury model, compound GA-8 had the strongest neuroprotective activity. Xu et al. [CHE X, et al. Molecules, 2016, 21(7): 923.] successfully synthesized a new derivative called methyl gardeniaside (MG) at the C3 position. Studies have shown that MG does not cause an increase in aspartate aminotransferase and alanine aminotransferase in rat liver indicators. In addition, oral administration of MG at a dose of 0.72 mmol / kg did not show obvious toxic effects on rats, reducing liver toxicity. In the 52-day forced swimming and tail suspension test, MG can reduce the immobility time of mice in a concentration-dependent manner, showing an antidepressant effect. In addition, HPLC analysis showed that the area under the curve of MG detected in the brain homogenate of mice after one day of oral treatment with 0.52mmol / kg / day of MG was 21.7 times that of GNP, proving that MG can cross the blood-brain barrier. In 2008, Shandong Target Drug Research Co., Ltd. disclosed a patent on "The use of geniposide and its derivatives and analogs in the preparation of drugs for the prevention and treatment of depression and anxiety." The invention involves patented compounds containing geniposide and its derivatives. Studies have found that gardenia glycoside and geniposide acid have effects similar to geniposide. In 2012, the Jinan University team disclosed a patent on "Geniposide derivatives and their use in the preparation of drugs for the prevention and treatment of neurodegenerative diseases."The patent discloses an N-substituted gardenamide A derivative with a specific chemical structure. The derivative uses GNP as the starting material, then selectively protects the hydroxyl group, converts it into a lactone with an oxidant, and then directly reacts with an amine compound to form an adduct, and finally obtains the target derivative by acid treatment. The derivative is of great significance in the preparation of drugs for the treatment of neurodegenerative diseases, especially drugs for the treatment of Alzheimer's disease. In 2017, the Shanxi Medical University team disclosed a patent entitled "Genipin derivatives and their use in the preparation of drugs for the prevention and treatment of neurodegenerative diseases". These genipin derivatives have been shown to have significant neuroprotective effects on PC12 cell oxidative damage and PD animal model experiments, and have important application prospects in the preparation of prevention and treatment of neurodegenerative diseases. In 2010, the Beth Israel Deaconess Medical Center in the United States disclosed a US patent entitled "Genipin derivatives and their uses". Studies have shown that GNPs can dimerize in the presence of nucleophiles, and the C7-C8 double bond and C10 primary alcohol of GNPs are necessary for the cross-linking process. Genipin derivatives lacking these two active sites have no cross-linking activity. Studies have found that the cross-linking activity of genipin seems to be unrelated to its UCP2 inhibitory activity, so the derivatives can become effective UCP2 inhibitors without the cross-linking activity of GNPs. Studies have shown that the lack of UCP2 gene or the removal of endogenously produced superoxide (UCP2 activator) can improve the glucose-stimulated insulin secretion reduction caused by hyperglycemia and obesity. GNPs can dimerize in the presence of nucleophiles such as primary amines, and their C7-C8 double bond and C10 primary alcohol play a key role in the cross-linking process. Genipin derivatives lacking these two active sites lose their cross-linking activity. The cross-linking of GNPs seems to be unrelated to their UCP2 inhibitory activity, and GNPs can act as effective UCP2 inhibitors even without cross-linking activity; despite years of research on AD, its pathophysiological mechanism is still incompletely understood. However, decreased mitochondrial function seems to be a common focus of many studies. In this prospect, mitochondria have great potential as a therapeutic target. Considering the important role of mitochondria in the pathophysiology of AD, the early occurrence of mitochondrial damage, late diagnosis, and other comorbidities also increase the complexity of exploring mitochondrial targeted therapeutic approaches. Although genipin-based derivatives have been widely used in the biological and medical fields for the treatment of AD, the development of derivatives based on the UCP2 target has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide geniposide derivatives and geniposide derivatives and their preparation methods and applications to solve the problems existing in the above-mentioned prior art. Target identification is very important for developing new drugs and understanding their side effects. Clarifying the binding strength and therapeutic mechanism of drugs and targets can provide guidance for drug evaluation, and determining the drug-target interaction pattern is crucial for developing new drugs. Based on the current research on anti-AD (Alzheimer's disease) drugs, it has been found that mitochondrial uncoupling protein 2 (UCP2) can protect hippocampal neurons from Aβ-induced oxidative stress and toxic damage, and the expression of UCP2 and UCP4 in brain samples of AD patients is abnormal. The effect of UCP2 is not limited to neurons, but can also affect microglia and astrocytes. Appropriate intervention of UCP2 can prevent hippocampal atrophy, neuronal dendrite shrinkage, metabolic changes and spatial memory disorders in AD mouse models. Therefore, based on the previous cognition of the covalent binding mode and mechanism of GNP targeting mitochondrial UCP2 protein, the present invention designs and develops a series of geniposide derivatives.

[0006] One of the technical solutions provided by the present invention:

[0007] A geniposide derivative is prepared by esterifying the unsaturated olefin side chain of the C6 carboxyl group of geniposide acid, wherein the esterification modification is geranyl modification or isopentenyl modification.

[0008] Preferably, the esterification modification is prenylation modification.

[0009] The second technical solution provided by the present invention is:

[0010] A method for preparing the above-mentioned geniposide derivatives comprises the following steps: adding geniposide acid to an alcohol solution under a nitrogen atmosphere, then adding an activator and a catalyst, heating for reaction, quenching the reaction with sodium bicarbonate after the reaction is complete, washing, drying, vacuum concentrating, and purifying by silica gel column chromatography to obtain the geniposide derivatives, wherein the alcohol solution is geraniol or isopentanol.

[0011] Preferably, the dosage ratio of the geniposide acid to the alcohol solution is 100 mg: 1 mL.

[0012] Preferably, the heating reaction is carried out at a temperature of 70° C. and for 24 hours.

[0013] Preferably, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0014] Preferably, the catalyst is 4-dimethylaminopyridine.

[0015] The third technical solution provided by the present invention is:

[0016] A genipin derivative is prepared by hydrolyzing the glycoside of the above geniposide derivative.

[0017] The fourth technical solution provided by the present invention is:

[0018] A method for preparing the above-mentioned genipin derivatives comprises hydrolyzing the glycoside of the above-mentioned geniposide derivatives by β-glucosidase to obtain the genipin derivatives.

[0019] The fifth technical solution provided by the present invention is:

[0020] A use of the above geniposide derivative in the preparation of a drug for treating neurodegenerative diseases, wherein the drug is in an oral dosage form, and the neurodegenerative diseases include Alzheimer's disease.

[0021] After an oral pharmaceutical dosage form is prepared with a geniposide derivative as the active ingredient, it is directly orally taken and transformed through intestinal bacterial metabolism and hydrolyzed to obtain a geniposide derivative, which then exerts its effect.

[0022] The sixth technical solution provided by the present invention is:

[0023] A use of the above genipin derivative in the preparation of a drug for treating neurodegenerative diseases, wherein the neurodegenerative diseases include Alzheimer's disease. The drug has a dosage form including tablets, capsules, granules, injections and other clinically acceptable dosage forms.

[0024] Technical principle of the present invention:

[0025] The C3 hemiacetal of geniposide participates in the condensation with the lysine of UCP2 protein, and this role provides a key guarantee for inhibiting proton reflux. The present invention, on the basis of retaining the C3 hemiacetal structure or glycoside protection, introduces an unsaturated olefin side chain substituent group at its C6 position to increase fat solubility and improve its membrane permeability. The present invention synthesizes the isopentenylated and geranyl-esterified derivatives IA-GNPS and GE-GNPS respectively by esterification modification of the carboxyl group of geniposide acid (GNPS) with an unsaturated olefin side chain, and further obtains IA-GNP and GE-GNP, two geniposide derivatives that can target UCP2 protein and have potential anti-AD therapeutic effects through β-glucosidase hydrolysis. The preparation method combines chemical synthesis and biocatalysis, and the preparation process is simple, that is, the hemiacetal pharmacophore is retained, and the drugability of GNP is also effectively improved.

[0026] According to the structural characteristics of GNP, the hemiacetal group at the C3 position is a necessary pharmacophore that can be protected by glucosidation, and the modification of the ester group at the C6 position can significantly enhance its binding ability with the UCP2 protein. Geniposide acid (GNPA) is used as a raw material to perform esterification modification of the unsaturated olefin side chain on its C6 carboxyl group, and geniposide derivatives IA-GNPS and GE-GNPS including geranylation and prenylation modification are synthesized, and the geniposide derivatives IA-GNPS and GE-GNPS are hydrolyzed by β-glucosidase to obtain geniposide derivatives IA-GNP and GE-GNP that can target the UCP2 protein.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] The present invention synthesizes isopentenylated and geranylated geniposide (GNPS) derivatives IA-GNPS and GE-GNPS respectively by esterification modification of the unsaturated olefin side chain of the carboxyl group of geniposide acid (GNPA), and further hydrolyzes them using β-glucosidase to obtain IA-GNP and GE-GNP, two geniposide derivatives that can target UCP2 protein and have potential anti-AD therapeutic effects. The preparation process of the above-mentioned compound is simple, and after oral administration, it can effectively alleviate D-galactose-induced AD, improve cognitive impairment and learning and social memory impairment, especially IA-GNP shows a more significant anti-AD effect, providing a new candidate drug molecule for AD treatment. Through animal behavioral experiments such as open field test, tail suspension test, water maze, etc., it is confirmed that in the AD model induced by D-galactose, GNP and its IA-GNP and IA-GNPS derivatives can improve the cognitive defects, learning and social memory impairment of AD mice to a certain extent, and significantly improve the despair of mice. The retention of the hemiacetal pharmacophore can effectively target the UCP2 protein, and the introduction of the isopentenyl side chain can increase the cell membrane permeability and improve the reaction efficiency. Among them, the effect of IA-GNP is better than that of GNP. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative labor.

[0030] Figure 1 The chemical structures and synthetic routes of GNP derivatives IA-GNP, IA-GNPS, GR-GNP and GE-GNPS based on GNPA;

[0031] Figure 2The structural characterization diagram of IA-GNPS prepared in Example 1, wherein A is the hydrogen nuclear magnetic resonance spectrum of IA-GNPS, B is the carbon nuclear magnetic resonance spectrum of IA-GNPS, and C is the high-resolution mass spectrum of IA-GNPS;

[0032] Figure 3 The structural characterization diagram of IA-GNP prepared in Example 1, wherein A is the hydrogen nuclear magnetic resonance spectrum of IA-GNP, B is the carbon nuclear magnetic resonance spectrum of IA-GNP, and C is the high-resolution mass spectrum of IA-GNP;

[0033] Figure 4 The structural characterization diagram of GE-GNPS prepared in Example 2, wherein A is the hydrogen nuclear magnetic resonance spectrum of GE-GNPS, B is the carbon nuclear magnetic resonance spectrum of GE-GNPS, and C is the high-resolution mass spectrum of GE-GNPS;

[0034] Figure 5 The structural characterization diagram of GE-GNP prepared in Example 2, wherein A is the hydrogen nuclear magnetic resonance spectrum of GE-GNP, B is the carbon nuclear magnetic resonance spectrum of GE-GNP, and C is the high-resolution mass spectrum of GE-GNP;

[0035] Figure 6 The results of the investigation of the reaction activities of genipin based on mitochondrial UCP2, IA-GNPS and IA-GNP prepared in Example 1, and GE-GNPS and GE-GNP prepared in Example 2, wherein A is the result of the investigation of the activity of genipin and the above derivatives with lysine cross-linking reaction (n=3); B is the result of the gel fluorescence imaging analysis to investigate the covalent binding ability of the above derivatives of genipin with brain mitochondrial UCP2 protein;

[0036] Figure 7 The open field experiment was used to investigate the effects of genipin and the IA-GNPS and IA-GNP prepared in Example 1 on the activity and central area activity time of AD mice, wherein A is the open field activity result and B is the central area activity time result;

[0037] Figure 8 Schematic diagram of the tail suspension experiment to investigate the effects of genipin and the IA-GNPS and IA-GNP prepared in Example 1 on the mood improvement of AD mice;

[0038] Fig. 9 Schematic diagram of the water maze experiment to investigate the effects of genipin and IA-GNPS and IA-GNP prepared in Example 1 on the learning and memory of AD mice. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The present invention targets mitochondrial UCP2 protein, takes the structural characteristics of GNP as a basis, uses GNPA as a raw material, and successfully develops geniposide derivatives IA-GNPS and GE-GNPS as well as geniposide derivatives IA-GNP and GE-GNP through a combination of chemical synthesis and biotransformation. Figure 1 The chemical structures and synthesis routes of GNP derivatives IA-GNP, IA-GNPS, GE-GNP and GE-GNPS based on GNPA as raw material.

[0045] The present invention confirms the efficacy of the above derivatives through comprehensive evaluation methods such as protein coupling and animal behavior evaluation. Through behavioral tests such as open field test, tail suspension test and water maze, the anti-AD effect of IA-GNP and IA-GNPS was evaluated and it was found that the genipin isopentenyl derivatives IA-GNP and IA-GNPS can significantly improve the cognitive function, learning ability and social memory of AD mice under the D-galactose model, and relieve anxiety. The effect is better than the prototype drug GNP, providing candidate drug molecules for the development of anti-AD drugs.

[0046] Example 1: Synthesis of prenyl geniposide and prenyl geniposide

[0047] (1) Synthesis of isopentenyl geniposide (IA-GNPS): Geniposide acid (GNPA, 800 mg, 2.14 mmol) was placed in a 25 mL round-bottom flask, 8 mL of isopentenol was added as a solvent and reactant under argon protection, and then the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 817 mg, 4.28 mmol) was added to the system, and then the catalyst 4-dimethylaminopyridine (DMAP, 16 mg, 0.21 mmol) was added, and the temperature was slowly raised to 70°C for 24 h. After the disappearance of the raw material by TLC detection, the reaction was quenched with 1 M sodium bicarbonate, and the organic phase was extracted and washed three times with dichloromethane: methanol = 4: 1 and saturated brine, respectively. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo, and purified by silica gel column chromatography (dry loading) to obtain 738 mg of light yellow solid IA-GNPS with a yield of 78%.

[0048] Compound IA-GNPS was detected by NMR and HRMS to confirm the correctness of the product. Figure 2 The structural characterization diagram of IA-GNPS prepared in this example, wherein A is the hydrogen nuclear magnetic resonance spectrum of IA-GNPS, B is the carbon nuclear magnetic resonance spectrum of IA-GNPS, and C is the high-resolution mass spectrum of LA-GNPS. The specific results of NMR and HRMS detection of compound IA-GNPS are as follows: 1H NMR (400MHz, DMSO-d6) δ7.44(d,J=1.1Hz,1H),5.68(s,1H),5.32(tt,J=7.1,1.5Hz,1H),5.11(d,J=6.9Hz,1H),5.0 4(d,J=5.2Hz,1H),4.97(d,J=4.9Hz,1H),4.94(d,J=5.2Hz,1H),4.71(t,J=5.4Hz,1H),4.60-4.57(m,1H),4.53(d, J=7.8Hz,1H),4.45(t,J=5.8Hz,1H),4.13(d,J=13.3Hz,1H),4.02-3.92(m,1H),3.66(ddd,J=11.8,6.2,2.0Hz,1H) ,3.42(dt,J=11.6,5.7Hz,1H),3.20-2.96(m,5H),2.72-2.61(m,2H),2.10-1.99(m,1H),1.72(s,3H),1.68(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.8,151.9,144.6,138.3,125.9,119.6,111.6,99.1,96.2 ,77.8,77.1,73.8,70.5,61.5,60.7,59.8,46.3,38.5,35.0,25.9,18.4.HRMS[M+Cl] - The calculated value is 477.1533, and the detected value is 477.1535. The data results show that the compound is the target compound IA-GNPS.

[0049] (2) Synthesis of isopentenyl genipin (IA-GNP): IA-GNPS (442 mg, 1 mmol) was placed in a 25 mL round-bottom flask, and 5 mL of an aqueous solution containing 0.5 mg / mL β-glucosidase (Shanghai Yuanye Biotechnology Co., Ltd., batch number: J14HS182613; enzyme activity unit: 12 U / mg) was added under argon protection, and the system was adjusted to pH 5.0 with sodium dihydrogen phosphate (NaH2PO4), 1 mL of ethyl acetate was added, and the reaction system was slowly heated to 50°C for 3 h. After the disappearance of the raw material by TLC detection, the system was extracted twice with ethyl acetate, the organic phase was concentrated in vacuo, and purified by silica gel column chromatography to obtain 215 mg IA-GNP with a yield of 76.8%.

[0050] Compound IA-GNP was tested by NMR and HRMS to confirm the correctness of the product. Figure 3The structural characterization diagram of IA-GNP prepared in this example, wherein A is the hydrogen nuclear magnetic resonance spectrum of IA-GNP, B is the carbon nuclear magnetic resonance spectrum of IA-GNP, and C is the high-resolution mass spectrum of IA-GNP. The specific results of NMR and HRMS detection of compound IA-GNP are as follows: 1 H NMR (400MHz, CDCl3) δ7.52(d,J=1.3Hz,1H),5.93-5.82(m,1H),5.40-5.31(m,1H),4.80(d,J=8.6Hz,1H),4.70-4.57(m,2H),4.43-4. 18(m,2H),3.29-3.16(m,1H),2.96-2.80(m,1H),2.53(td,J=8.2,1.9Hz,1H),2.15-1.97(m,1H),1.76(s,3H),1.72(d,J=1.4Hz,3H). 13 C NMR (100MHz, CDCl3) δ167.6,152.46,142.16,138.76,130.86,118.86,111.06,96.4,61.3,61.1,48.2,39.1,36.8,25.7,18.1.HRMS[MH] - The calculation result is: 279.1238, the detection result is: 279.1238, and the data results show that the compound is the target compound IA-GNP.

[0051] Example 2 Synthesis of Geranyl Geniposide and Geranyl Geniposide

[0052] (1) Synthesis of geranyl geniposide (GE-GNPS): Geniposide acid (GNPA, 800 mg, 2.14 mmol) was placed in a 25 mL round-bottom flask, 8 mL of geraniol was added as a solvent and reactant under argon protection, and then the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 817 mg, 4.28 mmol) was added to the system, and then the catalyst 4-dimethylaminopyridine (DMAP, 16 mg, 0.21 mmol) was added, and the temperature was slowly raised to 70°C for 24 h. After the disappearance of the raw material by TLC detection, the reaction was quenched with 1 M sodium bicarbonate, and the organic phase was extracted and washed three times with dichloromethane / methanol (4 / 1) and saturated brine, respectively. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo, and purified by silica gel column chromatography (dry loading) to obtain 677 mg of light yellow solid GE-GNPS with a yield of 63.8%.

[0053] The compound GE-GNPS was tested by NMR and HRMS. Figure 4The structural characterization diagram of GE-GNPS prepared in this example, wherein A is the hydrogen nuclear magnetic resonance spectrum of GE-GNPS, B is the carbon nuclear magnetic resonance spectrum of GE-GNPS, and C is the high-resolution mass spectrum of GE-GNPS. The NMR results are: 1 H NMR (400MHz, DMSO-d6) δ7.45(d,J=1.2Hz,1H),5.68(s,1H),5.31(t,J=7.0Hz,1H),5.08(td,J=10.4,9.9,6.5Hz,3H ),5.00(d,J=5.0Hz,1H),4.97(d,J=5.2Hz,1H),4.74(t,J=5.4Hz,1H),4.59(p,J=5.6Hz,2H),4.53(d,J=7.9Hz,1H), 4.48(t,J=5.8Hz,1H),4.18-4.08(m,1H),4.04-3.88(m,1H),3.65(ddd,J=11.9,6.2,2.0Hz,1H),3.42(dt,J=11.6,5 .7Hz,1H),3.22-2.92(m,5H),2.66(dq,J=21.9,8.2,7.5Hz,2H),2.11-1.97(m,5H),1.69-1.62(m,6H),1.56(s,3H). 13 C NMR (100MHz, DMSO-d6) δ166.8,152.0,144.6,141.5,131.5,125.9,124.2,119.4,111.6,99.0,96. 2,77.8,77.1,73.8,70.4,61.4,60.6,59.8,46.3,38.5,35.1,26.2,25.97,18.1,16.7.HRMS[M+Na] + The calculation result is: 533.2357, and the detection result is: 533.2357. The data results show that the compound is the target compound GE-GNPS.

[0054] (2) Synthesis of geranyl genipin (GE-GNP): Refer to the synthesis method of isopentenyl genipin IA-GNP in Example 1. GE-GNPS (510 mg, 1 mmol) was placed in a 25 mL round-bottom flask, and 5 mL of an aqueous solution containing 0.3 mg / mL β-glucosidase was added under argon protection, and the system was adjusted to pH 5.0 with sodium dihydrogen phosphate (NaH2PO4), 1 mL of ethyl acetate was added, and the reaction system was slowly heated to 50°C for 5 h. After the disappearance of the raw material by TLC detection, the system was extracted twice with ethyl acetate, the organic phase was concentrated in vacuo, and purified by silica gel column chromatography to obtain 217.5 mg GE-GNP with a yield of 62.5%.

[0055] The compound GE-GNP was tested by NMR and HRMS. Figure 5 The structural characterization diagram of GE-GNP prepared in this example, wherein A is the hydrogen nuclear magnetic resonance spectrum of GE-GNP, B is the carbon nuclear magnetic resonance spectrum of GE-GNP, and C is the high-resolution mass spectrum of GE-GNP. The compound GE-GNP was subjected to NMR detection, and the NMR results were: 1 H NMR (400MHz, CDCl3) δ7.53 (s, 1H), 5.88 (s, 1H), 5.36 (t, J = 7.1Hz, 1H), 5.08 ( t,J=6.8Hz,1H),4.80(d,J=8.7Hz,1H),4.67(h,J=6.3,5.9Hz,2H),4.31(q,J= 13.0Hz,2H),3.21(t,J=8.6Hz,1H),2.88(dd,J=16.9,8.7Hz,1H),2.53(t,J=8 .3Hz,1H),2.07(dd,J=13.7,6.8Hz,5H),1.69(d,J=12.9Hz,6H),1.60(s,3H). 13 C NMR (100MHz, CDCl3) δ167.6,152.4,142.1,142.0,131.8,131.0,123.8,118.5,11 1.0,96.4,61.3,61.1,48.2,39.5,39.1,36.8,26.3,25.7,17.7,16.5.HRMS[M+Na] + The calculation result is: 371.1829, and the detection result is: 371.1830. The data results show that the compound is the target compound GE-GNP.

[0056] Performance test

[0057] 1. Study on the Reactivity of Genipin and Its Derivatives Based on Lysine and Brain Mitochondrial UCP2

[0058] Figure 6 These are the results of investigating the reaction activities of genipin based on mitochondrial UCP2, the IA-GNPS and IA-GNPs prepared in Example 1, and the GE-GNPS and GE-GNPs prepared in Example 2.

[0059] The cross-linking degree of GNPs and their derivatives (IA-GNPS and IA-GNPs prepared in Example 1 and GE-GNPS and GE-GNPs prepared in Example 2) after reacting with lysine was evaluated by measuring the absorbance at 592 nm using an ELISA instrument to detect the cross-linking efficiency of the reaction. Figure 6A is the activity investigation result of the cross-linking reaction of genipin and the above derivatives with lysine (n=3). Figure 6 In Figure A, it can be seen that IA-GNPs and GE-GNPs have strong absorbance, while IA-GNPS and GE-GNPS do not, indicating that the sugar ring substitution of the C3 hydroxyl group will destroy its cross-linking effect.

[0060] In order to further evaluate the differences between the compounds, a protein binding experiment was also performed. The experimental operation reference [Chinese Chemical Letters, 2023, 34: 108203], using alkynyl-modified genipin probe Alk-GNP and fluorescent dye BDP TMR-N3, gel imaging analysis was performed with mouse mitochondria through click chemistry reaction. Figure 6 Figure B is the result of gel fluorescence imaging analysis to evaluate the covalent binding ability of the above derivatives of genipin with brain mitochondrial UCP2 protein. Alk-GNP can covalently bind to mouse brain mitochondrial UCP2 protein, showing a strong fluorescence band. GNPs at a concentration of 10 times can significantly compete with the binding of Alk-GNP to UCP2, weakening the protein fluorescence intensity. GNPS with a glycoside structure can hardly weaken the fluorescence intensity, while GNP derivatives (GE-GNP and IA-GNP) that expose the C3 hemiacetal site weaken the protein fluorescence intensity to varying degrees, especially after 10 times the dose of IA-GNP and GE-GNP competition, almost no fluorescence band can be observed. The results show that IA-GNPS and GE-GNPS are suitable for oral administration, and can only covalently bind to UCP2 protein after being metabolized into IA-GNP and GE-GNP in the body, and the effect of IA-GNP is better than that of GE-GNP.

[0061] 2. Construction of D-galactose-induced AD mouse model

[0062] D-galactose (D-gal) can cause many tissues to undergo degenerative changes similar to natural aging. The present invention uses mice to be subcutaneously injected with D-gal (500 mg / kg) daily for modeling, and at the same time, the following drugs are administered by gavage: donepezil (positive drug, Don 5 mg / kg); GNP prototype (GNP, 5, 15, 45 mg / kg); isopentenyl-genipin (IA-GNP, 5, 15, 45 mg / kg) prepared in Example 1, and isopentenyl-genipin (IA-GNPS, 5, 15, 45 mg / kg) prepared in Example 1 for 8 consecutive weeks, and after the experiment, the learning, cognition and memory of the mice are evaluated by behavioral experiments.

[0063] 3. Open field animal behavior

[0064] Figure 7The results of the open field experiment were used to investigate the effects of genipin and its derivatives (IA-GNPS and IA-GNP prepared in Example 1) on the activity and central area activity time of AD mice, where A is the open field activity result and B is the central area activity time result ( ## P<0.01vs.Con; *P<0.05, **P<0.01, vs.D-gal, n=5).

[0065] AD patients are often accompanied by emotional and behavioral problems, such as depression, anxiety, irritability, etc. The behavioral open field experiment can observe the effects of drugs on these emotional and behavioral problems, such as whether the drugs alleviate depression and anxiety symptoms. Refer to the dosing regimen provided in 2. Construction of D-galactose-induced AD mouse model for the behavioral open field experiment. The specific experimental setup and steps are to place the mice in a square open field with a side length of 50 cm and a height of 40 cm. Record the activity trajectory, standing time, and exploratory behavior of the mice for 5 minutes. Use behavioral automatic tracking software (Beijing Zhishu Duobao Biotechnology Co., Ltd.) to assist in data recording and result statistics. Finally, the experimental results are presented by calculating the activity level and the percentage of activity time in the central area. The results of the open field experiment are as follows: Figure 7 As shown in Figure 2, the activity of mice in the D-gal group was significantly lower than that in the Con group ( ## P<0.01), indicating that the activity of AD mice decreased. The positive drug Don group and each treatment group showed a concentration-dependent increase in the activity of mice. The low concentration of IA-GNP group (5mg / kg) can achieve the therapeutic effect of the high concentration of GNP group (45mg / kg). In addition, the time mice spend active in the central area is another indicator for evaluating the anxiety and stress response of mice in a new environment. The experimental results showed that the central area movement time of mice in the D-gal group was significantly lower than that of the Con group ( # P<0.05), while each treatment group increased the activity time of the central area in a concentration-dependent manner (*P<0.05, **P<0.01). The results of the open field test suggested that both IA-GNPs and IA-GNPS have an alleviating effect on depression and anxiety behaviors.

[0066] 4. Animal behavior in the tail suspension experiment

[0067] Figure 8 Schematic diagram of the tail suspension experiment to investigate the effects of genipin and the IA-GNPS and IA-GNP prepared in Example 1 on the mood improvement of AD mice ( # P<0.05vs.Con;*P<0.05,**P<0.01,***P<0.001vs.D-gal, & P<0.05vs.GNP 45mg / kg, n=5).

[0068] The principle of the tail suspension experiment is that mice give up struggling after their attempts to escape are futile. During the experiment, the immobility time of the animals is recorded to reflect their emotions and mental state, so as to evaluate whether drug intervention can improve this state. The tail suspension experiment was performed with reference to the dosing regimen provided in 2. Construction of D-galactose-induced AD mouse model. The specific experimental device and steps are to fix the mouse's tail on a specific device so that it is completely suspended in the air. The drug effect is evaluated by recording the immobility time of the mice during the 5-minute test period. The experimental results are shown in the figure. Figure 8 As shown, the immobility time of mice in the D-gal model group was significantly longer than that in the Con group, and they entered the despair state earlier. The positive drug Don group and each treatment group improved the despair time of mice and alleviated the degree of depression to varying degrees. Among them, the high concentration of IA-GN (45 mg / kg) had the best therapeutic effect (*P<0.05, **P<0.01), and the medium concentration of IA-GNP (15 mg / kg) could achieve the therapeutic effect of the high concentration of GNP group (15 mg / kg).

[0069] 5. Animal behavior in water maze experiment

[0070] Fig. 9 Schematic diagram of the water maze experiment to investigate the effects of genipin and the IA-GNPS and IA-GNP prepared in Example 1 on the learning and memory of AD mice ( ## P<0.01vs.Con; *P<0.05, **P<0.01vs.D-gal, & P<0.05vs.GNP45mg / kg, n=5).

[0071] The water maze test is a commonly used test for evaluating the spatial cognitive learning and memory ability of mice. It is used to evaluate the hippocampus-dependent spatial learning and long-term spatial memory of mice. Specifically, mice need to establish spatial memory by finding a hidden platform in the pool. By observing the escape latency and the number of times the mice cross the platform during the training and test periods, their learning and spatial memory abilities are evaluated. Refer to the dosing regimen provided in 2. Construction of D-galactose-induced AD mouse model for modeling, and conduct a water maze experiment after successful modeling. Specific experimental apparatus and steps: The MWM apparatus (Beijing Zhishu Duobao Biotechnology Co., Ltd.) includes a circular pool with a diameter of 120 cm and a height of 50 cm. The mice were pre-adapted to the experimental environment for 1 hour. The experiment is divided into a training period and a test period. The training period usually lasts 1 to 4 days, and the test period is conducted after the training period. The latency of the experimental group to find the platform on days 1-4 of the training period was significantly shorter than that of the D-gal modeling group, and as the number of training times increased, the latency gradually shortened, indicating that the training process was effective. The results of the number of times the platform was crossed on the 5th day of the test are as follows. Fig. 9As shown. The number of times the D-gal group crossed the platform was significantly lower than that of the Con group (##P<0.01), which indicates that the D-gal modeling caused damage to the learning and memory of mice. The positive drug Don group, the GNP high concentration group (45 mg / kg), the IA-GNP medium and high concentration groups (15, 45 mg / kg), and the IA-GNPS medium and high concentration groups (15, 45 mg / kg) were able to reverse the memory damage and cognitive decline caused by the D-gal modeling, and the IA-GNP medium concentration (15 mg / kg) could achieve the therapeutic effect of the GNP high concentration group (15 mg / kg) (*P<0.05, **P<0.01). However, there was no significant improvement in the behavior of the modeling mice treated with low and medium concentrations of GNP (5, 15 mg / kg).

[0072] The above results indicate that long-term subcutaneous injection of large amounts of D-gal causes learning and memory impairment in mice, while the genipin derivative IA-GNP and geniposide derivative IA-GNPS prepared in Example 1 of the present invention can reverse the memory impairment and cognitive decline caused by D-gal modeling.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a genipin derivative in the preparation of a drug for treating neurodegenerative diseases, characterized in that: The chemical structural formula of the genipin derivative is: ; The genipin derivatives target mitochondrial UCP2 protein to treat neurodegenerative diseases.

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