Compound as well as preparation method and application thereof

Abstract

The invention relates to a plant source compound as well as a preparation method and application thereof. According to the invention, hypericum perforatum (hypericum perforatum) is researched to obtain a new active component, and experiments show that the compound can specifically activate a TGR5 receptor, and can be used as a lead compound to be developed into drugs for treating obesity and other metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a plant-derived compound and a preparation method and application thereof. Background Art

[0002] Hypericumperforatum L., also known as Forsythia suspensa and St. John's wort, is a plant of the genus Hypericum of the family Garciniae. It is widely distributed in Jiangsu, Hebei, Sichuan, Guizhou, Shaanxi, Gansu and Xinjiang in my country. Hypericumperforatum has the effects of soothing the liver and relieving depression, clearing away heat and removing dampness, and reducing swelling and promoting lactation. It is mainly used to treat liver qi stagnation, emotional distress, depression, joint swelling and pain, mastitis, and lack of milk. As early as in the "Tang Materia Medica", there were records about the use of Hypericumperforatum as medicine. Hypericumperforatum has complex chemical components and a wide range of biological activities, including multiple structural types such as polycyclic polyisopentenyl-substituted acyl phloroglucinols, dianthrone, xanthones, and flavonoids.

[0003] TGR5 (also known as G protein-coupled bile acid receptor 1, or G-protein-coupledbile acid receptor 1) is a receptor that plays an important role in bile acid metabolism. It is mainly expressed in bile acid, intestine, liver and other tissues. The activation of TGR5 receptor can affect many physiological processes, so its relationship with various diseases has attracted widespread attention. Summary of the invention

[0004] The technical problem to be solved by the present invention is to further study the active components of Hypericum perforatum with TGR5 as the target and discover the active components.

[0005] In view of this, the present invention provides a compound having TGR5 receptor agonist activity isolated and prepared from Hypericum perforatum or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof, wherein the compound has a structure of Formula I:

[0006]

[0007] The present invention also proposes a method for preparing the above compound, which is characterized by comprising the following steps:

[0008] Step (1): extracting the Hypericum perforatum medicinal material with ethanol aqueous solution by reflux, and concentrating the extract to obtain an extract; the ethanol aqueous solution is preferably 75-95% ethanol aqueous solution, more preferably 95% ethanol aqueous solution;

[0009] Step (2): mixing the extract obtained in step (1) with water, extracting with dichloromethane, and concentrating the dichloromethane extraction portion to obtain a dichloromethane extract;

[0010] Step (3): decolorizing the dichloromethane extract obtained in step (2) through an MCI column, eluting with a 95% ethanol aqueous solution, collecting and concentrating the eluate;

[0011] Step (4): subjecting the concentrated solution obtained in step (3) to silica gel column chromatography, performing gradient elution with a petroleum ether-ethyl acetate mixed solvent, wherein the volume ratio of petroleum ether to ethyl acetate in the gradient elution is (30-1):1, and collecting the components separated at a volume ratio of petroleum ether to ethyl acetate of (10-5):1;

[0012] Step (5): subjecting the components separated in step (4) to MCI column chromatography for separation and purification, using a 40% to 100% methanol-water solution for gradient elution, and collecting the components separated by 80% to 90% methanol-water solution;

[0013] Step (6): The components separated in step (5) are loaded on an ODS-C8, C18 or AqC18 column, separated by a medium pressure chromatograph, gradient eluted with a 40% to 100% methanol-water solution, and the components separated by 80% to 90% methanol-water solution are collected;

[0014] Step (7): separating and purifying the components obtained in step (6) by gel column chromatography and eluting with an organic solvent;

[0015] Step (8): The eluted fraction obtained in step (7) is purified by semi-preparative liquid phase to obtain the compound represented by formula (I).

[0016] Furthermore, in the step (1), the weight ratio of Hypericum perforatum to ethanol aqueous solution is 1:(10-20).

[0017] Specifically, in the step (1), the ethanol aqueous solution is 95% ethanol, and reflux extraction is performed 2-3 times, each time for 1-2 hours.

[0018] Furthermore, in the step (4), before loading onto the column, the step also includes dissolving the concentrated solution obtained in step (3) with an organic solvent having a volume of 1.5 to 3 times the weight of the concentrated solution, and then adding 3 times the weight of the concentrated solution with 100 to 200, 200 to 300, or 300 to 400 mesh silica gel for sample mixing; the organic solvent is dichloromethane or methanol.

[0019] Specifically, in step (6), the chromatographic column used in the medium pressure chromatograph is octadecylsilane bonded silica gel, and the flow rate is 5 to 7 mL / min.

[0020] Specifically, the gel used in the gel column chromatography is selected from Sephadex LH-20 or Sephadex G-15.

[0021] Furthermore, in step (7), the organic solvent used is a mixed solvent of petroleum ether-dichloromethane-methanol, with a volume ratio of 5:5:1.

[0022] Specifically, in step (8), the chromatographic column used for the semi-preparative liquid phase is octadecylsilane bonded silica gel, the mobile phase is 80% to 90% acetonitrile aqueous solution, the flow rate is 4 mL / min, and the detection wavelength is 210 nm.

[0023] The present invention also provides a use of a compound of formula I or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof in the preparation of a TGR5 receptor agonist drug.

[0024] The present invention also provides a use of a compound of formula I or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof in the preparation of a drug for treating obesity, diabetes, or metabolic diseases.

[0025] The present invention also provides a drug comprising the aforementioned compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof.

[0026] Furthermore, the aforementioned medicine also includes a pharmaceutically acceptable carrier.

[0027] Furthermore, the pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, such as diluents, excipients and water, fillers such as starch, sucrose, lactose, microcrystalline cellulose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol and sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, micropowdered silica gel and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0028] Specifically, the drug can be any dosage form mentioned in pharmacy, including tablets, capsules, soft capsules, gels, oral preparations, suspensions, granules, patches, ointments, pills, powders, injections, infusions, lyophilized injections, intravenous emulsions, liposome injections, suppositories, sustained-release preparations or controlled-release preparations.

[0029] Specifically, the drug is selected from oral dosage forms, injection dosage forms or external administration preparations, preferably tablets, capsules, pills, granules, pastes, mixtures or suspensions.

[0030] The present invention studies Hypericum perforatum and obtains a new active component which has a significant effect of stimulating TGR5 receptors and can be further developed into a drug for treating obesity, diabetes and other metabolic diseases induced by them. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 HR-ESI-MS chart of the compound prepared in Example 1 of the present invention;

[0032] Figure 2 The compound prepared in Example 1 of the present invention 1 H NMR spectra;

[0033] Figure 3 The compound prepared in Example 1 of the present invention 13 C NMR spectra;

[0034] Figure 4 DEPT-135 diagram of the compound prepared in Example 1 of the present invention;

[0035] Figure 5 HSQC diagram of the compound prepared in Example 1 of the present invention;

[0036] Figure 6 is the HMBC diagram of the compound prepared in Example 1 of the present invention;

[0037] Figure 7 The compound prepared in Example 1 of the present invention 1 H- 1 H COSY diagram;

[0038] Figure 8 The NOESY diagram of the compound prepared in Example 1 of the present invention;

[0039] Fig. 9 The single crystal diffraction pattern of the compound prepared in Example 1 of the present invention;

[0040] Fig.10 Effect of compound 1 on body weight of diet-induced obese mice (Note: # P<0.05, ## P<0.01, ### P<0.001vs Chow; *P<0.05, **P<0.01, ***P<0.001vs HFD);

[0041] Fig.11 Effect of compound 1 on OGTT in diet-induced obese mice (Note: # P<0.05, ## P<0.01, ###P<0.001vs Chow; *P<0.05, **P<0.01, ***P<0.001vs HFD). DETAILED DESCRIPTION

[0042] The technical problem to be solved by the present invention is to further study the active ingredients of Hypericum perforatum with TGR5 as the target and discover its active ingredients. Takeda G-protein receptor 5 (TGR5) is a bile acid membrane receptor, also known as G-protein coupled bile acid receptor 1 (G protein-coupled bile acid receptor 1, GPBAR1) or G protein coupled receptor 19 (GPCR19), which is derived from the G-protein coupled receptor family and is the first known bile acid specific receptor. TGR5 activation produces a series of beneficial metabolic effects, including resistance to weight gain, protection of pancreatic and liver function, and maintenance of glucose homeostasis. The latest research has found that central administration of bile acids or specific TGR5 receptor agonists can reduce body weight and fat mass. This effect involves regulating food intake and energy expenditure through the sympathetic nervous system. On the contrary, downregulation of hypothalamic TGR5 gene expression in the basal region of the hypothalamus is conducive to the development of obesity. This study reveals that the hypothalamic TGR5 signaling pathway is a key mediator to offset diet-induced obesity.

[0043] The following is a more detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings and examples, so that the scheme of the present invention and its advantages in various aspects can be better understood. However, the specific embodiments and examples described below are only for the purpose of illustration, rather than for limiting the present invention.

[0044] It is particularly important to point out that similar substitutions and modifications made to the present invention are obvious to those skilled in the art, and they are all considered to be included in the present invention. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] Secondly, it should be noted that all unspecified concentrations involved in the present invention are by volume percentage (v / v). Unless otherwise specified, all percentages, ratios, proportions or parts are by weight. In addition, if the specific conditions are not specified in the present invention, they are all carried out according to conventional conditions or conditions recommended by the manufacturer, and the raw materials or excipients used, as well as the reagents or instruments used, whose manufacturers are not specified, are all conventional products that can be obtained through commercial purchase.

[0046] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the present invention. The present invention uses Hypericum perforatum medicinal material as raw material, and the Hypericum perforatum medicinal material can be Hypericum perforatum medicinal material known to those skilled in the art, and the present invention has no particular limitation on this.

[0047] Preparation of compounds

[0048] 1. Medicinal materials and reagents

[0049] Except for acetonitrile used in liquid chromatography, which was of chromatographic grade, all other reagents were of analytical grade. Hypericum perforatum was purchased from Bozhou, Anhui.

[0050] 2. Embodiment 1

[0051] 10 kg of Hypericum perforatum was crushed, and then 10 times the amount of 95% ethanol was added for reflux extraction twice, each time for 2 hours, filtered, and the filtrate was used to recover the ethanol and concentrated to obtain an extract. The extract was mixed with water, extracted with dichloromethane, and the dichloromethane extract was decolorized by MCI column, eluted with 95% ethanol aqueous solution, and the eluate was collected and concentrated. The concentrate was separated by silica gel column chromatography. Before loading on the column, 1.5 times the volume of the concentrated solution was dissolved in methanol, and then 3 times the weight of the concentrated solution was added with 100-200 mesh silica gel for sample mixing. Gradient elution was performed with petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1). One fraction was received for every 500 ml. The components of petroleum ether-ethyl acetate (10:1, 5:1) were collected. After reduced pressure concentration, the components were separated and purified by MCI column chromatography. Gradient elution was performed with 40% to 100% methanol-water solution. The components separated by 80% to 90% methanol-water solution were collected and concentrated. The components were separated by medium pressure chromatography (octadecylsilane bonded silica gel, flow rate of 6 mL / min), gradient eluted with 40% to 100% methanol-water solution, receiving one fraction for every 15 ml, and the components separated by 80% to 90% methanol-water solution were collected and concentrated, and then purified by Sephadex LH-20 gel column, eluted with petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction for every 5 ml, and concentrated under reduced pressure, and then purified by semi-preparative liquid phase (octadecylsilane bonded silica gel, flow rate of 4 mL / min, detection wavelength of 210 nm), eluted with 80% acetonitrile aqueous solution, and after the eluent was treated, 12 mg of the compound represented by formula (I) was obtained.

[0052] 3. Embodiment 2

[0053] 15 kg of Hypericum perforatum was crushed, and then 15 times the amount of 95% ethanol was added for reflux extraction 3 times, each time for 1 hour, filtered, and the filtrate was used to recover the ethanol and concentrated to obtain an extract. The extract was mixed with water, extracted with dichloromethane, and the dichloromethane extract was decolorized by MCI column, eluted with 95% ethanol aqueous solution, and the eluate was collected and concentrated. The concentrate is subjected to silica gel column chromatography, and before loading on the column, 2 times the volume of dichloromethane of the obtained concentrate is used to dissolve it, and then 3 times the weight of the concentrate is added with 200-300 mesh silica gel for sample mixing, and gradient elution is performed with petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1), and one fraction is received every 500 ml, and components of petroleum ether-ethyl acetate (10:1, 5:1) are collected. After reduced pressure concentration, the components are separated and purified by MCI column chromatography, and gradient elution is performed with 40%-100% methanol-water solution, and components separated by 80%-90% methanol-water solution are collected and concentrated. The component was separated by medium pressure chromatography (octadecylsilane bonded silica gel, flow rate of 5 mL / min), gradient eluted with 40% to 100% methanol-water solution, receiving one fraction for every 15 ml, and the components separated by 80% to 90% methanol-water solution were collected and concentrated, and then purified by Sephadex G-15 gel column, eluted with petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction for every 5 ml, and concentrated under reduced pressure, and then purified by semi-preparative liquid phase (octadecylsilane bonded silica gel, flow rate of 4 mL / min, detection wavelength of 210 nm), eluted with 85% acetonitrile aqueous solution, and after the eluent was treated, 13 mg of the compound represented by formula (I) was obtained.

[0054] 4. Embodiment 3

[0055] 5 kg of Hypericum perforatum was crushed, and then 20 times the amount of 95% ethanol was added for reflux extraction twice, each time for 2 hours, filtered, and the filtrate was used to recover the ethanol and concentrated to obtain an extract. The extract was mixed with water, extracted with dichloromethane, and the dichloromethane extract was decolorized by MCI column, eluted with 95% ethanol aqueous solution, and the eluate was collected and concentrated. The concentrate was separated by silica gel column chromatography. Before loading on the column, 3 times the volume of methanol of the obtained concentrate was used to dissolve it, and then 3 times the weight of the concentrate was added with 300-400 mesh silica gel for sample mixing. Gradient elution was performed with petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1). One fraction was received for every 500 ml. The components of petroleum ether-ethyl acetate (10:1, 5:1) were collected. After reduced pressure concentration, the components were separated and purified by MCI column chromatography. Gradient elution was performed with 40% to 100% methanol-water solution. The components separated by 80% to 90% methanol-water solution were collected and concentrated. The component was separated by medium pressure chromatography (octadecylsilane bonded silica gel, flow rate of 7 mL / min), gradient eluted with 40% to 100% methanol-water solution, receiving one fraction for every 15 ml, and the components separated by 80% to 90% methanol-water solution were collected and concentrated, and then purified by Sephadex LH-20 gel column, eluted with petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction for every 5 ml, and concentrated under reduced pressure, and then purified by semi-preparative liquid phase (octadecylsilane bonded silica gel, flow rate of 4 mL / min, detection wavelength of 210 nm), eluted with 90% acetonitrile aqueous solution, and after the eluent was treated, 10 mg of the compound represented by formula (I) was obtained.

[0056] Structural identification of compounds

[0057] The compounds obtained in Examples 1-3 were all colorless prismatic crystals. 1 HNMR, 13 CNMR, DEPT-135, HSQC, HMBC, 1 H- 1 The structures of the finished products obtained in Examples 1-3 were identified by H COSY, NOESY spectroscopy and X-ray single crystal copper-palladium diffraction experiments.

[0058] Figure 1 is the ESI-MS spectrum of the compound obtained in Example 1, Figure 2 The compound prepared in Example 1 1 H NMR spectrum, Figure 3 The compound prepared in Example 1 13 C NMR spectrum, Figure 4 DEPT-135 diagram of the compound prepared in Example 1, Figure 5is the HSQC spectrum of the compound obtained in Example 1, Figure 6 is the HMBC spectrum of the compound obtained in Example 1, Figure 7 The compound prepared in Example 1 1 H- 1 H COSY spectrum, Figure 8 is the NOESY spectrum of the compound obtained in Example 1, Fig. 9 This is the single crystal diffraction pattern of the compound prepared in Example 1.

[0059] HR-ESI-MS (positive) gave m / z 447.3110 [M+H] + (The theoretical calculated value is 447.3105), and then the molecular formula of the compound is determined to be C 27 H 42 O5, unsaturation is 7.

[0060] 1 The HNMR (600MHz, CD3OD) spectrum showed that the compound had 9 methyl singlet signals in the high field region: δ0.98 (3H, s, H-20), 1.06 (3H, s, H-23), 1.07 (3H, s, H-14), 1.26 (3H, s, H-27), 1.27 (3H, s, H-12), 1.27 (3H, s, H-22), 1.62 (3H, br.s, H-19), 1.68 (3H, br.s, H-26), 1.74 (3H, br.s, H-18), a proton signal δ4.16 (1H, dd, J=5.8, 10.9 Hz, H-11) appeared in the oxygen-linked zone, and a characteristic olefin proton signal δ5.10 (1H, br.t, J=7.2 Hz, H-16) on an isopentenyl-like fragment was observed in the unsaturated zone. 13 C NMR (150 MHz, CD3OD) spectrum showed 27 carbon signals, which can be classified into one carbonyl carbon signal δ207.3 (C-1) according to the experimental results of DEPT-135, two pairs of sp 2 Hybridized carbon-carbon bond connected carbon signals δ108.5 (C-25), 124.6 (C-16), 134.0 (C-17), 149.8 (C-24), sp 3 There are 22 carbon signals in the hybridized carbon atom region, including 9 methyl signals, 5 methylene signals, 3 methine signals and 5 quaternary carbon signals.

[0061] pass 1 H- 1The related signals of H COSY spectrum can be used to find the structural fragments formed by three proton spin coupling systems: H-10 / H-11 (fragment a), H-3 / H-4 / H-15 / H-16 (fragment b) and H-6 / H-7 / H-8 (fragment c). Through the long-range correlation signals of Me-20 and C-4 / C-5 / C-6 / C-9 in the HMBC spectrum, the above structural fragments b and c can be connected through C-4 / C-5 / C-6. According to the HMBC long-range correlation signals of H-4 and C-2, H-7 and C-9, and H-3 / H-8 and C-1, it is proved that the carbon skeleton core of the compound has a multi-substituted indene ring mother core structure. 1 H- 1 The allylic coupling visible in the H COSY spectrum, combined with the HMBC correlation points of H-18 and C-16 / C-17 / C-19, deduced the existence of the isopentenyl fragment, and proved that it was connected to the C-4 position of the indene ring mother nucleus based on the aforementioned correlation signals. Similarly, the correlation signals of H-14 and C-11 / C-12 / C-13 and H-10 and C-1 / C-2 in the HMBC spectrum proved the oxyisopentyl connected to the C-2 position, the correlation points of H-23 and C-8 / C-21 / C-22 proved the oxyisopropyl connected to the C-8 position, and H-8 and C-24, H-27 and C-24 / C-25 / C-26 indicated the existence of the 2-methyl-propenyl functional group connected to the C-9 position. The main part of the structure of the compound was deduced from this. In the above structural fragments, the chemical shifts of the double bond carbons of C-24 and C-25 are larger than the double bond carbon signals of the usual methyl substitution, respectively, and the quaternary carbon signal of the oxygen-linked region of the ortho-substituent of the indene ring parent core, δ84.0 (C-21), is also in a relatively low-field region. Therefore, it is inferred that C-24 and C-21 are connected through an oxygen bridge. Removing the above structural fragments, according to the calculation of unsaturation, the compound still has a ring structure. At the same time, removing the ketone carbonyl, the 11-position hydroxyl group and the ether oxygen atom between C-24 and C-21, the compound still has two oxygen atoms. Therefore, it is inferred that there is a peroxide bond connection between C-12 and C-2, forming a six-membered peroxide ring structure. In summary, the planar structure of the compound was determined. According to SciFinder, the compound is a PPAP compound with a 6 / 6 / 5 / 5 ring structure and a new skeleton with a rare peroxide group.

[0062] Its relative configuration was mainly deduced from the correlation signals obtained from the NOESY experiment. The NOE correlation between H-11 and H-14 showed that H-11 and Me-14 were located on the same side of the ring plane, which was determined to be β-oriented. The NOESY correlation points of H-20, H-27, and H-15, as well as the correlation signals of H-23 / H-26, confirmed that the substituents at C-4, C-5, C-8, and C-9 of the indene ring parent core of the compound were all oriented in the same direction. The NOESY correlation signals of H-10b (δ1.39) and Me-27 showed that the two were located on the same side of the indene ring, which was β-oriented, that is, the peroxide bond substitution at C-2 was located on the other side of the ring, which was α-oriented.

[0063] By growing a single crystal of the compound, a square crystal of the compound was obtained in an acetone solution of the compound. Using X-ray single crystal copper palladium diffraction experiments, the absolute configuration of the compound was determined to be (2R, 4S, 5R, 8S, 9R, 11S). The structure of the compound was finally determined and named Perforperoxytone A.

[0064] Table 1 NMR data of the compound represented by formula (I) provided by the present invention

[0065]

[0066]

[0067] Note: The test conditions are deuterated methanol. 1 HNMR 600MHz, 13 CNMR 150MHz.

[0068] HR-ESI-MS of Example 2-3, 1 H NMR, 13 C NMR, DEPT-135, HSQC, HMBC, 1 H- 1 The H COSY, NOESY spectra and single crystal diffraction patterns are the same as those in Example 1, indicating that the structural formulas are all as shown in Formula I.

[0069] In vitro TGR5 receptor agonism assay

[0070] 1. Test materials

[0071] 1.1 Cells: HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences and were cultured to the third generation for testing.

[0072] 1.2 Reagents: DMEM medium (Gibco), Opti-MEM medium (Gibco), fetal bovine serum (ThermoFisher Scientific), PBS buffer (Liop3000 transfection reagent), Liop3000 transfection reagent (Thermo Fisher Scientific), DMSO (Sigma), dual-luciferase reporter gene assay kit (Dalian Meilun Biotechnology Co., Ltd.); penicillin-streptomycin solution (Dalian Meilun Biotechnology Co., Ltd.), compound 1 (structural formula is shown in formula (I)).

[0073] 1.3 Instruments and equipment: CO2 incubator (Thermo scientific 3100), inverted microscope (OLYPUS), biological safety cabinet (Thermo Fisher Scientific), multifunctional microplate reader (TECAN), cell counter (Thermo Fisher Scientific), high-speed refrigerated centrifuge (Eppendorf), electronic analytical balance (Sartorius Stedim Biotech), G154DW vertical automatic pressure steam sterilizer (Schneider Electric), plate constant temperature oscillator (Hangzhou Aosheng Instrument Co., Ltd.).

[0074] 2. Test methods

[0075] 2.1 Preparation of test sample: Weigh the compound represented by formula (I) and dissolve it in DMSO to prepare a storage solution with a concentration of 20 mM. When testing, dilute it 1 / 1000 with serum-free DMEM culture medium as the test sample solution.

[0076] 2.2 Cell culture: After HEK293T cells were revived, they were routinely cultured in a 37°C, 5% CO2 incubator using DMEM complete medium containing 10% fetal bovine serum (FBS), 100 mg / L streptomycin and 100 U / L penicillin. The culture medium was replaced every 2 days, and cells in the logarithmic growth phase were used for experiments.

[0077] 2.3 TGR5 signaling pathway activation experiment: HEK293T cells were plated in 96-well cell culture dishes at a cell density of 2×10 cells / well. When the cell density reached 60%-80%, pCMV-TGR5, pCRE and Renilla plasmids were transfected at a ratio of 50:50:1 using Lipo3000 transfection reagent. 8 hours after transfection, the drug group was given 20 μM of compound 1. After 24 hours of treatment, 20 μL of cell lysate was aspirated according to the instructions of the dual luciferase reporter gene assay kit, and the fluorescence value was measured, and the effect of the drug on the TGR5 signaling pathway was analyzed.

[0078] 3. Statistical methods

[0079] Each experiment was repeated three times, and the data were processed using GraphPadprism 8.0.2 statistical software. The data were expressed as mean ± SD. The comparison among the three groups was analyzed by One-way ANOVA in the statistical software. If P < 0.05, the difference was statistically significant.

[0080] 4. Results

[0081] In the in vitro TGR5 signaling pathway activation experiment, compared with the blank group, the drug group had no significant effect on the growth of HEK293T cells at a concentration of 20 μM. Compared with the blank group, the compound represented by formula (I) showed a higher TGR5 receptor agonist efficiency at a concentration of 20 μM. The results are shown in Table 2.

[0082] Table 2 Evaluation of TGR5 receptor agonist activity of compound 1

[0083]

[0084]

[0085] (Note: Compared with the blank group, ***p<0.001, **p≤0.01, *p≤0.05)

[0086] In summary, the compound represented by formula (I) provided by the present invention has significant TGR5 receptor agonist activity and has potential for the development of metabolic disease drugs.

[0087] Pharmacodynamic evaluation in high-fat diet-induced obese mice

[0088] 1. Test materials

[0089] 1.1 Experimental animals: C57BL / 6J male mice, 5 weeks old, SPF grade, were purchased from Shanghai Slake Animal Experiment Co., Ltd. (Shanghai, China).

[0090] 1.2 Reagents: D-glucose (Sigma), physiological saline (Zhejiang Dubang Pharmaceutical Co., Ltd.), oleanolic acid (Shanghai Yuanye Biotechnology Co., Ltd.), compound 1 (structural formula is shown in formula (I)).

[0091] 1.3 Instruments and equipment: Roche Excellence blood glucose meter and blood glucose test strips (Roche), electronic analytical balance (Ohaus International Trading Co., Ltd.), surgical instruments (Suzhou Shiqiang Medical Equipment Co., Ltd.).

[0092] 2. Test methods

[0093] 2.1 Experimental animals and grouping: After 1 week of adaptive feeding, the mice were randomly divided into a normal group (Chow group, n = 10) and a high-fat group (n = 40), and fed with a basic diet and a high-fat diet, respectively. After 8 weeks, the 40 mice in the high-fat group were randomly divided into a high-fat model group (HFD), a low-dose group of compound 1 (PPAP-L group, 1.25 mg / kg), a high-dose group of compound 1 (PPAP-H group, 2.5 mg / kg), and a positive control drug group (OA group, 2.5 mg / kg), with 10 mice in each group. The mice in the normal control group and the experimental group continued to be fed with a normal diet and a high-fat diet for 8 weeks, respectively, and were intraperitoneally injected for 8 weeks.

[0094] 2.2 Body weight detection: The mice were weighed once before administration on the last day of each week and the body weight curve was drawn.

[0095] 2.3 Oral glucose tolerance test (OGTT): Oral glucose tolerance (OGTT) was performed on mice after 8 weeks of drug intervention. Mice were fasted for 14 hours, and after fasting blood glucose was measured, glucose (2 g / kg) was orally administered, and the blood glucose levels of mice were measured at 0, 15, 30, 60 and 120 minutes.

[0096] 3. Statistical methods

[0097] The experimental data were processed using GraphPadprism 8.0.2 statistical software. The data were expressed as mean ± SD. The comparison among the three groups was analyzed using One-way ANOVA in the statistical software. If P < 0.05, the difference was statistically significant.

[0098] 4. Results

[0099] 4.1 Effect of compound 1 on body weight in diet-induced obese mice

[0100] like Fig.10 The body weight changes of mice were monitored during the 8-week administration period, and it was found that the body weight of mice in the high-dose group of compound 1 (PPAP-H group) was significantly lower than that of mice in the HFD group, and the weight was statistically significant.

[0101] 4.2 Effect of compound 1 on oral glucose tolerance (OGTT) in diet-induced obese mice

[0102] like Fig.11 After 8 weeks of drug intervention, we measured the oral glucose tolerance (OGTT) of mice. The results showed that compared with the HFD group mice, the blood glucose regulation function of the mice in the high-dose group of compound 1 (PPAP-H group) was improved, and the area under the OGTT curve was reduced, with significant differences.

[0103] The above experiments show that the compound has the effect of treating obesity, regulating blood sugar and other metabolic diseases.

[0104] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods. For those skilled in the art, several improvements and modifications may be made without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof, wherein the compound has a structure of Formula I:

2. A method for preparing the compound according to claim 1, characterized in that: The steps include: Step (1): adding ethanol aqueous solution to the Hypericum perforatum medicinal material for reflux extraction, and concentrating the extract to obtain an extract; Step (2): mixing the extract obtained in step (1) with water, extracting with dichloromethane, and concentrating the dichloromethane extraction portion to obtain a dichloromethane extract; Step (3): decolorizing the dichloromethane extract obtained in step (2) through an MCI column, eluting with a 95% ethanol aqueous solution, collecting and concentrating the eluate; Step (4): subjecting the concentrated solution obtained in step (3) to silica gel column chromatography, performing gradient elution with a petroleum ether-ethyl acetate mixed solvent, wherein the volume ratio of petroleum ether to ethyl acetate in the gradient elution is (30-1):1, and collecting the components separated at a volume ratio of petroleum ether to ethyl acetate of (10-5):1; Step (5): subjecting the components separated in step (4) to MCI column chromatography for separation and purification, using a 40% to 100% methanol-water solution for gradient elution, and collecting the components separated by 80% to 90% methanol-water solution; Step (6): The components separated in step (5) are loaded on an ODS-C8, C18 or AqC18 column, separated by a medium pressure chromatograph, gradient eluted with a 40% to 100% methanol-water solution, and the components separated by 80% to 90% methanol-water solution are collected; Step (7): separating and purifying the components obtained in step (6) by gel column chromatography and eluting with an organic solvent; Step (8): The eluted fraction obtained in step (7) is purified by semi-preparative liquid phase to obtain the compound represented by formula (I).

3. The preparation method according to claim 2, characterized in that: In the step (1), the weight ratio of Hypericum perforatum to ethanol aqueous solution is 1:(10-20).

4. The preparation method according to claim 2, characterized in that: In the step (4), before loading onto the column, the step also includes dissolving the concentrated solution obtained in step (3) with an organic solvent having a volume of 1.5 to 3 times the weight of the concentrated solution, and then adding 100 to 400 mesh silica gel having a volume of 3 times the weight of the concentrated solution to mix the sample; the organic solvent is dichloromethane or methanol.

5. The preparation method according to claim 2, characterized in that: In the step (6), the chromatographic column used in the medium pressure chromatograph is octadecylsilane bonded silica gel, and the flow rate is 5 to 7 mL / min.

6. The preparation method according to claim 2, characterized in that: In the step (7), the gel used in the gel column chromatography is selected from Sephadex LH-20 or Sephadex G-15; the organic solvent used is a mixed solvent of petroleum ether-dichloromethane-methanol, with a volume ratio of 5:5:

1.

7. The preparation method according to claim 2, characterized in that: In the step (8), the chromatographic column used for the semi-preparative liquid phase is octadecylsilane bonded silica gel, with 80% to 90% acetonitrile aqueous solution as the mobile phase, a flow rate of 4 mL / min, and a detection wavelength of 210 nm.

8. Use of the compound of claim 1 or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite in the preparation of TGR5 receptor agonist drugs.

9. Use of the compound according to claim 1 or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite in the preparation of drugs for treating obesity, diabetes or metabolic diseases.

10. A drug comprising the compound of claim 1 or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof.

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