A compound, its preparation method and application

Abstract

This invention relates to a plant-derived compound, its preparation method, and its applications. The invention involves research on Hypericum perforatum (St. John's wort), yielding a novel active component. Experiments show that this compound can specifically activate the TGR5 receptor and can be developed as a lead compound into a drug for treating obesity and other metabolic diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a plant-derived compound, its preparation method, and its application. Background Technology

[0002] Hypericumpurium L., also known as St. John's wort or St. John's wort, is a plant belonging to the genus Hypericumpurium in the family Clusiaceae. It is widely distributed in Jiangsu, Hebei, Sichuan, Guizhou, Shaanxi, Gansu, and Xinjiang in my country. Hypericumpurium L. has the effects of soothing the liver and relieving depression, clearing heat and promoting diuresis, reducing swelling and promoting lactation. It is mainly used to treat liver qi stagnation, emotional distress, chest tightness, joint swelling and pain, mastitis, and insufficient lactation. Records of its medicinal use can be found as early as the Tang Dynasty medical text, *Tang Materia Medica*. Hypericumpurium L. has a complex chemical composition and broad biological activity, including various structural types such as polycyclic isopentenyl substituted acyl phloroglucinols, dianthrones, xanthones, and flavonoids.

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

[0004] The technical problem to be solved by this invention is to further study the active ingredients of Hypericum perforatum using TGR5 as a target, and to discover its active ingredients.

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

[0006]

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

[0008] Step (1): The Hypericum perforatum herb was extracted by reflux with an ethanol aqueous solution, and the extract was concentrated to obtain an extract; the ethanol aqueous solution is preferably a 75-95% ethanol aqueous solution, more preferably a 95% ethanol aqueous solution;

[0009] Step (2): Mix the extract obtained in step (1) with water, extract with dichloromethane, concentrate the dichloromethane extract to obtain dichloromethane extract;

[0010] Step (3): Decolorize the dichloromethane extract obtained in step (2) using an MCI column, elute with 95% ethanol aqueous solution, collect the eluent and concentrate it;

[0011] Step (4): Separate the concentrate obtained in step (3) by silica gel column chromatography, and perform gradient elution with a petroleum ether-ethyl acetate mixed solvent, wherein the volume ratio of petroleum ether-ethyl acetate in the gradient elution is (30-1):1, and collect the components obtained by separation with a volume ratio of petroleum ether-ethyl acetate of (10-5):1.

[0012] Step (5): The components obtained in step (4) are separated and purified by MCI column chromatography, using a gradient elution of 40% to 100% methanol-water solution, and the components obtained by separating with 80% to 90% methanol-water solution are collected.

[0013] Step (6): The components obtained in step (5) are packed into ODS-C8, C18 or AqC18 columns and separated using a medium-pressure chromatograph. Gradient elution is performed with 40% to 100% methanol-water solution, and the components separated by 80% to 90% methanol-water solution are collected.

[0014] Step (7): The components obtained in step (6) are separated and purified by gel column chromatography and eluted with organic solvents;

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

[0016] Further, in step (1), the weight ratio of St. John's wort to the ethanol aqueous solution is 1:(10-20).

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

[0018] Furthermore, in step (4), before loading the column, the sample is dissolved in an organic solvent at 1.5 to 3 times the weight of the concentrate obtained in step (3), and then mixed with 100 to 200, 200 to 300, or 300 to 400 mesh silica gel at 3 times the weight of the concentrate; 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 5-7 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 petroleum ether-dichloromethane-methanol mixed solvent with a volume ratio of 5:5:1.

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

[0023] This invention also proposes the application of a compound of Formula I or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite in the preparation of a TGR5 receptor agonist drug.

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

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

[0026] Furthermore, the aforementioned drugs also include pharmaceutically acceptable carriers.

[0027] Furthermore, the pharmaceutically acceptable carrier refers to conventional pharmaceutical carriers, such as diluents, excipients, and water; fillers such as starch, sucrose, lactose, and microcrystalline cellulose; binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as hexadecyl alcohol and sodium dodecyl sulfate; adsorbents such as kaolin and soap clay; and lubricants such as talc, calcium and magnesium stearate, micronized silica gel, and polyethylene glycol. Other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0028] Specifically, the drug can be any dosage form as defined in pharmaceutics, 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, injectable dosage forms, or topical dosage forms, preferably tablets, capsules, pills, granules, ointments, mixtures, or suspensions.

[0030] This invention studies Hypericum perforatum and obtains a new active component that has a significant effect on activating the TGR5 receptor, which can be further developed into a drug for treating obesity, diabetes and other metabolic diseases induced by it. Attached Figure Description

[0031] Figure 1 The above is an HR-ESI-MS chromatogram of the compound prepared in Example 1 of this invention;

[0032] Figure 2 The compound prepared in Example 1 of this invention 1 H NMR spectrum;

[0033] Figure 3 The compound prepared in Example 1 of this invention 13 C NMR spectrum;

[0034] Figure 4 The image shows the DEPT-135 of the compound prepared in Example 1 of this invention.

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

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

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

[0038] Figure 8 The NOESY diagram is shown for the compound prepared in Example 1 of this invention.

[0039] Figure 9 The single-crystal diffraction pattern of the compound prepared in Example 1 of this invention;

[0040] Figure 10 The effect of compound 1 on body weight 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);

[0041] Figure 11 The effect of compound 1 on the oral glucose tolerance test (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 Implementation

[0042] The technical problem this invention aims to solve is to further study the active components of Hypericum perforatum using TGR5 as a target, and to discover its active components. Takeda G-protein receptor 5 (TGR5) is a bile acid membrane receptor, also known as G protein-coupled bile acid receptor 1 (GPBAR1) or G protein-coupled receptor 19 (GPCR19), belonging to 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 resisting weight gain, protecting pancreatic and liver function, and maintaining glucose homeostasis. Recent 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. Conversely, downregulation of hypothalamic TGR5 gene expression in the basal region of the hypothalamus is conducive to obesity development. This study reveals that the hypothalamic TGR5 signaling pathway is a key mediator in counteracting diet-induced obesity.

[0043] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and examples, so as to better understand the solution of the present invention and its advantages in various aspects. However, the specific embodiments and examples described below are for illustrative purposes only and are not intended to limit the present invention.

[0044] It should be particularly noted that similar substitutions and modifications made to this invention are obvious to those skilled in the art, and they are all considered to be included in this invention. Those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0045] Secondly, it should be noted that all concentrations not specified in this invention are volume percentages (v / v). Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight. Furthermore, unless specific conditions are specified, this invention is carried out under conventional conditions or conditions recommended by the manufacturer. The active pharmaceutical ingredients or excipients used, as well as the reagents or instruments used, unless the manufacturer is specified, are all commercially available conventional products.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. This invention uses *Hypericum perforatum* medicinal material as raw material; the *Hypericum perforatum* medicinal material may be any *Hypericum perforatum* medicinal material known to those skilled in the art, and this invention does not have any particular limitations in this regard.

[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. The Hypericum perforatum medicinal material was purchased from Bozhou, Anhui Province.

[0050] 2. Example 1

[0051] 10 kg of St. John's wort herb was pulverized and extracted twice with 10 times its volume of 95% ethanol under reflux for 2 hours each time. The extract was filtered, and the ethanol was recovered from the filtrate and concentrated to obtain an extract. This extract was mixed with water and extracted with dichloromethane. The dichloromethane extract was decolorized by MCI column chromatography and eluted with 95% ethanol aqueous solution. The eluent was collected and concentrated. The concentrate was separated by silica gel column chromatography. Before loading the column, the concentrate was dissolved in 1.5 times its volume of methanol and then mixed with 100-200 mesh silica gel at 3 times its weight. The solution was eluted with a gradient of petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1), and one fraction was collected for every 500 ml. The petroleum ether-ethyl acetate (10:1, 5:1) fractions were collected, concentrated under reduced pressure, and then purified by MCI column chromatography with a gradient of 40%-100% methanol-water solution. The fractions separated by the 80%-90% methanol-water solution were collected and concentrated. The component was separated by medium-pressure chromatography (octadecylsilane-bonded silica gel, flow rate 6 mL / min), with gradient elution using 40%–100% methanol-water solution, collecting one fraction every 15 mL. The fraction obtained from the 80%–90% methanol-water solution was collected and concentrated, and then purified by Sephadex LH-20 gel column chromatography using petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction every 5 mL, concentrating under reduced pressure, and then purified by semi-preparative liquid chromatography (octadecylsilane-bonded silica gel, flow rate 4 mL / min, detection wavelength 210 nm), eluting with 80% acetonitrile aqueous solution. After treatment with this eluent, 12 mg of the compound shown in formula (I) was obtained.

[0052] 3. Example 2

[0053] 15 kg of St. John's wort herb was pulverized and extracted three times with 15 times the amount of 95% ethanol under reflux for 1 hour each time. The extract was filtered, and the ethanol was recovered from the filtrate and concentrated to obtain an extract. This extract was mixed with water and extracted with dichloromethane. The dichloromethane extract was decolorized by MCI column extraction and eluted with 95% ethanol aqueous solution. The eluent was collected and concentrated. The concentrate was separated by silica gel column chromatography. Before loading the column, the concentrate was dissolved in dichloromethane at a volume of 2:1, and then mixed with silica gel at a volume of 200-300 mesh at a volume of 3:1 (weight of the concentrate). The solution was eluted with a gradient of petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1), and one fraction was collected for every 500 ml. The petroleum ether-ethyl acetate (10:1, 5:1) fractions were collected, concentrated under reduced pressure, and then purified by MCI column chromatography with a gradient of 40%-100% methanol-water solution. The fractions separated by the 80%-90% methanol-water solution were collected and concentrated. The component was separated by medium-pressure chromatography (octadecylsilane-bonded silica gel, flow rate 5 mL / min), with gradient elution using 40%–100% methanol-water solution, collecting one fraction every 15 mL. The fraction obtained from the 80%–90% methanol-water solution was collected and concentrated, and then purified by Sephadex G-15 gel column chromatography using petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction every 5 mL, concentrating under reduced pressure, and then purified by semi-preparative liquid chromatography (octadecylsilane-bonded silica gel, flow rate 4 mL / min, detection wavelength 210 nm), eluting with 85% acetonitrile aqueous solution. After treatment with this eluent, 13 mg of the compound shown in formula (I) was obtained.

[0054] 4. Example 3

[0055] Five kilograms of St. John's wort herb were pulverized and extracted twice with 20 times the amount of 95% ethanol under reflux for 2 hours each time. The extract was filtered, and the ethanol was recovered from the filtrate and concentrated to obtain an extract. This extract was mixed with water and extracted with dichloromethane. The dichloromethane extract was decolorized by MCI column extraction and eluted with 95% ethanol aqueous solution. The eluent was collected and concentrated. The concentrate was separated by silica gel column chromatography. Before loading the column, the concentrate was dissolved in methanol at a volume of 3 times its weight and then mixed with silica gel at a volume of 300-400 mesh at a weight of 3 times its weight. The solution was eluted with a gradient of petroleum ether-ethyl acetate (30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1), and one fraction was collected for every 500 ml. The petroleum ether-ethyl acetate (10:1, 5:1) fractions were collected, concentrated under reduced pressure, and then purified by MCI column chromatography with a gradient of 40%-100% methanol-water solution. The fractions separated by the 80%-90% methanol-water solution were collected and concentrated. The component was separated by medium-pressure chromatography (octadecylsilane-bonded silica gel, flow rate 7 mL / min), with gradient elution using 40%–100% methanol-water solution, collecting one fraction every 15 mL. The fraction obtained from the 80%–90% methanol-water solution was collected and concentrated, and then purified by Sephadex LH-20 gel column chromatography using petroleum ether-dichloromethane-methanol (5:5:1), collecting one fraction every 5 mL, concentrating under reduced pressure, and then purified by semi-preparative liquid chromatography (octadecylsilane-bonded silica gel, flow rate 4 mL / min, detection wavelength 210 nm), eluting with 90% acetonitrile aqueous solution. After treatment with this eluent, 10 mg of the compound shown in formula (I) was obtained.

[0056] Structural identification of compounds

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

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

[0059] HR-ESI-MS (positive) gives an m / z value of 447.3110 [M+H]. + (The theoretical calculated value is 447.3105), thus determining the molecular formula of the compound to be C. 27 H 42 O5 has an unsaturation degree of 7.

[0060] 1 ¹H NMR (600 MHz, CD3OD) spectra showed nine methyl singlet signals in the high-field region of this compound: δ 0.98 (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, s, H-22), δ 0.98 (3H, s, H-20), δ 1.06 (3H, s, H-23), δ 1.07 (3H, s, H-14), δ 1.26 (3H, s, H-27), δ 1.27 (3H, s, H-12), δ 1 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=5.8,10.9Hz,H-11) appears in the oxygen-bound region, and a characteristic alkene proton signal δ5.10 (1H,br.t,J=7.2Hz,H-16) is visible on an isopentenyl-like fragment in the unsaturated region. 13 The C10 NMR (150 MHz, CD3OD) spectrum shows 27 carbon signals, which, based on the DEPT-135 experimental results, can be classified as a single carbonyl carbon signal δ207.3 (C-1), and two sp2 pairs. 2 The carbon signals of hybrid carbon-carbon bonds are δ108.5 (C-25), 124.6 (C-16), 134.0 (C-17), and 149.8 (C-24), sp. 3 The hybrid carbon atom region has 22 carbon signals, namely 9 methyl signals, 5 methylene signals, 3 methine signals and 5 quaternary carbon signals.

[0061] pass 1 H- 1The correlation signals from the H COSY spectrum revealed three structural fragments formed by proton spin-coupled 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). Long-range correlation signals between Me-20 and C-4 / C-5 / C-6 / C-9 in the HMBC spectrum indicated that fragments b and c were connected via C-4 / C-5 / C-6. Based on the HMBC long-range correlation signals between H-4 and C-2, H-7 and C-9, and H-3 / H-8 and C-1, it was confirmed that the carbon skeleton of this compound possesses a multi-substituted indene ring core structure. The correlation signals between H-16 and H-18, H-19... 1 H- 1 The allylic coupling visible in the HCl COSY spectrum, combined with the HMBC correlation points between H-18 and C-16 / C-17 / C-19, led to the deduction of the presence of an isopentenyl fragment, which, based on the aforementioned correlation signals, was linked to the C-4 position of the indene ring core. Similarly, the correlation signals between H-14 and C-11 / C-12 / C-13 and H-10 and C-1 / C-2 in the HMBC spectrum confirmed the oxoisopentyl group linked to the C-2 position, the correlation point between H-23 and C-8 / C-21 / C-22 confirmed the oxoisopropyl group linked to the C-8 position, and the correlations between H-8 and C-24, and H-27 and C-24 / C-25 / C-26 indicated the presence of a 2-methyl-propenyl functional group linked to the C-9 position. Thus, the main structural component of the compound was derived. In the above structural fragment, the chemical shifts of the double-bonded carbons at C-24 and C-25 show significant shifts to lower and higher fields, respectively, compared to the typical signals of double-bonded carbons substituted with methyl groups. Simultaneously, the quaternary carbon signal δ84.0 (C-21) of the oxygen-linked region of the ortho-substituent in the indene ring core is also located in a relatively low-field region. Therefore, it is inferred that C-24 and C-21 are connected via an oxygen bridge. Excluding the above structural fragment, based on unsaturation calculations, the compound also possesses a ring structure. Furthermore, after removing the ketone carbonyl group, the hydroxyl group at position 11, and the ether oxygen atom between C-24 and C-21, the compound has two remaining oxygen atoms. Therefore, it is inferred that a peroxy bond connects C-12 and C-2, forming a six-membered peroxy ring structure. In summary, the planar structure of this compound has been determined. SciFinder analysis indicates that this compound is a novel PPAP-type compound with a rare peroxy group and a 6 / 6 / 5 / 5 ring structure.

[0062] The relative configurations were mainly derived from the correlation signals obtained by the NOESY experiment. The NOESY correlation between H-11 and H-14 indicates that H-11 and Me-14 are located on the same side of the ring plane, which is defined as β orientation. The NOESY correlation points of H-20 with H-27 and H-15, as well as the correlation signals of H-23 / H-26, confirm that the substituents at C-4, C-5, C-8, and C-9 of the indene ring core of this compound have the same orientation. The NOESY correlation signals of H-10b (δ1.39) and Me-27 indicate that they are located on the same side of the indene ring, which is β orientation, meaning that the peroxide bond substitution at C-2 is located on the other side of the ring, which is α orientation.

[0063] Single-crystal cultivation of this compound yielded quadrangular crystals in an acetone solution. X-ray single-crystal copper-palladium diffraction experiments determined the absolute configuration of the compound to be (2R, 4S, 5R, 8S, 9R, 11S). Based on this, the structure of the compound was finally determined, and it was named Perforperoxytone A.

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

[0065]

[0066]

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

[0068] HR-ESI-MS in Examples 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 in Example 1, indicating that their 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 passaged to the third generation for experiments.

[0072] 1.2 Reagents: DMEM medium (Gibco), Opti-MEM medium (Gibco), fetal bovine serum (Thermo Fisher 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 as shown in formula (I)).

[0073] 1.3 Instruments and Equipment: Carbon dioxide incubator (Thermo Scientific 3100), inverted microscope (OLYPUS), biosafety cabinet (Thermo Fisher Scientific), multi-functional 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 shaker (Hangzhou Aosheng Instrument Co., Ltd.).

[0074] 2. Test methods

[0075] 2.1 Preparation of test sample: Weigh the compound shown in formula (I) and dissolve it in DMSO to prepare a stock 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 resuscitation, HEK293T cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS), 100 mg / L streptomycin and 100 U / L penicillin at 37°C in a 5% CO2 incubator. The culture medium was changed every 2 days, and cells in the logarithmic growth phase were used for experiments.

[0077] 2.3 TGR5 Signaling Pathway Activation Assay: HEK293T cells were seeded at a density of 2 × 10⁶ cells / well in 96-well cell culture dishes. When the cell density reached 60%-80%, pCMV-TGR5, pCRE, and Renilla plasmids were transfected using Lipo3000 transfection reagent at a ratio of 50:50:1. Eight hours after transfection, the treatment group was given 20 μM of compound 1. After 24 hours of treatment, 20 μL of cell lysate was collected according to the instructions of the Dual-Luciferase Reporter Gene Assay Kit for fluorescence measurement, and the effect of the drug on the TGR5 signaling pathway was analyzed.

[0078] 3. Statistical methods

[0079] Each experiment was repeated three times. Data were processed using GraphPadprism 8.0.2 statistical software and expressed as mean ± standard deviation (mean ± SD). One-way ANOVA analysis was used to compare the three groups. A p < 0.05 was considered statistically significant.

[0080] 4. Results

[0081] In in vitro TGR5 signaling pathway activation experiments, the drug-treated group at a concentration of 20 μM had no significant effect on the growth of HEK293T cells compared to the control group. Compared to the control group, the compound shown in formula (I) exhibited 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 control group, ***p<0.001, **p≤0.01, *p≤0.05)

[0086] In summary, the compound shown in formula (I) provided by this invention has significant TGR5 receptor agonist activity and has the potential to be developed into a drug for metabolic diseases.

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

[0088] 1. Test materials

[0089] 1.1 Experimental animals: C57BL / 6J male mice, 5 weeks old, SPF grade, purchased from Shanghai Slack 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 as shown in formula (I)).

[0091] 1.3 Instruments and equipment: Roche Advanced Blood Glucose Meter and Blood Glucose Test Strips, Electronic Analytical Balance (Ohaus International Trading Co., Ltd.), Surgical Instruments (Suzhou Shiqiang Medical Instrument Co., Ltd.).

[0092] 2. Test methods

[0093] 2.1 Experimental Animals and Grouping: After one week of acclimatization, mice were randomly divided into a normal group (Chow group, n=10) and a high-fat group (n=40), fed with basal and high-fat diets, 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 compound 1 group (PPAP-L group, 1.25 mg / kg), a high-dose compound 1 group (PPAP-H group, 2.5 mg / kg), and a positive control group (OA group, 2.5 mg / kg), with 10 mice in each group. Mice in the normal control group and experimental group continued to be fed with normal and high-fat diets, respectively, for 8 weeks, while simultaneously receiving intraperitoneal injections of the compound 1 for 8 weeks.

[0094] 2.2 Weight monitoring: The mice were weighed once on the last day of each week before administration, and a weight curve was plotted.

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

[0096] 3. Statistical methods

[0097] Experimental data were processed using GraphPadprism 8.0.2 statistical software. Data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA analysis was used to compare the three groups. A p < 0.05 was considered statistically significant.

[0098] 4. Results

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

[0100] like Figure 10 During the 8-week administration period, the body weight of mice was monitored, 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 this was statistically significant.

[0101] 4.2 Effect of Compound 1 on Oral Glucose Tolerance Test (OGTT) in Diet-Induced Obese Mice

[0102] like Figure 11 Eight weeks after drug intervention, we measured the oral glucose tolerance test (OGTT) in mice. The results showed that, compared with the HFD group, the high-dose compound 1 group (PPAP-H group) had improved glycemic regulation, a reduced area under the OGTT curve, and the difference was statistically significant.

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

[0104] Obviously, the above embodiments are merely examples for clearly illustrating the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that various improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A compound or its pharmaceutically acceptable salt, tautomer, or stereoisomer, having the structure of Formula I:

2. A method for preparing the compound according to claim 1, characterized in that, Includes the following steps: Step (1): St. John's wort is extracted by reflux with an ethanol aqueous solution. The extract is concentrated to obtain an extract. The weight ratio of St. John's wort to the ethanol aqueous solution is 1:(10-20). Step (2): Mix the extract obtained in step (1) with water, extract with dichloromethane, concentrate the dichloromethane extract to obtain dichloromethane extract; Step (3): Decolorize the dichloromethane extract obtained in step (2) using an MCI column, elute with 95% ethanol aqueous solution, collect the eluent and concentrate it; Step (4): Separate the concentrate obtained in step (3) by silica gel column chromatography, using a 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 collect the components obtained by separation with a petroleum ether-ethyl acetate volume ratio of (10-5):1; wherein, before loading the column, the sample is further mixed by dissolving it in 1.5-3 times the weight of the concentrate obtained in step (3) with organic solvent and then adding 3 times the weight of the concentrate in 100-400 mesh silica gel; wherein the organic solvent is dichloromethane or methanol; Step (5): The components obtained in step (4) are separated and purified by MCI column chromatography, using a gradient elution of 40% to 100% methanol-water solution, and the components obtained by separating with 80% to 90% methanol-water solution are collected. Step (6): The components obtained in step (5) are packed into an ODS-C8, C18 or AqC18 column and separated using a medium-pressure chromatograph. Gradient elution is performed with 40% to 100% methanol-water solution, and the components separated by 80% to 90% methanol-water solution are collected. The chromatographic column used in the medium-pressure chromatograph is octadecylsilane-bonded silica gel, and the flow rate is 5 to 7 mL / min. Step (7): The components obtained in step (6) are separated and purified by gel column chromatography and eluted with an organic solvent; the gel used in the gel column chromatography is selected from Sephadex LH-20 or Sephadex G-15; the organic solvent used is a petroleum ether-dichloromethane-methanol mixed solvent with a volume ratio of 5:5:

1. Step (8): The eluted fraction obtained in step (7) is purified by semi-preparative liquid chromatography to obtain the compound shown in formula (I); the chromatographic column used in the semi-preparative liquid chromatography 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.

3. The use of the compound of claim 1 or its pharmaceutically acceptable salts, tautomers, or stereoisomers in the preparation of TGR5 receptor agonist drugs.

4. The use of the compound of claim 1 or its pharmaceutically acceptable salts, tautomers, or stereoisomers in the preparation of a drug for treating obesity or diabetes.

5. A drug comprising the compound of claim 1 or its pharmaceutically acceptable salt, tautomer, or stereoisomer.

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