Application of Naphthoquinone Compounds Derived from Intestines

Abstract

The present invention provides a class of naphthoquinone compounds FF-C1-FF-C3 derived from intestinal fungi for preparing a ceramide synthase inhibitor, or for preparing a drug for treating and / or preventing steatohepatitis, or for preparing a drug for treating and / or preventing metabolic dysfunction and / or its complications associated with steatohepatitis, or for preparing a drug with hepatoprotective effect. The inhibitor and drug prepared by the naphthoquinone compounds of the present invention have no obvious toxic and side effects and have a significant therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of compound applications, and particularly to the applications of naphthoquinone compounds. Background Art

[0002] Metabolic diseases are a group of metabolic disorder syndromes with complex manifestations, including obesity, diabetes, cardiovascular and cerebrovascular diseases, and metabolic dysfunctions related to steatohepatitis. With the changes in nutritional conditions and lifestyles, the incidence of metabolic diseases has gradually increased in the past decade. Among the chronic diseases that account for more than 80% of the current global disease burden, metabolic diseases already account for more than half, posing a serious threat to the lives and health of patients and causing a great psychological and economic burden on their families.

[0003] As a bridge connecting the internal and external environments of the human body, the imbalance of the gut microbiota is closely related to the occurrence of various metabolic diseases. In recent years, many studies have reported that gut microbiota regulate the occurrence and development of host metabolic diseases through various mechanisms. The intestinal FXR signaling pathway in type 2 diabetes patients is activated, inducing insulin resistance by promoting the production of ceramide. Metformin targets the Bacteroides fragilis-bile acid GUDCA-intestinal FXR axis, improves insulin sensitivity, and effectively ameliorates metabolic diseases. Research by the National Cancer Institute of the United States indicates that the gut microecology improves the metabolic regulation pathways of obesity, insulin resistance, and fatty liver through fermentation products. More complex regulatory pathways, such as hyocholic acid HCA, upregulate the production and secretion of intestinal endocrine cell GLP-1 by simultaneously activating the G protein-coupled receptor TGR5 and inhibiting FXR, mediating the intestine-liver interaction, and improving metabolic diseases. Therefore, it is of great scientific and social significance to identify the new functions and action mechanisms of the gut microbiota, develop novel targets, and search for new treatment strategies for metabolic diseases.

[0004] Ceramide synthase (CERS) is a key enzyme in lipid metabolism that can catalyze the formation of ceramide from sphingosine and fatty acids. Ceramide is an important component of cell membranes and is also involved in various signal transduction processes, affecting cell growth, differentiation, apoptosis, and inflammatory responses. Therefore, ceramide synthase plays a crucial role in maintaining the normal physiological functions of organisms. Ceramide synthase has become an important direction in drug research as a key target for disease treatment. Specific inhibitors targeting ceramide synthase 6 can significantly reduce the activation of T cells, reduce asthma-related inflammatory responses, and significantly improve the lung function of asthma patients. In a study on an obese mouse model, regulating the expression of ceramide synthase 1 can significantly reduce adipocyte accumulation and improve the metabolic situation. The effects and mechanisms of action of targeting ceramide synthase in more metabolic diseases need to be analyzed.

[0005] FusariumFusarium spp. ) is an intestinal symbiotic fungus isolated from feces. It has been found in this application that its fermentation extract can inhibit the activity of ceramide synthase in mice, improve hepatocyte injury and inflammation, and improve lipid metabolism. Based on activity-based metabolite separation, a new class of naphthoquinone compounds has been identified, and their role in metabolic diseases has not been reported in the literature. It is a potential therapeutic drug for anti-metabolic diseases. Summary of the Invention

[0006] The present invention provides a naphthoquinone compound derived from intestinal fungi and its medicinal salts as a ceramide synthase inhibitor, or for preparing a drug for treating and / or preventing steatohepatitis, or for preparing a drug for treating and / or preventing metabolic dysfunction and / or its complications associated with steatohepatitis, or for preparing a drug with hepatoprotective effects.

[0007] To this end, in the first aspect of the present invention, there is provided an application of a naphthoquinone compound FF-C1, FF-C2, FF-C3 or its medicinal salt in any one of the following (1) to (5):

[0008] (1) Preparing a ceramide synthase inhibitor;

[0009] (2) Preparing a drug for treating and / or preventing liver diseases;

[0010] (3) Preparing a drug for treating and / or preventing metabolic dysfunction associated with liver diseases;

[0011] (4) Preparing a drug for treating and / or preventing hyperlipidemia;

[0012] (5) Preparing a drug for treating and / or preventing diabetes or obesity;

[0013] The structural formula of the naphthoquinone compound is shown as follows:

[0014] .

[0015] In the second aspect of the present invention, there is provided an application of a composition in any one of the following (1) to (5), characterized in that the composition comprises at least one of the naphthoquinone compounds FF-C1, FF-C2, FF-C3 and their medicinal salts:

[0016] (1) Preparing a ceramide synthase inhibitor;

[0017] (2) Preparing a drug for treating and / or preventing liver diseases;

[0018] (3) Preparing a drug for treating and / or preventing metabolic dysfunction associated with liver diseases;

[0019] (4) Preparing a drug for treating and / or preventing hyperlipidemia;

[0020] (5) Prepare a medicament for treating and / or preventing diabetes or obesity.

[0021] In the above technical solution, the liver disease is steatohepatitis, preferably, the liver disease is steatohepatitis.

[0022] In the above technical solution, the inhibitor or medicament is an injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream or spray.

[0023] In the above technical solution, the inhibitor or medicament includes one or more pharmaceutically acceptable excipients.

[0024] In the above technical solution, the excipients include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and sustained release agents in the pharmaceutical field.

[0025] In the above technical solution, the inhibitor or medicament is administered by methods such as oral administration, intraluminal administration, injection, spraying, physical or chemical mediation, or is administered after being mixed or encapsulated with other substances.

[0026] The present invention isolates three naphthoquinone compounds from the fermentation extract of Fusarium isolated from the intestine. It is demonstrated in vitro and in vivo that the compounds can effectively inhibit mammalian ceramide synthase and have obvious improvement effects on the liver weight / body weight ratio, liver function, inflammation and liver fibrosis of mice with metabolic dysfunction related to steatohepatitis. It can be used as a ceramide synthase inhibitor and can be used for liver protection; in addition, it also has significant effects in reducing blood sugar and blood lipids. Description of the Drawings

[0027] Figure 1 is the 1 1H NMR spectrum of FF-C1 (acetone- d 6, 600 MHz);

[0028] Figure 2 is the 13 13C NMR spectrum of FF-C1 (acetone- d 6, 125 MHz);

[0029] Figure 3 is the HSQC spectrum of FF-C1 (acetone- d 6);

[0030] Figure 4 is the 1 1H- 1 1H-1H COSY spectrum of FF-C1 (acetone- d 6);

[0031] Figure 5 is the HMBC spectrum of FF-C1 (acetone- d 6);

[0032] Figure 6 is the ECD spectrum of FF-C1;

[0033] Figure 7 is the infrared spectrum of FF-C1:

[0034] Figure 8 is that of FF-C2 1 1H NMR spectrum (CDCl3, 600 MHz);

[0035] Figure 9 is that of FF-C2 13 13C NMR spectrum (CDCl3, 125 MHz);

[0036] Figure 10 is the HSQC spectrum of FF-C2 (CDCl3);

[0037] Figure 11 is that of FF-C2 1 1H- 1 1H COSY spectrum (CDCl3);

[0038] Figure 12 is the HMBC spectrum of FF-C2 (CDCl3);

[0039] Figure 13 is that of FF-C3 1 1H NMR spectrum (CDCl3, 600 MHz);

[0040] Figure 14 is that of FF-C3 13 13C NMR spectrum (CDCl3, 125 MHz);

[0041] Figure 15 is the HSQC spectrum of FF-C3 (CDCl3);

[0042] Figure 16 is that of FF-C3 1 1H- 1 1H COSY spectrum (CDCl3);

[0043] Figure 17 is the HMBC spectrum of FF-C3 (CDCl3). Detailed implementation manners

[0044] The present invention will be described in detail below with reference to embodiments, but the following embodiments should not be construed as limiting the scope of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0046] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial sources.

[0047] Example 1. Preparation of Compounds

[0048] (1) Preparation of an extract from Fusarium sp. isolated from the intestine ( Fusarium spp. )

[0049] Fusarium sp. ( Fusarium spp. ) was pre-activated in a potato dextrose broth (PDB) medium, and after activation, it was inoculated into a rice medium (total amount 40 kg) and cultured at 28 °C for 30 days.

[0050] The medium was extracted three times with 40 L of ethyl acetate, with each extraction being sonicated for one hour. The ethyl acetate was evaporated to dryness using a rotary evaporator to obtain 150 g of an extract, denoted as Fr.

[0051] (2) Preparation of compounds

[0052] The Fr prepared in step (1) was separated by silica gel column chromatography, and gradient elution was carried out using a petroleum ether:ethyl acetate system (volume ratio of petroleum ether to ethyl acetate was 10:1, 4:1, 1:1) and a dichloromethane:methanol system (volume ratio of dichloromethane to methanol was 1:0, 100:1, 20:1, 0:1). Each gradient was washed for 3 retention volumes, with each volume being 500 mL. A total of 7 fractions were obtained, denoted as Fr 1 - 7, and each fraction was freeze-dried.

[0053] Fraction Fr-3 was further separated using an ODS reversed-phase silica gel column. Elution was carried out successively using a petroleum ether:ethyl acetate system (volume ratio of petroleum ether to ethyl acetate was 6:1, 4:1, 2:1, 1:1). Each elution system was washed for 3 retention volumes, with each retention volume being 500 mL. A total of 4 sub-fractions were obtained, denoted as Fr-3-1 - 4, and each fraction was freeze-dried.

[0054] Fraction Fr-3-1 was further separated by HPLC. An acidified water solution (here the acidified water is an aqueous solution of 0.01% trifluoroacetic acid by volume percentage) with 5% - 100% acetonitrile by volume percentage was used as the eluent for HPLC preparation. The gradient change time was 30 minutes, the flow rate was 1 mL / min, and the chromatographic peaks at 19.6, 20.8, and 22.8 min were collected to obtain compounds FF-C1, FF-C2, and FF-C3 respectively.

[0055] The HPLC chromatographic conditions are as follows: The sample is prepared into a 10 mg / mL solution with chromatographically pure methanol, the sample injection volume is 10 μL each time, the chromatographic column is a Kromasil 10×250mm C18 semi-preparative column, the column temperature is 25°C, and detection is carried out at a wavelength of 210 nm.

[0056] The prepared compounds are respectively subjected to nuclear magnetic resonance, infrared, mass spectrometry and other detections (see Figures 1-17 ), and the structures of the compounds are determined by comparing with the literature data and through the structural identification of the compounds.

[0057] The nuclear magnetic resonance spectrometers used are Bruker Mercury-500 and Bruker Mercury-600 MHz (Bruker Spectral Instruments), the infrared chromatograph is Nicolet IS5 FT-IR (Thermo Fisher Scientific), and the mass spectrometers are Bruker APEX III 7.0 T and APEX II FT-ICR (Bruker Spectral Instruments).

[0058] Among them, FF-C1 is a brown powder; +12.0 (c 0.1, CH3OH); UV (CH3Cl) λ max (log ε ) 231 (1.92), 298 (1.71), 482 (1.51) nm; IR (neat) ν max 1664, 1629, 1585, 1407, 1382, 1350, 1240, 1207, 724 cm -1 ; ECD (2.9×10 -3 M) λ max (Δ ε ) 224 (+3.90), 245 (+0.73), 257 (+1.12), 302 (–0.68); positive HRESIMS at m / z 275.0920 [M+H] + (calcd for C 15 H 15 O5 m / z 275.0914).

[0059] Example 2. In vitro assay for the inhibitory activity of compounds against murine ceramide synthase 6

[0060] Accurately weigh 3 compounds prepared in Example 1, and prepare them into 1 mM with DMSO for activity testing (the final concentration ranges from 1 - 100 μM. When preparing, dissolve in a small amount of DMSO and then dilute to the corresponding concentration with distilled water, controlling the final volume fraction of DMSO < 0.1%); when preparing, dissolve in a small amount of DMSO and then dilute to the corresponding concentration with distilled water, controlling the final volume fraction of DMSO < 0.1%).

[0061] The plasmid expressing mouse ceramide synthase 6 was transfected into HEK293 cells. After 24 h, the cells were harvested, sonicated on ice for 5 minutes, and centrifuged at 800 g for 10 min. The protein concentration of the supernatant was determined using the BCA method. The reaction buffer included: 20 mM Hepes (pH 7.4), 25 mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.1% fatty acid - free BSA, and 10 μM dihydrosphingosine. C16:0 - CoA was used as the fatty acyl substrate for CerS6. The tested inhibitors (fermentation extracts or fractions of the strain or compounds shown in Formula 1 - 3) were added respectively, and then 2.5 - 25 μg of lysed protein was added for the reaction. The enzyme activity was measured at 37 °C for 30 min, stopped with 400 μL of chloroform and methanol (volume ratio 2:1), and the internal standard was 1 μM of LM6002. Oscillate at room temperature for 20 min, centrifuge at 13000 rpm for 20 min, transfer the lower layer liquid to a new tube and dry it, and dissolve it with 100 μL of methanol and isopropanol (volume ratio 4:1). The detection of ceramide was performed using a liquid chromatography - tandem mass spectrometry (LC - MS / MS) system. Chromatographic separation was carried out using ACQUITY CSH C18 (2.1×100 mm, 1.7 μm, Waters), at a temperature of 40 °C and a flow rate of 0.2 mL / min. The injection volume was 5 μL. Mobile phase A was 0.4% FA (70% water + 30% methanol), and mobile phase B was 0.4% FA (70% methanol + 30% isopropanol). Chromatographic separation was carried out using a linear gradient: 0.00 - 0.50 min, 40% B; 0.50 - 1.50 min, 40 - 60% B; 1.50 - 6.00 min, 60 - 80% B; 6.00 - 8.00 minutes, 80 - 90% B; 8.00 - 9.50 minutes, 90 - 95% B; 9.50 - 16.00 min, 95% - 98% B; 16.00 - 17.00, 98 - 40% B; LC - MS / MS operation control and quantitative analysis were performed using Analyst version 1.7.3.

[0062] Statistical analysis was performed on the experimental data, and the enzyme activity inhibition rates of each tested sample were calculated as shown in Table 1. It can be seen that the shown compounds all have certain inhibitory activities against mouse ceramide synthase 6.

[0063] Table 1 Detection Results of the Inhibitory Activity of Compounds on Mouse Ceramide Synthase 6

[0064]

[0065] Example 3: Effects of Compounds on Liver Weight / Body Weight Ratio, Liver Function, Inflammation and Liver Fibrosis in Mice with Metabolic Dysfunction Associated with Steatohepatitis Fed a High-Fat Diet

[0066] Materials: Accurately weigh 3 compounds prepared in Example 1, and prepare a 10 mg / mL solution with DMSO for activity testing. C57BL / 6J Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The temperature was 20 - 24 °C, the humidity was kept at 50 - 60%, the light was on for 12 hours (8:00 - 20:00), the environment was soundproof, and the mice had free access to food and water. After adapting to the environment for one week, the experiment was carried out. The detection kits for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Zhong Sheng Bei Kong) were purchased from Beijing Boyu Zhongkang Trading Co., Ltd.

[0067] Methods: C57BL / 6J Six 16-week-old C57BL / mice with a body weight of 28 - 30 g were used as the normal control group (Group 1). Male hepatitis model mice were induced with a choline-deficient, amino acid-defined and high-fat diet (CDAA-HFD). Eight-week-old C57BL / 6J mice were fed a CDAA-HFD diet for 5 weeks. After successful modeling, they were continued to be fed a CDAA-HFD diet for 3 weeks and were simultaneously divided into 4 groups: (2) the PBS administration group, i.e., the model group, (3) the FF-C1 administration group (10 mg / kg), (4) the FF-C2 administration group (10 mg / kg), (5) the FF-C3 administration group (10 mg / kg), with 6 mice in each group. The normal control group was given an equal amount of PBS for 3 consecutive weeks. One week after the last administration, blood was taken and centrifuged at 3000 rpm at 4 °C to measure the liver weight, body weight, alanine aminotransferase and aspartate aminotransferase of mice in each group. The results are shown in Tables 2 - 6.

[0068] Table 2 Effects of Compounds on the Body Weight of CDAA-HFD-Induced Hepatitis Mouse Model

[0069]

[0070] Table 3 Effects of Compounds on the Liver Weight of CDAA-HFD-Induced Hepatitis Mouse Model

[0071]

[0072] Table 4 Effects of Compounds on Liver Weight / Body Weight Ratio in CDAA-HFD Induced Hepatitis Mouse Model

[0073]

[0074] Table 5 Effects of Compounds on Alanine Aminotransferase in CDAA-HFD Induced Hepatitis Mouse Model

[0075]

[0076] Table 6 Effects of Compounds on Aspartate Aminotransferase in CDAA-HFD Induced Hepatitis Mouse Model

[0077]

[0078] Note: In Tables 1-6, compared with the control group, P<0.01; compared with the model group, ## P<0.01.

[0079] Example 4: Acute Toxicity Test in Mice

[0080] Materials: 40 C57BL / 6J mice, 6-8 weeks old, weighing 20-22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0081] Methods:

[0082] (1) Preparation of Compounds

[0083] Accurately weigh the 3 compounds prepared in Example 1 and dissolve them in DMSO to prepare a 0.2 g / mL solution.

[0084] (2) Animal Grouping and Administration

[0085] The mice were adaptively fed for one week, fasted at 18:00 on the day before the experiment, and allowed free access to water. During the experiment, the mice were randomly grouped by body weight, with 10 mice in each group. They were administered by gavage, with a dosage of 0.25 mL per 10 g body weight, that is, the dosage was 5 g / kg. The control group was given DMSO by gavage, with a dosage of 0.25 mL per 10 g body weight.

[0086] (3) Evaluation Index

[0087] After administration, observe the general condition of the animals, such as appearance, behavior, response to stimuli, secretions and excretions, and record the occurrence time of abnormal phenomena. Observe whether there is death, record the occurrence time of death, conduct autopsy, and observe whether there are abnormal phenomena.

[0088] In the acute toxicity test, the toxicity grading of gavage in rats and mice is based on LD50 It is divided into five levels: LD 50 1 mg / kg is highly toxic, LD 50 Between 1 - 50 mg / kg is extremely toxic, LD 50 Between 51 - 500 mg / kg is moderately toxic, LD 50 Between 501 - 5000 mg / kg is slightly toxic, and greater than 5000 mg / kg is practically non - toxic.

[0089] Results:

[0090] No obvious abnormal phenomena were found after administration. On the 30th day, the mice were decapitated and autopsied, and no obvious abnormal phenomena were found. The LD of compounds FF - C1, FF - C2, and FF - C3 50 All are greater than 5000 mg / kg, and there are no obvious abnormalities in the autopsy, belonging to "practically non - toxic".

[0091] It should be understood that within the scope of the present invention, each of the above - mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

Claims

1. Use of a naphthoquinone compound FF-C1, FF-C2, FF-C3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing liver disease, wherein the liver disease is fatty hepatitis; the naphthoquinone compound has the following structural formula: 。 2. Use of a composition in the preparation of a medicament for treating and / or preventing liver disease, wherein the liver disease is fatty hepatitis, characterized in that: The composition comprises at least one compound as set forth in claim 1 or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1 or 2, characterized in that: The medicine is in the form of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

4. The use according to claim 1 or 2, characterized in that: The drug comprises one or more pharmaceutically acceptable excipients.

5. The use according to claim 1 or 2, characterized in that: The drug is administered orally, enterally, by injection, spraying, by physical or chemical mediation, or after being mixed or encapsulated with other substances.

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