Application of naphthoquinone compound from intestinal tract

Abstract

The invention provides preparation of a ceramide synthase inhibitor from naphthoquinone compounds FF-C1-FF-C3 derived from intestinal fungi, or preparation of a medicine for treating and / or preventing fatty degenerative liver diseases, or preparation of a medicine for treating and / or preventing fatty degenerative liver diseases related metabolic dysfunction and / or complications thereof, and application of the naphthoquinone compounds FF-C1-FF-C3 to preparation of a medicine for treating and / or preventing fatty degenerative liver diseases related metabolic dysfunction and / or complications thereof. The inhibitor prepared from the naphthoquinone compound disclosed by the invention is free from obvious toxic and side effects and remarkable in treatment effect.

Description

Technical Field

[0001] The present invention relates to the application field of compounds, and in particular to the application of naphthoquinone compounds. Background Art

[0002] Metabolic diseases are a group of complex metabolic disorders, including obesity, diabetes, cardiovascular and cerebrovascular diseases, and metabolic dysfunction associated with fatty liver disease. With the changes in nutritional conditions and lifestyles, the incidence of metabolic diseases has gradually increased over the past decade. Among the chronic diseases that currently account for more than 80% of the global disease burden, metabolic diseases account for more than half, posing a serious threat to the life and health of patients and placing a great psychological and economic burden on their families.

[0003] As a bridge connecting the internal and external environment of the human body, the imbalance of intestinal flora is closely related to the occurrence of various metabolic diseases. In recent years, many studies have reported that intestinal microorganisms regulate the occurrence and development of host metabolic diseases through various mechanisms. The intestinal FXR signaling pathway is activated in patients with type 2 diabetes, inducing insulin resistance by promoting the production of ceramide. Metformin improves insulin sensitivity and effectively improves metabolic diseases by targeting the intestinal Bacteroides fragilis-bile acid GUDCA-intestinal FXR axis. Research from the National Cancer Center of the United States pointed out that the intestinal microecology improves the metabolic regulation pathway of obesity, insulin resistance and fatty liver through fermentation products. More complex regulatory pathways, such as porcine bile acid HCA, upregulates the production and secretion of GLP-1 in enteroendocrine cells by activating the G protein-coupled receptor TGR5 and inhibiting FXR at the same time, mediates intestinal-liver interactions, and improves metabolic diseases. Therefore, it is of great scientific and social significance to identify new functions and mechanisms of action of intestinal flora, develop novel targets, and find new strategies for the treatment of metabolic diseases.

[0004] Ceramide synthase (CERS) is a key enzyme in the lipid metabolism process that catalyzes sphingosine and fatty acids to produce ceramide. Ceramide is an important component of the cell membrane and is involved in a variety of signal transduction processes, affecting cell growth, differentiation, apoptosis and inflammatory response. Therefore, ceramide synthase plays a vital role in maintaining the normal physiological function of organisms. As a key target for disease treatment, ceramide synthase has become an important direction for drug research. Inhibitors that specifically target ceramide synthase 6 can significantly reduce T cell activation, reduce asthma-related inflammatory responses, and significantly improve lung function in asthma patients. In a study of an obese mouse model, regulating the expression of ceramide synthase 1 can significantly reduce fat cell accumulation and improve metabolism. The effects and mechanisms of ceramide synthase as a target in more metabolic diseases need to be analyzed.

[0005] FusariumFusarium spp. ) is an intestinal symbiotic fungus isolated from feces. The present application found that its fermented extract can inhibit the activity of mouse ceramide synthase, improve liver cell damage and inflammation, and improve lipid metabolism. Based on the separation of active metabolites, a new class of naphthoquinone compounds was identified. Its role in metabolic diseases has not been reported in the literature and 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 a pharmaceutical salt thereof as a ceramide synthase inhibitor, or a drug for preparing a drug for treating and / or preventing fatty liver disease, or a drug for preparing a drug for treating and / or preventing metabolic dysfunction and / or its complications associated with fatty liver disease, or a drug with a liver protective effect.

[0007] To this end, the first aspect of the present invention provides a naphthoquinone compound FF-C1, FF-C2, FF-C3 or a pharmaceutically acceptable salt thereof for use in any one of the following (1) to (5): (1) Preparation of ceramide synthase inhibitors; (2) Preparation of drugs for the treatment and / or prevention of liver diseases; (3) Preparation of drugs for treating and / or preventing metabolic dysfunction associated with liver disease; (4) Preparation of drugs for treating and / or preventing hyperlipidemia; (5) Preparation of drugs for treating and / or preventing diabetes or obesity; The naphthoquinone compound structural formula is as follows: .

[0008] The second aspect of the present invention provides a composition for use in any one of the following (1) to (5), characterized in that the composition comprises at least one of naphthoquinone compounds FF-C1, FF-C2, FF-C3 and pharmaceutically acceptable salts thereof: (1) Preparation of ceramide synthase inhibitors; (2) Preparation of drugs for the treatment and / or prevention of liver diseases; (3) Preparation of drugs for treating and / or preventing metabolic dysfunction associated with liver disease; (4) Preparation of drugs for treating and / or preventing hyperlipidemia; (5) Preparation of drugs for treating and / or preventing diabetes or obesity.

[0009] In the above technical solution, the liver disease is fatty liver disease, preferably, the liver disease is fatty hepatitis.

[0010] In the above technical solution, the inhibitor or drug is in the form of injection, tablet, powder, granule, pill, capsule, oral solution, ointment, cream or spray.

[0011] In the above technical solution, the inhibitor or drug includes one or more pharmaceutically acceptable excipients.

[0012] In the above technical solution, the auxiliary materials include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and sustained-release agents in the pharmaceutical field.

[0013] In the above technical solution, the inhibitor or drug is administered orally, enterally, by injection, spraying, physical or chemical mediation, or is administered after being mixed or encapsulated with other substances.

[0014] The present invention separates three naphthoquinone compounds from a fermentation extract of intestinal Fusarium, and proves in vitro and in vivo that the compounds can effectively inhibit mammalian ceramide synthase, and have a significant improvement effect on the liver weight / body weight ratio, liver function, inflammation and liver fibrosis of mice with metabolic dysfunction associated with fatty liver disease. They can be used as ceramide synthase inhibitors and can be used for liver protection; in addition, they also have significant effects in lowering blood sugar and blood lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is FF-C1 1 H NMR spectrum (acetone- d 6 , 600 MHz); Figure 2 It is FF-C1 13 C NMR spectrum (acetone- d 6 , 125 MHz); Figure 3 This is the HSQC spectrum of FF-C1 (acetone- d 6 ); Figure 4 It is FF-C1 1 H- 1 H COSY spectrum (acetone- d 6 ); Figure 5 This is the HMBC spectrum of FF-C1 (acetone- d 6 ); Figure 6 is the ECD spectrum of FF-C1; Figure 7 This is the infrared spectrum of FF-C1: Figure 8 It is FF-C2 1 H NMR spectrum (CDCl 3 , 600 MHz); Fig. 9 It is FF-C2 13 C NMR spectrum (CDCl 3 , 125 MHz); Fig.10 This is the HSQC spectrum of FF-C2 (CDCl 3 ); Fig.11 It is FF-C2 1 H- 1 H COSY spectrum (CDCl 3 ); Fig.12 This is the HMBC spectrum of FF-C2 (CDCl 3 ); Fig.13 It is FF-C3 1 H NMR spectrum (CDCl 3 , 600 MHz); Fig.14 It is FF-C3 13 C NMR spectrum (CDCl 3 , 125 MHz); Fig.15 This is the HSQC spectrum of FF-C3 (CDCl 3 ); Fig.16 It is FF-C3 1 H- 1 H COSY spectrum (CDCl 3 ); Fig.17 This is the HMBC spectrum of FF-C3 (CDCl 3 ). DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with examples, but the following examples should not be construed as limiting the scope of the present invention.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0019] Example 1. Preparation of Compounds (1) Preparation of intestinal Fusarium ( Fusarium spp. ) Extract Fusarium Fusarium spp. ) were pre-activated in potato dextrose broth (PDB) medium and then inoculated into rice medium (total amount 40 kg) and cultured at 28 degrees Celsius for 30 days.

[0020] The culture medium was extracted three times with 40 L of ethyl acetate, each time with ultrasonic treatment for one hour. The ethyl acetate was evaporated with a rotary evaporator to obtain 150 g of extract, which was recorded as Fr.

[0021] (2) Preparation of compounds The Fr prepared in step (1) was separated by silica gel column chromatography, and gradient elution was performed with a petroleum ether: ethyl acetate system (the volume ratio of petroleum ether: ethyl acetate was 10: 1, 4: 1, 1: 1) and a dichloromethane: methanol system (the volume ratio of dichloromethane: methanol was 1: 0, 100: 1, 20: 1, 0: 1), and each gradient elution had 3 retention volumes, each volume was 500 mL. A total of 7 fractions were obtained, recorded as Fr 1-7, and each fraction was freeze-dried.

[0022] Fraction Fr-3 was further separated using an ODS reverse phase silica gel column. The fraction was eluted sequentially using a petroleum ether: ethyl acetate system (volume ratio of petroleum ether: ethyl acetate was 6:1, 4:1, 2:1, 1:1), and each elution system was eluted with 3 retention volumes, each retention volume was 500 mL. A total of 4 sub-fractions were obtained, recorded as Fr-3-1~4, and each fraction was freeze-dried.

[0023] The fraction Fr-3-1 was further separated by HPLC. The HPLC preparation was carried out using 5% to 100% by volume acetonitrile in acid water (the acid water here is an aqueous solution with a volume percentage of 0.01% trifluoroacetic acid) as the eluent, 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.

[0024] The above HPLC chromatographic conditions are as follows: the sample is prepared into a 10 mg / mL solution with chromatographically pure methanol, the sample load is 10 μL each time, the chromatographic column is a Kromasil 10×250mm C18 semi-preparative column, the column temperature is 25°C, and the detection is performed at a wavelength of 210nm.

[0025] The prepared compounds were tested by nuclear magnetic resonance, infrared, mass spectrometry, etc. (see Figure 1-17 ), the structure of each compound was determined by comparing it with literature data and identifying the structure of the compound.

[0026] The nuclear magnetic resonance instruments used were Bruker Mercury-500 and Bruker Mercury-600 MHz (Bruker Optics), the infrared chromatograph was Nicolet IS5 FT-IR (Nicolet Instruments, Inc., USA), and the mass spectrometers were Bruker APEX III 7.0 T and APEX II FT-ICR (Bruker Optics).

[0027] Among them, FF-C1 is a brown powder; +12.0 (c 0.1, CH 3 OH); UV (CH 3 Cl) 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 O 5 m / z 275.0914).

[0028] Example 2: In vitro test on the inhibition of mouse ceramide synthase 6 activity by compounds The three compounds prepared in Example 1 were accurately weighed and prepared into 1 mM with DMSO for activity test (the final concentration range was 1-100 μM, and they were dissolved in a small amount of DMSO during preparation and diluted with distilled water to the corresponding concentration, and the final volume fraction of DMSO was controlled to be <0.1%). When preparing, they were dissolved in a small amount of DMSO and diluted with distilled water to the corresponding concentration, and the final volume fraction of DMSO was controlled to be <0.1%).

[0029] The plasmid expressing mouse ceramide synthase 6 was transfected into HEK293 cells. After 24 hours, the cells were harvested, sonicated on ice for 5 minutes, and centrifuged at 800 g for 10 minutes. 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 MgCl 2 , 0.5 mM dithiothreitol, 0.1% fatty acid-free BSA and 10 μM dihydrosphingosine. C16:0-CoA is used as the fatty acyl substrate of CerS6. The inhibitors tested (strain fermentation extracts or fractions or compounds shown in formula 1-3) were added respectively, and then 2.5-25 μg of lysate protein was added for reaction. The enzyme activity was measured at 37°C for 30 minutes, stopped with 400 μL chloroform and methanol (volume ratio of 2:1), and the internal standard was 1 μM LM6002. Oscillate at room temperature for 20 minutes, centrifuge at 13000 rpm for 20 minutes, transfer the lower layer of liquid to a new tube and dry it, and dissolve it with 100 μL methanol and isopropanol (volume ratio of 4:1). Ceramide was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) system, and chromatographic separation was performed by ACQUITYCSH C18 (2.1×100 mm, 1.7 μm, Waters), temperature was 40°C, flow rate was 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). A linear gradient was used for chromatographic separation: 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 min, 80-90% B; 8.00-9.50 min, 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.

[0030] The experimental data were statistically analyzed, and the enzyme activity inhibition rate of each test sample was calculated as shown in Table 1. It can be seen that the compounds shown have certain mouse ceramide synthase 6 inhibitory activity.

[0031] Table 1 Results of mouse ceramide synthase 6 inhibitory activity test of the compounds

[0032] Example 3 Effects of Compounds on Liver Weight / Body Weight Ratio, Liver Function, Inflammation and Liver Fibrosis in Mice Fed a High-Fat Diet and Suffering from Metabolic Dysfunction Associated with Steatotic Liver Disease Materials: Accurately weigh the three compounds prepared in Example 1 and prepare them into 10 mg / mL solutions with DMSO for activity testing. 6J Mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with a temperature of 20-24 degrees Celsius, constant humidity of 50-60%, light for 12 hours (8:00-20:00), soundproofing, free access to food and water, and experiments were conducted after one week of adaptation. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits (Zhongsheng Beikong) were purchased from Beijing Boyu Zhongkang Trading Co., Ltd.

[0033] Method: C57BL / 6J Six mice, 16 weeks old, weighing 28-30 g, were used as the normal control group (Group 1). Male hepatitis model mice were induced by choline-deficient, amino acid-defined and high-fat diet (CDAA-HFD), 8-week-old C57BL / 6J Mice were fed with CDAA-HFD for 5 weeks. After successful modeling, they were fed with CDAA-HFD for another 3 weeks and divided into 4 groups: (2) PBS group (model group), (3) FF-C1 group (10 mg / kg), (4) FF-C2 group (10 mg / kg), (5) FF-C3 group (10 mg / kg), 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 collected and centrifuged at 3000 rpm at 4 degrees Celsius 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.

[0034] Table 2 Effects of compounds on body weight in CDAA-HFD-induced hepatitis mouse model

[0035] Table 3 Effects of compounds on liver weight in CDAA-HFD-induced hepatitis mouse model

[0036] Table 4 Effects of compounds on liver weight / body weight ratio in CDAA-HFD-induced hepatitis mouse model

[0037] Table 5 Effects of compounds on alanine aminotransferase in CDAA-HFD-induced hepatitis mouse model

[0038] Table 6 Effects of compounds on aspartate aminotransferase in CDAA-HFD-induced hepatitis mouse model

[0039] Note: In Table 1-6, compared with the control group, P<0.01; compared with the model group, ## P<0.01.

[0040] Example 4: Acute toxicity test in mice Materials: 6-8 weeks old C57BL / 6J Mice, 40 in number, weighing 20-22 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0041] method: (1) Preparation of compounds The three compounds prepared in Example 1 were accurately weighed and prepared into 0.2 g / mL solutions with DMSO.

[0042] (2) Animal grouping and drug administration The mice were fed adaptively for one week and fasted from 18:00 the day before the experiment, with free access to water. During the experiment, the mice were randomly divided into groups according to body weight, with 10 mice in each group, and the drug was administered by gavage, with 0.25 mL per 10 g body weight, that is, the dosage was 5 g / kg. The control group was gavaged with DMSO, with 0.25 mL per 10 g body weight.

[0043] (3) Evaluation indicators After administration, observe the general condition of the animals, such as appearance, behavior, response to stimulation, secretions and excretions, and record the time of occurrence of abnormal phenomena. Observe whether there is death, record the time of death, and perform autopsy to observe whether there are any abnormal phenomena.

[0044] In the acute toxicity test, the toxicity classification of rats and mice by oral administration is based on LD 50 Divided into five levels: LD 50 <1 mg / kg is extremely toxic, LD 50 1-50 mg / kg is highly toxic, LD 50 51-500 mg / kg is moderately toxic, LD 50 The toxicity is low between 501-5000 mg / kg and practically non-toxic when it is greater than 5000 mg / kg.

[0045] result: No obvious abnormality was found after administration. The mice were killed by dislocation of the neck on the 30th day. No obvious abnormality was found in the autopsy. LD 50Both are greater than 5000 mg / kg, and there are no obvious abnormalities in the autopsy, belonging to "practically non-toxic".

[0046] It should be understood that within the scope of the present invention, 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 any one of the following (1) to (5): (1) Preparation of ceramide synthase inhibitors; (2) Preparation of drugs for the treatment and / or prevention of liver diseases; (3) Preparation of drugs for treating and / or preventing metabolic dysfunction associated with liver disease; (4) Preparation of drugs for treating and / or preventing hyperlipidemia; (5) Preparation of drugs for treating and / or preventing diabetes or obesity; The naphthoquinone compound structural formula is as follows: 。 2. Use of a composition in any one of the following (1) to (5), characterized in that: The composition comprises at least one compound as shown in claim 1 or a pharmaceutically acceptable salt thereof: (1) Preparation of ceramide synthase inhibitors; (2) Preparation of drugs for the treatment and / or prevention of liver diseases; (3) Preparation of drugs for treating and / or preventing metabolic dysfunction associated with liver disease; (4) Preparation of drugs for treating and / or preventing hyperlipidemia; (5) Preparation of drugs for treating and / or preventing diabetes or obesity.

3. The use according to claim 1 or 2, characterized in that: The liver disease is fatty liver disease.

4. The use according to claim 1 or 2, characterized in that: The liver disease is steatotic hepatitis.

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

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

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

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