Pregnane compound containing aromatic ring as well as preparation method and application of pregnane compound

Abstract

The invention provides a pregnane compound containing an aromatic ring, and a stereoisomer, a tautomer, a geometric isomer or a pharmaceutically acceptable salt thereof, the structure of the pregnane compound containing the aromatic ring is shown as a formula I: # imgabs0 #, the # imgabs1 # represents a single bond or a double bond, and the # imgabs2 # represents a single bond or a double bond; r1 and R2 are respectively and independently selected from hydrogen, hydroxyl, carbonyl and C1-6 alkyl. The invention also provides an application of the pregnane compound containing the aromatic nucleus in preparation of antibacterial drugs.

Description

Technical Field

[0001] The patent of the present invention relates to the field of biomedicine technology, and in particular refers to a pregnane compound containing an aromatic ring. Background Art

[0002] Antibiotic resistance (AR) affects countries in all regions and income levels, and many antimicrobial drugs are losing their effectiveness due to misuse and overuse of antibiotics.

[0003] Against this backdrop, China has made significant progress in the research and development of antibacterial drugs. For example, the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, has successfully developed and marketed antofloxacin hydrochloride, which is China's first Class 1.1 chemical new drug with a novel chemical structure and independent intellectual property rights. In addition, China has 20 new antibacterial drugs entering the clinical research stage, of which 2 have been marketed and 18 are in different clinical stages.

[0004] However, the global research and development of new antibacterial drugs faces many challenges, including high research and development costs, long investment returns, and the rapid development of drug resistance. According to statistics, on average, only six new clinically meaningful antibacterial drugs are developed every 10 years, and each new drug development costs an average of US$2 billion. These factors have led to a lack of enthusiasm for capital investment, affecting the progress of new drug research and development.

[0005] Therefore, there is an urgent need to develop new antibacterial drugs with good antibacterial activity. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, in the first aspect of the present invention, the present invention provides a pregnane compound containing an aromatic ring, and a stereoisomer, a tautomer, a geometric isomer or a pharmaceutically acceptable salt thereof. The structure of the pregnane compound containing an aromatic ring is shown in Formula I:

[0007]

[0008] in, represents a single bond or a double bond; R1 and R2 are independently selected from hydrogen, hydroxyl, carbonyl and C1-6 alkyl.

[0009] In one or more embodiments of the present invention, the structure of the aromatic ring-containing pregnane compound is as shown in Formula II:

[0010]

[0011] Wherein, R1 and R2 are independently selected from hydrogen, hydroxyl and C1-6 alkyl.

[0012] In one or more embodiments of the present invention, R1 is a hydroxyl group, and R2 is a C1-3 alkyl group, or R1 is a C1-3 alkyl group, and R2 is a hydroxyl group.

[0013] In one or more embodiments of the present invention, the aromatic ring-containing pregnane compound is selected from the following structures:

[0014]

[0015] In a second aspect of the present invention, the present invention provides a method for preparing the above-mentioned aromatic ring-containing pregnane compound, characterized in that it comprises the following steps:

[0016] 1): Cut the soft coral into pieces, extract with acetone by ultrasonic, and concentrate under reduced pressure to obtain a crude acetone extract;

[0017] 2) extracting the crude acetone extract obtained in step 1) with ether until the supernatant is colorless, and concentrating under reduced pressure to obtain ether extract;

[0018] 3): The ether extract obtained in step 2) is subjected to silica gel column, gel column chromatography, and high performance liquid chromatography separation to obtain the aromatic ring-containing pregnane compound.

[0019] In one or more embodiments of the present invention, the soft coral is Scleronephthya gracillimum.

[0020] In one or more embodiments of the present invention, in step 3), the silica gel column is eluted with a mixed solvent of petroleum ether and diethyl ether, and the gel column chromatography is eluted with a mixed solvent of petroleum ether, dichloromethane and methanol.

[0021] In one or more embodiments of the present invention, in step 3), the ether extract obtained in step 2) is subjected to silica gel column chromatography, gradient eluted with a mixed solvent of petroleum ether and diethyl ether, and the eluent is collected to obtain fractions A, B, C, D, E, F, and G; fractions C and D are subjected to gel column chromatography, respectively, and eluted with an eluent composed of petroleum ether, dichloromethane and methanol in a volume ratio of 2:1:1 to obtain subcomponents CB and DD; subcomponent CB is separated and purified by high performance liquid chromatography to obtain compounds sclerogra A and sclerogra D, and subcomponent DD is separated and purified by high performance liquid chromatography to obtain compounds sclerogra B and sclerogra C.

[0022] In one or more embodiments of the present invention, the subcomponent CB obtained in step 3) is separated and purified by high performance liquid chromatography, and the chromatographic column used in the high performance liquid chromatography is Agilent Eclipse XDB-C18, and the mobile phase is composed of acetonitrile and water in a volume ratio of 85:15, and the elution is isocratic; the subcomponent DD obtained in step 3) is separated and purified by high performance liquid chromatography, and the chromatographic column is Agilent Eclipse XDB-C18, and the mobile phase is composed of acetonitrile and water in a volume ratio of 90:10, and the elution isocratic.

[0023] In the third aspect of the present invention, the present invention provides a pharmaceutical composition, comprising the above-mentioned aromatic ring-containing pregnane compound, or the stereoisomer, tautomer, geometric isomer, and pharmaceutically acceptable salt of the above-mentioned aromatic ring-containing pregnane compound.

[0024] In one or more embodiments of the present invention, the pharmaceutical composition further includes pharmaceutical excipients.

[0025] In one or more embodiments of the present invention, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral solutions or injections.

[0026] In the fourth aspect of the present invention, the present invention provides the use of the above-mentioned aromatic ring-containing pregnane compounds, or the stereoisomers, tautomers, geometric isomers, pharmaceutically acceptable salts and / or the above-mentioned pharmaceutical compositions of the above-mentioned aromatic ring-containing pregnane compounds in the preparation of antibacterial drugs.

[0027] In one or more embodiments of the present invention, the antibacterial drugs include antibacterial drugs that inhibit at least one of vancomycin-resistant Enterococcus faecium, multidrug-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Candida albicans, oxytetracycline-resistant Streptococcus parauberis, Streptococcus parauberis, Streptococcus agalactiae, Photorhabdus mermaids, Photorhabdus halophilus, Aeromonas salmonicida, and Lactococcus gesneri.

[0028] The beneficial effects of the present invention are:

[0029] 1. The present invention provides a pregnane compound containing an aromatic ring, which is a new compound and has antibacterial activity.

[0030] 2. The present invention also provides the use of the aromatic ring-containing pregnane compound in the preparation of antibacterial drugs, including antibacterial drugs that inhibit at least one of vancomycin-resistant Enterococcus faecium, multidrug-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Candida albicans, oxytetracycline-resistant Streptococcus parauberis, Streptococcus parauberis, Streptococcus agalactiae, Photobacterium mermaidense, Photobacterium halophilum, Aeromonas salmonicida, and Lactococcus gesneri. The research and development of such drugs will help reduce dependence on existing antibiotics, delay the development of drug resistance, and provide new treatment options for treating drug-resistant infections. At the same time, this will also enhance China's competitiveness in the global pharmaceutical market and promote the innovative development of the domestic pharmaceutical industry. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples, but the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be applied to the present invention.

[0032] Some of the reagents and equipment used in the present invention are shown in Table 1 below:

[0033] Table 1

[0034]

[0035] Example 1 Preparation of aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D

[0036] (1) The soft coral collected from the waters of Ximao Island, Hainan Province, China in 2018 (the soft coral was identified by experts as Scleronephthya gracillimum) was thawed, cut into pieces, and ultrasonically extracted four times with acetone (1 L × 4) (each ultrasonic time was 30 min, and the water temperature was 25-30°C). The extracts were combined and concentrated under reduced pressure to obtain a crude acetone extract. The crude acetone extract was extracted with anhydrous ether until the supernatant was colorless, and then concentrated under reduced pressure to obtain an anhydrous ether extract.

[0037] (2) The anhydrous ether extract was subjected to silica gel column chromatography (200-300 mesh), and gradient elution was performed with organic solvents of petroleum ether and ether (0-100%), and the eluent was collected to obtain 7 fractions (AG); components C and D were subjected to Sephadex LH-20 gel column chromatography, and the volume ratio of petroleum ether: dichloromethane: methanol was 2:1:1 to obtain subcomponents CB and DD, respectively.

[0038] (3) Subcomponent CB was separated and purified by reversed-phase high-performance liquid chromatography to obtain compounds sclerogra A and sclerogra D. The chromatographic conditions were as follows: the chromatographic column was Agilent Eclipse XDB-C18 column (5mm, 9.4×250mm); the mobile phase was acetonitrile: water = 85:15, isocratic elution, flow rate 3mL / min; the detection wavelength was 210nm, the retention time of sclerogra A was 22.0min, and the retention time of sclerogra D was 21.0min; subcomponent DD was separated and purified by reversed-phase high-performance liquid chromatography to obtain compounds sclerogra B and sclerogra C. The chromatographic conditions were as follows: the chromatographic column was Agilent Eclipse XDB-C18 column (5mm, 9.4×250mm); the mobile phase was acetonitrile:water = 90:10, isocratic elution, flow rate 3mL / min; the detection wavelength was 210nm, the retention time of sclerogra B was 15.0min, and the retention time of sclerogra C was 14.2min.

[0039] (4) Through spectral analysis, the chemical structures of the aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C, and sclerogra D were determined to be:

[0040]

[0041] Example 2 Study on the antibacterial activity of aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D, pregna-1,4,20-trien-3-one (abbreviated E)

[0042] 1. Source of strains

[0043] The strains included five human pathogens [Vancomycin-Resistant Enterococcus faecium, Multidrug-Resistant Enterococcus faecium, Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Candida albicans ATCC 76485] and seven fish pathogens [Oxytetracycline-Resistant Streptococcus parauberis, Streptococcus parauberis, Streptococcus agalactiae, Photobacterium damselae, Photobacterium halophilum halotolerans), Aeromonas salmonicida, Lactococcus garvieae], human pathogens were from Ruijin Hospital affiliated to Shanghai Jiaotong University School of Medicine, and fish pathogens were from the Chinese Academy of Tropical Agricultural Sciences. Among them, vancomycin-resistant Enterococcus faecium and multidrug-resistant Enterococcus faecium were resistant strains isolated from the hospital. After antibiotic sensitivity testing, the MIC value of vancomycin-resistant Enterococcus faecium to vancomycin was greater than 256μg / mL. The MIC value of multidrug-resistant Enterococcus faecium to erythromycin and gentamicin was greater than 128μg / mL, the MIC value to ampicillin and tetracycline was 128μg / mL, the MIC value to norfloxacin and quinupristin was 8μg / mL, and the MIC value to teicoplanin was 1μg / mL. Oxytetracycline-resistant Streptococcus parauberis is a resistant bacterium isolated from an aquaculture base. Its MIC value to tetracycline is greater than 24.05 μg / mL, and its MIC value to oxytetracycline is 12.42 μg / mL.

[0044] 2. Experimental Methods

[0045] The MIC value of the test substance was determined by the modified 0.5 McFarland standard method. The test solution was a 5wt% DMSO-dissolved test compound (sclerogra A, sclerogra B, sclerogra C and sclerogra D, pregna-1,4,20-trien-3-one and positive control). The turbidity of the bacterial suspension was measured at 600nm, and the culture medium was adjusted to fit the 0.5 McFarland standard. In a 96-well plate, 190μL of bacterial culture and 10μL of test solution were inoculated into each well, incubated at 37°C for 12h, and the MIC value was determined, n=3. The blank control group was added with 10μL of the compound's solvent carrier (DMSO) and 190μL of bacterial culture to exclude that the solvent carrier had no significant effect on bacterial growth.

[0046] 3. Experimental Results

[0047] As shown in Table 1, the aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D showed inhibitory activity against 12 strains of bacteria, with minimum inhibitory concentrations (MIC) ranging from 1.9 to 29.6 μg / mL. The minimum inhibitory concentrations of the aromatic ring-containing pregnane compounds sclerogra A (abbreviated as A), sclerogra B (abbreviated as B), sclerogra C (abbreviated as C) and sclerogra D (abbreviated as D), and the compound pregna-1,4,20-trien-3-one (abbreviated as E) are shown in Table 2. The positive controls were TC: tetracycline; OT: oxytetracycline hydrochloride; LF: levofloxacin hydrochloride; AMP: ampicillin sodium; VAN: vancomycin hydrochloride.

[0048] The structural formula of pregna-1,4,20-trien-3-one (abbreviated as E) is as follows:

[0049]

[0050] Table 2 Minimum inhibitory concentration (MIC) of aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D (μg / mL)

[0051]

[0052] TC: tetracycline; OT: oxytetracycline; LF: levofloxacin; AMP: ampicillin; VAN: vancomycin; '-': negative result at a concentration of 100 μΜ; NT: not tested.

[0053] From the above data results, it can be seen that the aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D have broad-spectrum antibacterial effects, and the compounds are effective against drug-resistant infections, so they can reduce dependence on existing antibiotics.

[0054] In summary, the aromatic ring-containing pregnane compounds sclerogra A, sclerogra B, sclerogra C and sclerogra D have the potential to be developed into new antibacterial drugs, and it is worthy of further in-depth research to develop the compounds into antibacterial drugs or lead compounds.

[0055] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, which shall all be included in the scope of protection of the present invention.

Claims

1. A pregnane compound containing an aromatic ring, and its stereoisomers, tautomers, geometric isomers or pharmaceutically acceptable salts, characterized in that: The structure of the aromatic ring-containing pregnane compound is shown in Formula I: in, represents a single bond or a double bond; R1 and R2 are independently selected from hydrogen, hydroxyl, carbonyl and C1-6 alkyl.

2. The aromatic ring-containing pregnane compound according to claim 1, characterized in that: The structure of the aromatic ring-containing pregnane compound is shown in Formula II: Wherein, R1 and R2 are independently selected from hydrogen, hydroxyl and C1-6 alkyl.

3. The aromatic ring-containing pregnane compound according to claim 1, characterized in that: R1 is hydroxyl, R2 is C1-3 alkyl, or R1 is C1-3 alkyl, R2 is hydroxyl.

4. The aromatic ring-containing pregnane compound according to claim 1, characterized in that: The aromatic ring-containing pregnane compound is selected from the following structures:

5. A method for preparing an aromatic ring-containing pregnane compound according to any one of claims 1 to 4, characterized in that: The steps include: 1): Cut the soft coral into pieces, extract with acetone by ultrasonic, and concentrate under reduced pressure to obtain a crude acetone extract; 2) extracting the crude acetone extract obtained in step 1) with ether until the supernatant is colorless, and concentrating under reduced pressure to obtain ether extract; 3): The ether extract obtained in step 2) is subjected to silica gel column, gel column chromatography, and high performance liquid chromatography separation to obtain the aromatic ring-containing pregnane compound.

6. The method for preparing the aromatic ring-containing pregnane compound according to claim 5, characterized in that: In the step 3), the silica gel column is eluted with a mixed solvent of petroleum ether and ether, and the gel column chromatography is eluted with a mixed solvent of petroleum ether, dichloromethane and methanol.

7. A pharmaceutical composition, characterized in that The invention comprises the aromatic ring-containing pregnane compound according to any one of claims 1 to 4, or its stereoisomers, tautomers, geometric isomers, pharmaceutically acceptable salts and pharmaceutical excipients.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition is tablet, capsule, granule, oral solution or injection.

9. Use of the aromatic ring-containing pregnane compound according to any one of claims 1 to 4, or its stereoisomers, tautomers, geometric isomers, pharmaceutically acceptable salts and / or the pharmaceutical composition according to claim 7 or 8 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drugs include antibacterial drugs that inhibit at least one of vancomycin-resistant Enterococcus faecium, multidrug-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Candida albicans, oxytetracycline-resistant Streptococcus parauberis, Streptococcus parauberis, Streptococcus agalactiae, Photobacterium mermanii, Photobacterium halophilum, Aeromonas salmonicida, and Lactococcus gesneri.

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