Marine bacterium-derived pyrazine compound as well as preparation method and application thereof

Abstract

The invention discloses a pyrazine compound derived from marine bacteria. The structural formula of the pyrazine compound is as shown in a formula A: # imgabs0. The invention discloses the pyrazine compound derived from the marine bacteria as well as a preparation method and application of the pyrazine compound. The pyrazine compound (Vibripyrazine A) is obtained by fermenting a marine bacterium (Vibrio ruber ZXR-93), and the preparation method of the pyrazine compound (Vibripyrazine A) comprises the following steps: fermenting the marine bacterium (Vibrio ruber ZXR-93); marine bacteria have the advantages of being easy to culture, short in growth cycle and the like, and a method for extracting compounds from microorganisms is safe, simple, convenient and environmentally friendly, so that pyrazine compounds are rich in source and low in preparation cost; in addition, the pyrazine compound has a good anti-cancer effect, and provides a new choice and approach for research and development of new anti-tumor drugs.

Description

Technical Field

[0001] The present application relates to the technical field of medicinal chemistry, and particularly relates to a pyrazine compound derived from marine bacteria, a preparation method thereof, and an application thereof. Background Art

[0002] The ocean is a treasure trove of chemical and biological resources. Due to its special characteristics such as high pressure, high salt, low temperature, anoxia, darkness, and oligotrophy, combined with the complex and extensive ecological interactions among biological species brought about by species diversity, marine organisms have unique metabolic pathways and adaptation mechanisms different from those of terrestrial organisms. Therefore, the secondary metabolites they produce often have characteristics such as novel structures, rich varieties, and significant biological activities, and are important sources of new drugs and their lead compounds.

[0003] Marine bacteria reproduce quickly and are easy to culture. This biosynthetic method also has advantages such as simple method, environmental friendliness, and low risk, which lay the foundation for large-scale industrial production in the future. Therefore, the research on extracting new compounds from marine bacterial metabolites is of great significance. The present invention provides a pyrazine-derived compound from marine bacteria, a preparation method thereof, and an application thereof. Summary of the Invention

[0004] Aiming at the disadvantages and deficiencies existing in the prior art, the primary object of the present invention is to provide a pyrazine compound (Vibripyrazine A) derived from marine bacteria.

[0005] Another object of the present invention is to provide a preparation method of the above-mentioned pyrazine compound derived from marine bacteria.

[0006] Another object of the present invention is to provide the application of the above-mentioned pyrazine compound derived from marine bacteria in anti-tumor.

[0007] A pyrazine compound (Vibripyrazine A) derived from marine bacteria, the structural formula of which is shown in Formula A:

[0008]

[0009] The pyrazine compound is isolated from the fermentation broth of marine bacterium Vibrio ruber ZXR-93; the marine bacterium Vibrio ruber ZXR-93 is isolated from the seawater of the coastal area of Xinbu Island, Haikou City, Hainan Province, China. Through ITS rRNA identification, this marine bacterium belongs to the genus Vibrio (Vibrio ruber), so it is named VibrioruberHKZ-93, and this bacterium is preserved in the Key Laboratory of Tropical Molecular Pharmacology and Micro-Nano Diagnosis Innovation, Tropical Medicine College, Hainan Medical College.

[0010] The preparation method of the pyrazine compounds derived from the above-mentioned marine bacteria comprises the following operating steps:

[0011] S1. Inoculate the marine bacterium Vibrio ruber ZXR-93 into a seed culture medium and culture it in a shaker to obtain a seed culture solution;

[0012] S2. Inoculate the seed solution into a fermentation culture medium and culture it in a shaker to obtain a fermentation broth;

[0013] S3. Separate the thalli and the bacterial solution obtained in the culture of S2. Extract the separated thalli with methanol 3 to 5 times and concentrate the extract; extract the separated bacterial solution with ethyl acetate 3 to 5 times and concentrate the extract; combine the collected and concentrated extracts and separate them by silica gel column chromatography; then separate them by silica gel column chromatography and preparative thin layer chromatography to obtain the compound with the structural formula shown in Formula A.

[0014] The present invention has found through research that the IC 50 values of the pyrazine compound Vibripyrazine A against human gastric cancer cell line SGC-7901 and cervical cancer cell line HeLa are 178.8±15.39 and 351.43±77.41 μg / mL respectively, and it can be used for the prevention and treatment of cancer diseases such as gastric cancer and cervical cancer.

[0015] Therefore, the application of the pyrazine compound Vibripyrazine A, or its pharmaceutically acceptable salt, or its prodrug compound in the preparation of anti-tumor drugs is also within the protection scope of the present invention.

[0016] The pharmaceutically acceptable salt of the pyrazine compound is its inorganic acid salt, inorganic base salt, or double salt.

[0017] The acid of the inorganic acid salt can be selected from any one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid, propionic acid, malonic acid, butyric acid, lactic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, maleic acid, benzoic acid, succinic acid, picric acid, tartaric acid, citric acid, fumaric acid.

[0018] The base of the inorganic base salt is selected from any one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, trimethylamine, triethylamine, pyridine.

[0019] The prodrug compound of the pyrazine compound Vibripyrazine A refers to a substance that can be converted into the pyrazine compound Vibripyrazine A or its salt in vivo.

[0020] The present invention further provides an anti-tumor drug comprising the pyrazine compound, or its pharmaceutically acceptable salt, or its prodrug compound.

[0021] Preferably, the drug further comprises pharmaceutically acceptable excipients and is formulated into different dosage forms.

[0022] The pharmaceutically acceptable excipients include, but are not limited to, diluents, lubricants, binders, disintegrants, stabilizers or solvents.

[0023] The diluents include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar or glucose.

[0024] The lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate or poloxamer.

[0025] The binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, sodium alginate or polyvinylpyrrolidone.

[0026] The disintegrants include, but are not limited to, starch effervescent mixtures, namely sodium bicarbonate and citric acid, tartaric acid or low-substituted hydroxypropyl cellulose.

[0027] The stabilizers include, but are not limited to, polysaccharides such as acacia gum, agar, alginic acid, cellulose ethers or carboxymethyl chitin ester.

[0028] The solvents include, but are not limited to, water or balanced salt solutions.

[0029] Preferably, the drug dosage forms include, but are not limited to, injections, oral preparations, sprays, inhalants.

[0030] The injections include, but are not limited to, small volume injections, infusions or freeze-dried powder injections.

[0031] The oral preparations are solid oral preparations or liquid oral preparations. Among them, the solid oral preparations include, but are not limited to, ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills or powders; the liquid oral preparations include, but are not limited to, oral liquids or emulsions.

[0032] The drug can also be a spray or an inhalant. Drugs for inhalation or insufflation include pharmaceutically acceptable aqueous solvents or organic solvents, or their solutions and suspensions, as well as powders. They can be made into aerosol inhalants or sprayed with the aid of equipment.

[0033] Preferably, the pyrazine compound or its pharmaceutically acceptable salt or its prodrug compound in the drug accounts for 1% - 90% of the mass of the drug.

[0034] The pyrazine compound Vibripyrazine A claimed by the present invention, or its pharmaceutically acceptable salt, or its prodrug compound is applied to the preparation of anti-tumor drugs, including but not limited to, using the compound of the present invention, or its pharmaceutically acceptable salt, or its prodrug compound for preventing or treating diseases caused by gastric cancer tumor cells and cervical cancer tumor cells, and the uses of drugs for alleviating disease symptoms or relieving the development or onset of diseases.

[0035] The pyrazine compound Vibripyrazine A claimed by the present invention, or its pharmaceutically acceptable salt, or its prodrug compound can be applied to veterinary treatment of pets, introduced breed animals and farm animals, including mammals, rodents, etc. in addition to being beneficial to human treatment.

[0036] A pyrazine compound derived from marine bacteria of the present invention is isolated from a marine bacterium. Marine bacteria are widely distributed and have a large number in the marine environment. The method for extracting compounds from marine bacteria is simple, making the source of pyrazine compounds rich and the cost low; and this pyrazine compound has strong anti-tumor activity, providing new options and ways for the research and development of new anti-tumor drugs, and having good research and development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 1H NMR spectrum of a pyrazine compound derived from marine bacteria of the present invention;

[0038] Figure 2 13C NMR spectrum of a pyrazine compound derived from marine bacteria of the present invention;

[0039] Figure 3 1H-1H COSY spectrum of a pyrazine compound derived from marine bacteria of the present invention;

[0040] Figure 4 HSQC spectrum of a pyrazine compound derived from marine bacteria of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0041] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0043] Strain background: The marine bacterium Vibrio ruber ZXR-93 involved in the following examples was isolated from seawater on the shore of Bihai Yintan Park, Xinbudao, Haikou City, Hainan Province, China (110°35'496”E, 200°7'776”N). It is a bacterium with antibacterial and anti-tumor activities. Identified by ITS rRNA, this marine bacterium belongs to the genus Vibrio, so it is named Vibrio ruber ZXR-93. This strain has been preserved in the Key Laboratory of Tropical Molecular Pharmacology and Micro-Nano Diagnosis Innovation, Hainan Medical College. The preservation date is March 10, 2022, and the preservation number is ZXR-93. The present invention will be further described in detail below through specific embodiments in conjunction with the drawings.

[0044] Example 1: Isolation and identification of compound A

[0045] In the present invention, compound A was isolated from the culture of the marine bacterium Vibrio ruber ZXR-93 through fermentation culture. The preparation method of the compound A includes the following steps:

[0046] 1. Fermentation of the marine bacterium Vibrio ruber ZXR-93

[0047] S1. Take out the preserved strain of the marine bacterium Vibrio ruber ZXR-93 from the -80°C refrigerator and quickly melt it in a 37°C water bath. Use an inoculation loop to pick up a small amount of bacterial liquid and streak it on a solid seed medium for zoning, and place it in an incubator for cultivation;

[0048] S2. Pick a single colony into the seed liquid medium and obtain a seed culture solution after shaking culture;

[0049] S3. Inoculate the seed liquid into the fermentation medium and obtain a fermentation broth after shaking culture;

[0050] The components of the seed solid medium in step S1 are: peptone 5 g / L, yeast powder 2 g / L, FePO4 0.05 g / L, agar 18 g / L, crude sea salt 35 g / L, adjust the pH to 7.5 - 7.7; the conditions of the incubator in step S1 are: temperature 26 - 28°C, culture time 12 - 24 h.

[0051] The components of the seed liquid medium in step S2 are: peptone 5 g / L, yeast powder 2 g / L, FePO4 0.05 g / L, crude sea salt 35 g / L, adjust the pH to 7.5 - 7.7; the shaking culture conditions in step S2 are: temperature 26 - 28°C, rotation speed 180 - 220 rpm, fermentation time 6 - 12 h.

[0052] In step S3, the components of the fermentation medium are: peptone 5 g / L, yeast powder 2 g / L, maltose 5 g / L, and crude sea salt 35 g / L; the shaking culture conditions in step S3 are: temperature 26 - 28 °C, rotation speed 180 - 220 rpm, and fermentation time 24 - 48 h.

[0053] 2. Crude extraction, separation, purification and identification of compound A

[0054] Separate the bacterial cells from the fermentation broth, extract the separated bacterial cells with methanol 3 - 5 times, and concentrate the extraction solution; extract the separated fermentation broth with ethyl acetate 3 - 5 times, and concentrate the extraction solution; combine the collected and concentrated extracts, and separate them by silica gel column chromatography; use dichloromethane - methanol gradient elution of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, 90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 0:100 respectively, merge similar fractions after detection by thin - layer chromatography; collect the third fraction when eluting with 95:5 dichloromethane - methanol, and then perform preparative thin - layer chromatography separation twice with developing agents of 1:20 methanol - dichloromethane and 5:5:1 ethyl acetate - chloroform - n - butanol respectively to obtain compound A (Vibripyrazine A).

[0055] Perform structural test and analysis on the obtained compound A, and obtain the following test data:

[0056] Compound A: C 12 H 18 O3N2, yellow powder, HR ESIMS: 238.2435[M - H] + (Theoretical calculated value 238.1317). The specific NMR data are shown in Table 1.

[0057] Table 1 NMR spectrum of compound A (Vibripyrazine A) (CDOD, 150 MHz / 600 MHz, ppm)

[0058]

[0059] After identification, compound A (Vibripyrazine A) is a new pyrazine compound, and its structural formula is shown as formula A below:

[0060]

[0061] Example 2: Antitumor activity test of compound A

[0062] Use the MTT method to test the antitumor activity of the pyrazine compound of the present invention.

[0063] Preparation materials: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT for short), using 10 mM PBS as the solvent, prepare an MTT solution with a concentration of 5 mg / mL, then filter and sterilize it with a 0.22 μm pore size filter membrane, aliquot and store it in the -20 °C refrigerator in the dark.

[0064] Cell culture medium: Add 10% inactivated (heated at 56 °C for 30 min) calf serum and 1% double antibody (100× penicillin and streptomycin) to RPMI-1640 (Roswell Park Memorial Institute 1640) medium, and mix gently.

[0065] PBS buffer (pH 7.4): 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 0.2 g of KCl, 8 g of NaCl, dissolve in distilled water and make up to 1000 mL, autoclave at 121 °C and 0.1 MPa for 20 min.

[0066] Cell line selection: Human gastric cancer cell line SGC-7901, human cervical cancer cell line HeLa.

[0067] Operation steps:

[0068] Collect two types of tumor cells in the logarithmic growth phase, adjust the cell suspension concentration to 1×10 4 cells / mL after cell counting, add 100 μL to each well of a 96-well plate, add 200 μL of complete medium to the blank control, and culture in a 37 °C, 5% CO2 incubator for 4 h. Dissolve the test sample with chromatographic grade methanol and dilute it with complete medium, add 100 μL of medium with different concentrations of the compound to each well, so that the final concentration in each well is 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL respectively, set three replicates for each concentration, add 100 μL of negative control to complete medium without the sample, and continue to culture in the incubator for 24 h. Add 20 μL of 5 mg / mL MTT solution to each well and continue to culture for 4 h. Aspirate the medium in the wells, add 150 μL of DMSO to each well, shake on a shaker at low speed for 10 min, and then measure the absorbance at a wavelength of 570 nm with an enzyme-linked immunosorbent assay reader. The calculation formula for cell inhibition rate: Inhibition rate = (OD value of the experimental group - OD value of the blank control group) / (OD value of the negative control group - OD value of the blank control group) × 100%. The sample concentration when the inhibition rate is 50% is the IC 50 50, calculate the IC 50 50 value, and the results are expressed as mean ± standard deviation.

[0069] The pyrazine compounds of the present invention show inhibitory effects in the activity tests on two types of cancer cells. Among them, the smaller the IC 50 value, the better the inhibitory effect of the pyrazine compounds of the present invention on the cancer cells. The test results are shown in Table 2.

[0070] Table 2. Inhibitory effect of compound A (Vibripyrazine A) on tumor cells

[0071]

[0072] The above content is a further detailed description of the present invention in combination with specific implementation manners, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can also be made.

Claims

1. A pyrazine compound derived from marine bacteria, characterized in that: Its structural formula is shown in Formula A:

2. The preparation method of the pyrazine compound Vibripyrazine A described in claim 1, characterized in that: S1: Seed culture of the marine bacterium Vibrio ruber ZXR-93: Components of the seed culture medium: peptone 5 g / L, yeast extract 2 g / L, FePO4 0.05 g / L, crude sea salt 35 g / L, adjust the pH to 7.5 - 7.7; pick the strain and inoculate it into the seed culture solution, at a temperature of 26 - 28 °C, a rotation speed of 180 - 220 rpm, and culture for 6 - 12 h. S2: Fermentation culture of the marine bacterium Vibrio ruber ZXR-93: Components of the liquid culture medium used for fermentation culture: peptone 5 g / L, yeast extract 2 g / L, maltose 5 g / L, crude sea salt 35 g / L; inoculate the seed strain obtained from the culture in S1 into the liquid culture medium, at a temperature of 26 - 28 °C, a rotation speed of 220 rpm, and ferment for 24 - 48 h. S3: Crude extraction and separation and purification: Separate the thalli and the bacterial liquid obtained from the culture in S2, extract the separated thalli with methanol 3 - 5 times, and concentrate the extraction solution; extract the separated bacterial liquid with ethyl acetate 3 - 5 times, and concentrate the extraction solution; combine the collected and concentrated extracts, and separate them using silica gel column chromatography; use dichloromethane-methanol gradient elution of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, 90:10, 80:20, 70:30, 60:40, 50:50, 30:70, 0:100 respectively, merge the similar fractions after detection by thin layer chromatography; collect the second component when eluting with 95:5 dichloromethane-methanol, and then perform preparative thin layer chromatography separation twice with developing agents of 1:20 methanol-dichloromethane and 5:5:1 ethyl acetate-chloroform-n-butanol respectively to obtain the compound of Formula A.

3. The application of the pyrazine compound Vibripyrazine A described in claim 1, or its pharmaceutically acceptable salt, or its prodrug compound in the preparation of anti-tumor drugs.

4. The application according to claims 3 to 4, characterized in that The pharmaceutically acceptable salt of the pyrazine compound Vibripyrazine A is its inorganic acid salt, inorganic base salt or double salt.

5. The application according to claims 3 to 4, characterized in that, The prodrug compound of the pyrazine compound Vibripyrazine A refers to a substance that can be converted into the pyrazine compound Vibripyrazine A or its salt in vivo.

6. An anti-tumor drug, characterized in that, Containing the pyrazine compound Vibripyrazine A described in claims 1 - 5, or its pharmaceutically acceptable salt, or its prodrug compound.

7. The drug according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients and is made into different dosage forms.

8. The drug according to claim 7, wherein The pharmaceutically acceptable excipients include diluents, lubricants, binders, disintegrants, stabilizers or solvents.

9. The drug according to claim 7, characterized in that, The dosage forms include injections, oral preparations, sprays or inhalants.

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