CN114349767B - Sulfur-containing compound and use thereof - Google Patents

Abstract

The invention discloses a compound, which has the activity of resisting staphylococcus aureus, escherichia coli and mycobacterium. The compound has the following structural formula:

Description

Sulfur-containing compound and use thereof

Technical Field

The invention belongs to the technical field of compound preparation.

Background

Since penicillin was discovered, human beings have obtained many important natural products, such as erythromycin, streptomycin, rifamycin, and the like, from secondary metabolites of microorganisms, which are important sources of drug production, and have contributed greatly to the health industry of human beings.

Among the drugs currently on the market, more than 120 important drugs come from microorganisms, including penicillin, cyclosporin a, doxorubicin, etc., and especially in anti-infective and antitumor drugs, the proportion of microbial drugs exceeds 50%.

The ocean is a high-salt, oligotrophic, even low-temperature, high-pressure and non-lighting environment, the particularity of the ecological environment causes the biosynthesis pathway and enzyme reaction system of secondary metabolites generated by ocean microorganisms to have great difference compared with terrestrial microorganisms, endows the ocean microorganisms with unique metabolic pathway, causes some novel strains exclusive to the ocean and the generation of ocean drug lead compounds with peculiar and novel chemical structures and remarkable diversity of biological activity, and provides a large amount of strain resources, mode structures and drug precursors for the research and development of new drugs. Therefore, in order to open up new medicine sources, all countries in the world move to the "dark blue march" and search new medicine resources from marine microorganisms.

Disclosure of Invention

The invention aims to provide a compound, which has the following structural formula:

the invention also provides application of the compound in preparing anti-mycobacterial medicaments.

The medicament may further include pharmaceutically acceptable diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, synergists, additives, solvents, and the like. In the preparation of the antibacterial agent, an effective dose of the compound may be mixed with pharmaceutically acceptable diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, synergists, additives, solvents, etc. to prepare various pharmaceutical preparations. The medicine can be in the form of oral preparation such as tablet, capsule, soft capsule, powder, granule, fine granule, liquid, pill, emulsion or suspension, or non-oral preparation such as injection (such as powder, water solution or oil) suppository, ointment, plaster, patch, spray, tincture or eye drop. These preparations can be obtained by conventional methods well known to those skilled in the art. The administration route can be oral, transdermal, intravenous or intramuscular injection.

Drawings

FIG. 1 is a phylogenetic tree.

FIG. 2 is a mass spectrum of a compound.

FIG. 3 shows compounds dissolved in DMSO-d6 ¹ H-NMR spectrum.

FIG. 4 shows compounds dissolved in DMSO-d6 ¹³ C-NMR spectrum.

Detailed Description

The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Mycobacterium tuberculosis (ATCC 27294)

Example 1

Isolation and identification of Strain BTBU20218885

1. Isolation of Strain BTBU20218885

Taking 1g of sea mud sample (collected from coastal sea mud of China Fujian Xiamen in 2018 in 10 months), putting the sea mud sample into a 50ml centrifugal tube filled with 9ml of sterile water, and shaking the sea mud sample for 2 hours at 20KHz and 100W power ultrasonic for 2min and 200rpm; taking 1ml of the suspension, putting the suspension into a 50ml centrifuge tube filled with 9ml of sterile water, and fully shaking and uniformly mixing; taking 1ml of the suspension, putting the suspension into a 50ml centrifuge tube filled with 9ml of sterile water, and fully shaking and uniformly mixing; and taking 1ml of suspension, putting the suspension into a 50ml centrifuge tube filled with 9ml of sterile water, fully shaking and uniformly mixing, placing at 60 ℃ for 1 hour, taking 0.2ml of suspension, and coating the suspension on a strain isolation culture medium to obtain a strain, wherein the strain is named as strain BTBU20218885.

The components of the strain isolation culture medium are as follows (all percentage by mass): 2 percent of soluble starch, 0.05 percent of L-aspartyl and KNO ₃ 0.1％、K ₂ HPO ₄ ·H ₂ O 0.05％、NaCl 0.05％、MgSO ₄ ·7H ₂ O 0.05％、CaCO ₃ 0.1%, agar 2% and water, pH7.2-7.5.

2. Identification of Strain BTBU20218885

The strain BTBU20218885 grows for 10 days at 28 ℃ on a slant culture medium, the coding sequence of 16S rRNA of the strain BTBU20218885 is shown as a sequence 1 (SEQ ID NO. 1) in a sequence table, and sequence information is submitted to an NCBI database for sequence alignment. The similarity of the coding sequence of the 16S rRNA of the strain BTBU20218885 with the model strain Streptomyces purpureus NBRC 13927 (T) was 98.68%. The 16S rRNA sequences obtained were subjected to multiple sequence alignment using CLSSTAL W sequence analysis software, and phylogenetic trees were generated using the neighbor joining method in MEGA8.0 software, as shown in FIG. 1 (default values were set to 1000).

According to the morphology of the colonies and the results of the sequence alignment, the strain BTBU20218885 belongs to Streptomyces (Streptomyces sp.). The strain BTBU20218885 has been deposited at 11.5.2021 in China general microbiological culture Collection center (CGMCC, address: no. 3 Xilu 1 Beijing, chaoyang, north Chen) with a collection number of CGMCC No.23712. Streptomyces sp. BTBU20218885 No.23712 is abbreviated as Streptomyces BTBU20218885.

Example 2

Compound produced and prepared by streptomycete BTBU20218885

1. Preparation of seed liquid

1. Streptomyces BTBU20218885 is streaked and inoculated on a plate culture medium, and cultured for 7 days at 28 ℃ (aerial hypha grow well) to obtain a plate strain.

The preparation method of the plate culture medium comprises the following steps: 4 g of yeast extract, 10g of malt extract, 4 g of glucose and 20g of agar powder are dissolved in water, the pH is adjusted to 7.0-7.2, the volume is adjusted to 1L with water, and the mixture is sterilized at 115 ℃ for 30 minutes.

2. Sub-packaging the seed culture medium in 500ml Erlenmeyer flask (100 ml/bottle), inoculating slant culture to the seed culture medium, performing shake culture at 28 deg.C and 200rpm for 5 days to obtain OD _600nm Seed liquid of = 1.2-1.4.

The preparation method of the seed culture medium comprises the following steps: 4 g of yeast extract, 10g of malt extract and 4 g of glucose were dissolved in water, the pH was adjusted to 7.0 to 7.2, the volume was adjusted to 1L with water, and sterilization was performed at 115 ℃ for 30 minutes.

2. Fermentation of

5ml of the seed solution prepared in the first step was added to 100ml of the fermentation medium, and cultured at 28 ℃ for 10 days with shaking at 200 rpm.

The preparation method of the fermentation medium comprises the following steps: 10g of glucose, 20g of millet powder (millet meal), 20g of medical media, 20g of MOPS, 1L of distilled water, adjusting the pH to 7.0-7.5, diluting to 1L with water, and sterilizing at 115 ℃ for 30 minutes.

3. Separation and purification of compounds

1. And (4) centrifuging the fermentation system obtained in the step two at 20 ℃ and 10000rpm for 10 minutes, and respectively collecting supernatants.

2. Taking the supernatant obtained in the step 1, extracting with ethyl acetate, standing until a water phase and an ethyl acetate phase are layered, and collecting the ethyl acetate phase on the upper layer; extracting the rest water phase with ethyl acetate again, standing until the water phase and the ethyl acetate phase are layered, and collecting the ethyl acetate phase on the upper layer; extracting the residual water phase with ethyl acetate again, standing until the water phase and the ethyl acetate phase are layered, and collecting the ethyl acetate phase on the upper layer; and (4) combining ethyl acetate phases obtained by three times of extraction, and distilling under reduced pressure to remove the organic solvent to obtain a crude product.

3. The crude product was dissolved in ethyl acetate, and the organic solvent was removed by distillation under reduced pressure to obtain a sample.

4. The sample was redissolved with methanol, filtered to remove insoluble material and subjected to reverse phase high performance liquid chromatography.

Conditions of reverse phase high performance liquid chromatography: an Agilent Eclipse XDB C-18 reverse phase chromatography column (9.4X 250 mm) was used; the mobile phase is acetonitrile or a mixture of acetonitrile and water; the elution time was 10min and the flow rate was 3.0 ml/min; during elution, the volume percentage of acetonitrile in the mobile phase increased linearly from 20% to 90%; the detection wavelength was 254 nm.

And collecting the eluent of the peak with the retention time of 4.67min, and evaporating to dryness under reduced pressure to obtain the compound.

4. Structural characterization of the Compounds

1. Mass spectrometry

The mass spectrometry adopts high-resolution electrospray ionization mass spectrometry HRESIMS, and methanol is a compound solvent in mass spectrometry detection.

The mass spectrum of the compound is shown in FIG. 2, which shows [ M + H ]] ⁺ Peak 621.0712m/z.

5. Nuclear magnetic resonance spectroscopy

Compounds dissolved in DMSO-d ₆ In (1) ¹ The H-NMR spectrum is shown in FIG. 3.

The compound is dissolved in DMSO-d ₆ In (1) ¹³ The C-NMR spectrum is shown in FIG. 4.

Assignment of Compounds to Table 1

The compound has the following structural formula:

example 3

Detection of the anti-mycobacterial Activity of Compounds

7H9 Medium: 4.7g of Middlebrook 7H9 Broth medium powder (BD Co., USA), 2mL of glycerol, 0.5mL of Tween80, 900mL of water and 100mL of Middlebrook OADC Enrichment (BD Co., USA), were thoroughly mixed and sterilized by filtration through a sterile filter having a pore size of 0.22. Mu.m.

Isoniazid: purchased from Sigma-Aldrich.

Rifampicin: purchased from Sigma-Aldrich.

1. Preparation of bacterial liquid

The Mycobacterium was inoculated into 7H9 medium, cultured with shaking at 37 ℃ and 60rpm until OD _600nm Is 0.50-0.55. Such as Mycobacterium tuberculosis.

2. Preparation of the solutions to be tested

The compound was prepared as a 4mg/mL stock solution in sterile DMSO, and then diluted sequentially in sterile DMSO to give dilutions of 2mg/mL, 1mg/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, and 31.25. Mu.g/mL. The following compounds were used respectively: compound a, compound B, compound C, compound D, compound E, compound F, compound G, and compound J.

The positive control drug was prepared as a 320. Mu.g/mL stock solution in sterile DMSO, and then sequentially diluted with sterile DMSO to give dilutions of 160. Mu.g/mL, 80. Mu.g/mL, 40. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, and 2.5. Mu.g/mL.

The following positive control drugs were used, respectively: isoniazid (as positive control drug in the detection of mycobacterium bovis and mycobacterium smegmatis), rifampicin (as positive control drug in the detection of mycobacterium tuberculosis).

3. Determining the minimum inhibitory concentration of a compound against mycobacteria

1. A96-well cell culture plate was taken and 40. Mu.L of 7H9 medium was added to each well.

2. The 96-well cell culture plate which completes the step 1 is taken and processed in groups as follows:

positive control group (7 wells per positive control drug): respectively adding 2 mu L of the positive control drug diluent with 7 dilutions prepared in the step two;

experimental groups (7 wells per compound): respectively adding 2 mu L of the compound diluent prepared in the second step;

negative control group (7 wells): add 2. Mu.L of sterile DMSO each.

3. And (3) taking a 96-well cell culture plate which finishes the step 2, adding 40 mu L of the bacterial liquid obtained in the step one into each well, culturing for 96 hours at 37 ℃, and observing the growth condition of the mycobacteria in each well: if the well is turbid, the corresponding concentration of compound is indicated to have no antimycobacterial activity; if the wells are clear, the corresponding concentrations of compound are indicated to have anti-mycobacterial activity.

For each compound, the final concentration of the compound (concentration of compound in the added dilution/40) for the well in which growth of mycobacteria is completely inhibited is the minimum inhibitory concentration, MIC value, of the compound against mycobacteria.

The activity of the compound against mycobacteria was 10. Mu.g/ml.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> Sulfur-containing Compound and use thereof

<130> P21054

<141> 2021-12-21

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1379

<212> DNA

<213> Streptomyces sp

<400> 1

agtcgaacga tgaacccact tcggtggggg attagtggcg aacgggtgag taacacgtgg 60

gcaatctgcc cttcactctg ggacaagccc tggaaacggg gtctaatacc ggataacacc 120

ggccttcgca tggaggctgg ttgaaagctc cggcggtgaa ggatgagccc gcggcctatc 180

agcttgttgg tggggtaatg gcccaccaag gcgacgacgg gtagccggcc tgagagggcg 240

accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300

attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg 360

ttgtaaacct ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg 420

gctaactacg tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt 480

gggcgtaaag agctcgtagg cggcttgtca cgtcggatgt gaaagcccgg ggcttaaccc 540

cgggtctgca ttcgatacgg gcaggctaga gtgtggtagg ggagatcgga attcctggtg 600

tagcggtgaa atgcgcagat atcaggagga acaccggtgg cgaaggcgga tctctgggcc 660

attactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 720

cacgccgtaa acgttgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct 780

aacgcattaa gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 840

cgggggcccg cacaagcagc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta 900

ccaaggcttg acatcgcccg gaaagcatca gagatggtgc cccccttgtg gtcgggtgac 960

aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020

gcgcaaccct tgttctgtgt tgccagcatg cccttcgggg tgatggggac tcacaggaga 1080

ctgccggggt caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc 1140

ttgggctgca cacgtgctac aatggccggt acaaagagct gcgatgccgt gaggcggagc 1200

gaatctcaaa aagccggtct cagttcggat tggggtctgc aactcgaccc catgaagtcg 1260

gagttgctag taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca 1320

caccgcccgt cacgtcacga aagtcggtaa cacccgaagc cggtggccca accccttgt 1379

Claims (3)

1. A compound having the formula:

2. use of a compound according to claim 1 for the manufacture of an antimycobacterial medicament.

3. Use of a compound according to claim 2 for the preparation of an antimycobacterial agent, wherein the antimycobacterial activity is 10 μ g/ml.

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