CN114315741A

CN114315741A - Thio compound and preparation method and application thereof

Info

Publication number: CN114315741A
Application number: CN202210061429.6A
Authority: CN
Inventors: 许言超; 王立平; 刘�文; 朱伟明
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2022-04-12
Anticipated expiration: 2042-01-19
Also published as: CN114315741B

Abstract

The invention discloses a thio compound, a preparation method and application thereof, wherein the compound has a structure shown in any one of formulas 1-6 and is obtained by separating and purifying a fermentation product of cavernous fungi. The preparation method comprises the steps of adopting the Aspergillus fumigatus GZWMJZ-152, fermenting in a solid state fermentation mode, extracting, and separating and purifying monomers to obtain the thio compound. Pharmacological experiments show that the compounds have antioxidant effect and can be applied to preparation of antioxidant stress pharmaceutical preparations.

Description

Thio compound and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a thio compound, and a preparation method and application thereof.

Background

Sulfur atoms are widely present in polypeptides, proteins and small molecule compounds and have important biological effects. Natural products containing sulfur as a special active compound have been isolated from plants, microorganisms and animals, have a wide range of biological activities, and some compounds have been approved for clinical use, such as tenoxicam, trabectedin, edoxaban, etc. Recently, the isolation of some sulfur-containing compounds with new backbones from secondary metabolites of microorganisms has greatly enriched the structural types of natural products. The cavern microorganisms have special living environments which are not possessed by general soil microorganisms, are free of illumination, moist and relatively nutrient-poor, and have high methane content in the cavern atmosphere, and the extreme environments can influence metabolic pathways of the cavern microorganisms to generate active compounds with novel structures. The key point of the patent is that 6 sulfur-containing compounds with novel structures are obtained by separation and identification in the continuous research of the cavernous fungus Aspergillus fumigatus GZWMJZ-152 and have remarkable antioxidant activity. Oxidative stress is a negative effect produced in vivo by free radicals and is considered to be an important factor in aging and diseases. Therefore, the discovery of the antioxidant active molecules has important significance for resisting oxidative stress diseases.

Disclosure of Invention

The invention aims to extract, separate and purify a thio compound from a cave fungus fermentation product, has an antioxidant effect and can be applied to an antioxidant stress medicament. Provides a thio compound and a preparation method and application thereof. The purpose of the invention and the main technical problem of solving the invention are realized by adopting the following technical scheme: a thio compound having the structure of any one of formulae 1-6:

the preparation method of the compounds 1-6 comprises the following steps:

preparing a strain A: the method comprises the following steps of adding 300 parts by weight of potato 200-25 parts by weight of glucose and 15-20 parts by weight of agar into 1L of purified water, adjusting the pH value to 6.5-7.5, preparing a slope, inoculating mycelium of Aspergillus fumigatus GZWMJZ-152 at normal temperature, and culturing at 28-35 ℃ for 3 days to serve as a strain;

b, inoculation: b, adopting a solid fermentation mode, filling 50-100 parts of rice and 50-100 parts of purified water into each conical flask, inoculating the strain obtained in the step A, and standing and culturing at 28 ℃ for 30 days to obtain a fermentation culture medium;

c, extraction: soaking the fermentation medium in the step B in ethyl acetate for 48-72 hours, stirring and extracting for 3-5 times by using a stirrer, wherein each time is 30-40 minutes, combining the supernate, and recovering ethyl acetate in a rotary evaporator to obtain a crude extract;

d, monomer separation and purification: c, performing column chromatography separation on the crude extract obtained in the step C to obtain compounds 1-6; the chromatographic separation method comprises silica gel column chromatography, gel column chromatography and semi-preparative high performance liquid chromatography.

The thio compound is applied to the drugs for resisting oxidative stress.

A pharmaceutical composition comprising a thio compound having any one of structural formulae 1-6 and a pharmaceutically acceptable adjuvant.

The pharmaceutical composition contains 0.1-99% of thio compound with any one of organic structure formulas 1-6 by mass fraction, and the balance of pharmaceutical carrier or excipient.

The inventor carries out more systematic research on the fermentation product in the aspects of extraction, separation and purification in the continuous research on the cavernous fungus Fumigatus GZWMJZ-152, and obtains 6 novel thio compounds 1-6. The prepared thio compound has an antioxidant effect and can be applied to an antioxidant stress medicament.

Drawings

FIG. 1 is a structural formula of compounds 1 to 6 of the present invention

FIG. 2 is a high-resolution mass spectrum of Compound 1 of the present invention

FIG. 3 is a high resolution mass spectrum of Compound 2 of the present invention

FIG. 4 is a high resolution mass spectrum of Compound 3 of the present invention

FIG. 5 is a high resolution mass spectrum of Compound 4 of the present invention

FIG. 6 is a high resolution mass spectrum of Compound 5 of the present invention

FIG. 7 is a high resolution mass spectrum of Compound 6 of the present invention

FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of Compound 1 of the present invention

FIG. 9 is a nuclear magnetic resonance hydrogen spectrum of Compound 2 of the present invention

FIG. 10 is a nuclear magnetic resonance hydrogen spectrum of Compound 3 of the present invention

FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of Compound 4 of the present invention

FIG. 12 is a nuclear magnetic resonance hydrogen spectrum of Compound 5 of the present invention

FIG. 13 is a NMR spectrum of Compound 6 of the present invention

FIG. 14 is a nuclear magnetic resonance carbon spectrum of Compound 1 of the present invention

FIG. 15 is a nuclear magnetic resonance carbon spectrum of Compound 2 of the present invention

FIG. 16 is a nuclear magnetic resonance carbon spectrum of Compound 3 of the present invention

FIG. 17 is a NMR carbon spectrum of Compound 4 of the present invention

FIG. 18 is a nuclear magnetic resonance carbon spectrum of Compound 5 of the present invention

FIG. 19 is a NMR carbon spectrum of Compound 6 of the present invention

Detailed Description

Example 1:

preparation of Compound 1-6

(1) Preparation of the Strain

The purification medium used had the following composition: 200 parts of potato, 20 parts of glucose, 20 parts of agar and 1L of purified water. Sterilizing at high temperature, making into slant, inoculating mycelia of Aspergillus fumigatus GZWMJZ-152, and standing at 28 deg.C for culturing when there is no bacteria growing.

(2) Inoculation of

Preparing 100 conical bottles by adopting a solid fermentation mode, filling 100 parts of rice and 100 parts of purified water into each conical bottle, sterilizing at 121 ℃ for 30min at high temperature, inoculating the strain, and standing and culturing for 30 days at 28 ℃.

(3) Extraction of

Soaking the fermented liquid and mycelium in ethyl acetate for 48 hr, and extracting with stirrer for 30min three times. After standing, the supernatant was combined and put in a rotary evaporator to recover ethyl acetate, and 620.1 g of crude extract was obtained.

(4) Separation and purification of monomers

Dissolving the crude extract, mixing with 100-mesh 200-mesh silica gel, performing silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate (0-100%) and dichloromethane/methanol (0-50%), detecting by TLC, and mixing the same components to obtain 7 components. And (3) performing silica gel column chromatography on the component 3, performing gradient elution (25-50%) by using petroleum ether/dichloromethane, detecting by TLC, and combining the same components to obtain 5 components. Fractions 3-3 were subjected to gel column chromatography (methanol column), TLC detection, and the same fractions were combined to give 3 fractions, and fractions 3-3-2 were separated by semi-preparative high performance liquid chromatography (60% methanol/water) to give compound 2(10.8 mg). And (4) performing silica gel column chromatography, performing dichloromethane/methanol gradient elution (0-60%), performing TLC detection, and combining the same components to obtain 7 components. Fraction 4-2 was subjected to gel column chromatography (methanol column), TLC detection, and the same fractions were combined to give 7 fractions, and fractions 4-2-7 were separated by semi-preparative high performance liquid chromatography (65% methanol/water) to give compound 3(5.2mg) and compound 4(4.5 mg). Fractions 4-7 were subjected to gel column chromatography (methanol column), TLC detection, and the same fractions were combined to give 10 fractions, and fractions 4-7-10 were separated by semi-preparative high performance liquid chromatography (45% methanol/water, 0.15% trifluoroacetic acid) to give compound 5(9.6mg) and compound 6(3.2 mg). Fraction 7 was subjected to gel column chromatography (methanol column), TLC detection, and the same fractions were combined to give 4 fractions, and 7-4 fractions were separated by semi-preparative high performance liquid chromatography (55% methanol/water) to give compound 1(14.2 mg).

Structure identification of (di) Compounds 1-6

The structure of the compound 1-6 is determined by comprehensively analyzing data such as high-resolution mass spectrum, ultraviolet spectrum, infrared spectrum, optical rotation, nuclear magnetic resonance and the like, and the physicochemical properties are as follows:

compound 1: a light yellow powder; molecular formula C₂₃H₂₄O₉N₂S; a molecular weight of 504; UV (MeOH) lambda_max(logε)207(3.29),288(2.75)nm；IR(KBr)ν_max 3356,2357,2335,1739,1692,1632,1580,1436,1344,1261,1213,1105,1089,1024,821,669cm^-1；HRESIMS m/z 527.1098[M+Na]⁺(calculated for C₂₃H₂₄O₉N₂NaS,527.1095)；[α]_D ²⁰60.3(c 0.7), and the NMR hydrogen spectrum and NMR carbon spectrum data are shown in Table 1.

Compound 2: light yellow crystals; molecular formula C₁₉H₂₀O₈S; a molecular weight of 408; UV (MeOH) lambda_max(logε)220(3.22),290(2.95)nm；IR(KBr)ν_max 3421,2357,2341,1725,1625,1592,1454,1367,1336,1284,1110,1094,1025,965,821,667cm^-1；HRESIMS m/z 407.0807[M–H]^–(calculated for C₁₉H₁₉O₈S,407.0795), whose nmr hydrogen spectrum and nmr carbon spectrum data are shown in table 1.

Compound 3:colorless crystals; molecular formula C₂₁H₂₂O₈S; a molecular weight 434; UV (MeOH) lambda_max(logε)202(3.81),287(3.31),350(2.94)nm；IR(KBr)ν_max 3436,2357,2339,1719,1629,1584,1461,1367,1309,1270,1220,1193,1112,1085,1037,987,835,667cm^-1；HRESIMS m/z 457.0925[M+Na]⁺(calculated for C₂₁H₂₂O₈NaS,457.0928), the NMR hydrogen spectrum and NMR carbon spectrum data are shown in Table 1.

Compound 4: colorless crystals; molecular formula C₂₁H₂₂O₉S; a molecular weight of 450; UV (MeOH) lambda_max(logε)208(3.64),288(3.14)nm；IR(KBr)ν_max 3127,2357,2342,1724,1669,1633,1585,1436,1367,1321,1220,1151,1110,1093,1022,927,825,669cm^-1；HRESIMS m/z 473.0879[M+Na]⁺(calculated for C₂₁H₂₂O₉NaS,473.0877), the NMR hydrogen spectrum and NMR carbon spectrum data are shown in Table 1.

Compound 5: colorless crystals; molecular formula C₉H₉O₃NS; a molecular weight of 211; UV (MeOH) lambda_max(logε)215(3.12),230(3.07),280(2.71)nm；IR(KBr)ν_max 3270,1587,1494,1449,1429,1342,1276,1115,1071,1005,919,901,830,749cm^-1；HRESIMS m/z 210.0217[M-H]^-(calculated for C₉H₈O₃NS,210.0219), whose nmr hydrogen spectrum and nmr carbon spectrum data are shown in table 2.

Compound 6: a white powder; molecular formula C₁₀H₁₁O₄NS; a molecular weight of 241; UV (MeOH) lambda_max(logε)220(3.11),230(3.09),285(2.78)nm；IR(KBr)ν_max 3362,1593,1494,1461,1432,1352,1217,1183,1127,1083,1054,1006,869,756cm^-1；HRESIMS m/z 240.0330[M-H]^-(calculated for C₁₀H₁₀O₄NS,240.0325), whose nmr hydrogen spectrum and nmr carbon spectrum data are shown in table 2.

TABLE 1 NMR data (DMSO-d) for Compounds 1-4₆)

TABLE 2 NMR hydrogen and carbon spectra data (DMSO-d) for Compounds 5 and 6₆)

Example 2

To further verify the beneficial effects of the synthesized compounds of the present invention, the compounds 1-6 were tested for antioxidant activity, and the specific experiments were as follows:

this experiment used the DPPH method to test the compound for its free radical scavenging ability, the ORAC method to test the compound's antioxidant capacity index, and the ability of the compound to protect against oxidative damage on PC12 cells.

The DPPH method comprises the following steps: a DPPH solution a (0.15mmol/L) and a sample solution b were prepared using methanol as a solvent. Set "sample control wells" (160 μ Lb +40 μ L methanol), "negative control wells" (160 μ L methanol +40 μ L a), "sample + DPPH wells" (160 μ Lb +40 μ L a), positive control Vit C; the OD value is measured by a microplate reader under the wavelength of 517nm, and the calculation formula of the clearance rate is as follows: clearance rate [ OD ]_{Negative control}-(OD_{Sample + DPPH}-OD_{Sample controls})]/OD_{Negative control}×100％

The ORAC method comprises the following steps: the main solutions used in the experiment comprise 0.153 mol/L2, 2' -azobisisobutylamidine hydrochloride solution (AAPH), 81.6nmol/L fluorescein solution (FL), 5-series concentration positive control drug Trolox (concentration 50, 25, 12.5, 6.25, 3.125 mu mol/L) and samples to be tested, the solvents are all dissolved by phosphate buffer solution (PBS buffer solution) with concentration 75mmol/LpH being 7.4, the test method is that in a 96-well plate, a negative control group, a blank control group, a positive control group and samples to be tested are arranged in the experimentAnd (3) groups, each group comprises 3 parallel samples, 25 mu L of PBS buffer solution, Trolox and a sample to be detected are correspondingly added into each group, 150 mu L of FL is added into each hole, the 96-hole plate after sample addition is placed into an incubator at 37 ℃, incubation is carried out for 10min, the samples are taken out, 25 mu L of PBS buffer solution is added into a negative control group, 25 mu L of AAPH is added into the other groups, the groups are immediately placed into a multifunctional microplate reader to detect fluorescence, the excitation wavelength of the instrument is set to be 485nm, the emission wavelength of the instrument is 530nm, detection is carried out for 1 time per minute, and the detection time is about 1 h. The ratio of the measured fluorescence value to the initial fluorescence value when the relative fluorescence intensity f is equal to the initial fluorescence value is calculated by the approximate integration method using the relative fluorescence intensity, and the area under the fluorescence decay curve (AUC) is calculated. The formula is as follows: AUC 0.5+ f1+. fi +. f59+0.5 × f60, where f_iRepresents the relative fluorescence intensity measured at time i min. The net area netAUC is the difference between the sample AUC and the blank AUC. The regression equation is made by the time t of Trolox fluorescence measurement to netAUC, Y: (_netAUC)＝aX(_μM) + b. The final ORAC value was calculated using the regression equation between Trolox concentration and net AUC and expressed as Trolox equivalent per micromolar sample in μmol TE/μmol. The formula is as follows:

relative ORAC value ═ AUC [ ("AUC")_{Sample (I)}-AUC_{Blank space})/(AUC_Trolox-AUC_{Blank space})](Trolox molarity. mu. mol/L)/(sample molarity. mu. mol/L)

The test method for the oxidative damage protection of the PC12 cells comprises the following steps: preparing cell suspension from PC-12 cells with culture medium containing 10% fetal calf serum, inoculating 100 μ L of 1 × 10 cells to 96-well plate⁵Cells in/mL, at 5% CO₂Preculture was carried out at 37 ℃ for 24 h. Dissolving the sample by DMSO, diluting the sample (10 mu M) by a basal medium (RPMI-1640), sucking out the old medium, adding 100 mu L of sample solution into each well, and setting 3 multiple wells for each concentration, wherein the final concentration of each well is 10 mu M; placing in an incubator for 16 h. The old medium was aspirated, 100. mu.L of basal medium (RPMI-1640) was added to each well of the drug-toxic well, 100. mu.L of hydrogen peroxide solution (5. mu.M) was added to each well of the oxidative stress well, and 5% CO was added at 37 ℃ to₂The cultivation was continued for 4 h. Then, the old medium was aspirated, and 100. mu.L of CCK-8 solution diluted ten times with DMEM was added directly to each well at 37 ℃ with 5% CO₂The cultivation was continued for 2h (operation in the dark). Using enzyme-linked immunosorbent assayAbsorbance at 450nm was measured and the results were recorded, with + -SD. The results of the antioxidant experiments are shown in Table 3

TABLE 3 antioxidant Activity (IC) of Compounds 1-6₅₀)

^aThe cell survival rate of the hydrogen peroxide injury model group is 46.91 +/-3.74%

These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

Claims

1. A thio compound characterized in that it has a structure represented by any one of formulas 1 to 6

2. The thio compound according to claim 1, wherein any one of the compounds 1 to 6 is isolated and purified from the fermentation product of Aspergillus fumigatus GZWMJZ-152.

3. The thio compound according to claim 1, wherein the process for the preparation of any one of the compounds 1 to 6 comprises the following steps:

4. Use of a thio compound as claimed in any one of claims 1 to 3 in a medicament for combating oxidative stress.

5. A pharmaceutical composition comprising the thio compound of any one of claims 1 to 3 and a pharmaceutically acceptable adjuvant.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition comprises the thio compound according to any one of claims 1 to 3 in an amount of 0.1 to 99% by weight, with the remainder being pharmaceutically acceptable carriers or excipients.