CN107746422B - Ergosta-7, 22-diene-3-ketaminothiohydrazone, preparation method thereof and application thereof in preparation of antibacterial drugs - Google Patents

Abstract

The invention discloses an ergosta-7, 22-diene-3-ketal thizone, a preparation method and an application in the preparation of antibacterial drugs. Described ergosta-7, 22-diene-3-ketal thione ketal is a kind of new ergosteroid natural product derivative, can pass through ergosta-7, 22-diene-3-ketone and aminothiohydrazone. Urea produced by condensation reaction. The in vitro antibacterial test results show that ergosta-7, 22-diene-3-ketathizone has good anti-pathogenic activity and can be used for preparing antibacterial drugs.

Description

Ergosta-7,22-dien-3-ketothiazide and its preparation method and application in the preparation of antibacterial drugs

technical field

The present invention relates to an antibacterial compound, more particularly, the present invention relates to ergosta-7,22-dien-3-ketide thizone and its preparation method and application in the preparation of antibacterial drugs.

Background technique

In recent years, due to the abuse of antibiotics, many pathogenic bacteria have developed drug resistance, and super bacteria have appeared. At the same time, new pathogens have been emerging, which seriously threatens human health. There is an urgent need to develop new antibacterial drugs. Structural modification of natural product leads is one of the important methods for screening new drugs. Steroids are widely present in animals, plants and microorganisms, and have a variety of biological activities. Structural modification of steroids has led to the discovery of many steroid derivatives with good antibacterial, antitumor and other activities. Ergosta-7,22-dien-3-one widely exists in the fruiting bodies of various higher fungi, and has antibacterial activity, anti-complement activity and platelet aggregation enhancer activity. But so far, there is no report on ergosta-7,22-dien-3-ketothione and its preparation method and application in the preparation of antibacterial drugs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel ergosteroid derivative, namely ergosta-7,22-dien-3-ketide thizone, hereinafter referred to as compound A.

Another object of the present invention is to provide a preparation method of compound A.

A further object of the present invention is to provide the application of compound A in the preparation of antibacterial drugs.

The technical solution adopted by the present invention in order to achieve the above object is as follows:

The structural formula of the compound A of the present invention is:

The present invention provides a kind of preparation method of compound A, and this method comprises the following steps:

(1) get ergosta-7,22-dien-3-one and dissolve with petroleum ether;

(2) Add thiosemicarbazide and absolute ethanol in the reaction flask, insert a magnetic stirring bar, install a spherical condenser, connect with cooling water, heat to a slight boiling under stirring to make thiosemicarbazide completely dissolve, then add ergot The petroleum ether solution of ster-7,22-dien-3-one is added, and a small amount of concentrated hydrochloric acid is added, and the reaction is continued with stirring and heating to reflux until the reaction is complete;

(3) the reaction is completed, most of the solvent is recovered by vacuum rotary evaporation, then distilled water is added, and extracted three times with equal volume of ethyl acetate;

(4) Combine the ethyl acetate extracts, wash with saturated sodium carbonate solution and saturated brine solution successively, and finally dry with anhydrous magnesium sulfate, remove the solid by filtration, and recover ethyl acetate from the filtrate by vacuum rotary evaporation;

(5) The solid after recovering ethyl acetate is recrystallized with a mixed solvent of petroleum ether and ethanol to obtain compound A in the form of colorless crystals.

Meanwhile, the present invention also provides the application of the compound A in the preparation of antibacterial drugs.

When the compound A of the present invention is used in medicine, it can be used directly or in the form of a pharmaceutical composition. It can be administered by spray, oral, injection (intravenous, intramuscular).

In order to better understand the essence of the present invention, the in vitro antibacterial activity test results of the compound of formula (A) of the present invention will be used to illustrate its application in the preparation of antibacterial drugs.

1. Materials and methods

1.1 Reagents: dimethyl sulfoxide (DMSO), p-iodonitrotetrazolium violet (INT).

1.2 Medium: M-H broth medium.

1.3 Compound: The detected compound is Compound A.

1.4 Test pathogens: Staphylococcus aureu and Escherichia coli.

1.5 In vitro antibacterial activity test of compound A

The antibacterial activity of compound A against the tested pathogens in vitro was determined by the microdilution method, and penicillin sodium was used as the positive control. First make the test bacteria suspension. The pathogenic bacteria to be tested were inoculated into M-H broth medium, and cultured at 30°C for 4 hours to obtain bacterial suspension, which was diluted with M-H broth medium to obtain a 0.5 McFarland turbidity inoculum. Compound samples were then dissolved in DMSO and formulated to a starting concentration of 12.80 μg/μL, and filtered through a syringe filter. Antibacterial assays were performed in sterile 96 microwell plates. First, add 200 μL sterile water to the peripheral wells of the 96-well plate, and add 100 μL M-H broth medium to the remaining wells. Then, 100 μL of the filtered drug solution was added to the first well of each row, and the drug solution was doubling and half-diluted. Finally, 100 μL of inoculum was added to each test well and no-drug control well. The final drug solution detection concentration range is 0～3.20μg/μL. After the culture plate was incubated at 30°C for 18 hours, 20 μL of 5% INT solution was added to each well, and the incubation was continued for 1 hour before observation. Pink wells are positive growth. The minimum inhibitory concentration (MIC) is defined as the lowest drug concentration that prevents the color from changing to pink. The test was repeated three times.

2. Test results

The MIC values of compound A against Staphylococcus aureu and Escherichia coli were both 1.60 μg/μL and 3.20 μg/μL, respectively.

Compared with the prior art, the present invention has the following beneficial effects:

1. So far, there is no report about ergosta-7,22-dien-3-ketothione and its preparation method and application in the preparation of antibacterial drugs.

2. The in vitro antibacterial test results showed that Compound A could significantly inhibit the growth of the tested pathogens, and the MIC values for Staphylococcus aureu and Escherichia coli were 1.60μg/μL and 3.20μg/μL, respectively. It shows that compound A has good antibacterial activity and can be used to prepare antibacterial drugs.

3. The raw material ergosta-7,22-dien-3-one of the compound A of the present invention is rich in sources, and can be obtained not only by artificial synthesis, but also by isolation from higher fungi. Compound A has a simple preparation process, and can be administered by spraying, orally or by injection, and provides a new candidate drug for human treatment of bacterial infections.

Description of drawings

Fig. 1 is the electrospray ionization mass spectrogram of ergosta-7,22-dien-3-ketal thizone;

Fig. 2 is ergosta-7, the hydrogen nuclear magnetic resonance spectrogram of 22-dien-3-ketal thizone;

Figure 3 is a hydrogen nuclear magnetic resonance spectrum of ergosta-7,22-dien-3-one.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

Example 1

1. Preparation of Compound A:

(1) get 0.50mmol ergosta-7,22-dien-3-one, after dissolving with 10mL petroleum ether for subsequent use;

(2) Add 0.60 mmol thiosemicarbazide and 50 mL absolute ethanol to the two-necked round-bottomed flask, insert a magnetic stirring bar, install a spherical condenser, plug the other port with a hollow glass stopper, connect to cooling water, Heat to slight boiling under magnetic stirring to completely dissolve thiosemicarbazide, then add the petroleum ether solution of ergosta-7,22-dien-3-one, add 5 drops of concentrated hydrochloric acid, continue to stir and heat under reflux for 4 hours, and use silica gel TLC followed until the reaction was complete;

(3) the reaction is completed, most of the solvent is recovered by vacuum rotary evaporation at 50 ° C, then 50 mL of distilled water is added, and extracted three times with equal volume of ethyl acetate;

(4) Combine the ethyl acetate extracts, wash with saturated sodium carbonate solution and saturated brine solution successively, and finally dry with anhydrous magnesium sulfate, remove the solid by filtration, and recycle ethyl acetate by vacuum rotary evaporation of the filtrate at 50°C;

(5) The solid after recovering ethyl acetate is recrystallized with a mixed solvent of petroleum ether and ethanol to obtain compound A in the form of colorless crystals.

2. Structure identification of compound A:

The molecular formula of Compound A is C ₂₉ H ₄₇ N ₃ S, and the calculated relative molecular weight is 469.7696. The electrospray ionization mass spectrum (Fig. 1) showed its quasi-molecular ion peaks at 492.5 [M+Na] ⁺ (calcd: 492.7594) and 470.5 [M+H] ⁺ (calcd: 470.7775), respectively.

Using deuterated chloroform as the solvent and tetramethylsilane as the internal standard, the BRUKER-400 nuclear magnetic resonance spectrometer was used to analyze the compound A and the raw material ergosta-7,22-dien-3-one by hydrogen nuclear magnetic resonance spectroscopy. The hydrogen NMR spectrum of A (Fig. 2) shows three alkene proton signals at the 7-position, 22-position and 23-position of compound A at the chemical shift δ _H value of 5.20 (m, 3H), while at the chemical shift δ _H The values of 8.58(br s), 7.21(br s) and 6.16(br s) are more than the hydrogen NMR spectrum of the raw material ergosta-7,22-dien-3-one (Fig. 3). 3 NH active proton signals.

Comprehensive mass spectrometry and hydrogen nuclear magnetic resonance spectral information shows that compound A is the product of the condensation reaction between the raw material ergosta-7,22-dien-3-one and thiosemicarbazide, namely ergosta-7,22-dien-3-one Thiazone.

Example 2

Compound A was prepared according to Example 1, and excipients were added in the ratio of compound crystals to excipients in a mass ratio of 1:1, and granulated and pressed into tablets.

Example 3

Compound A was prepared according to Example 1, and excipients were added in the ratio of compound crystals to excipients in a mass ratio of 1:2, and granulated and pressed into tablets.

Example 4

Compound A was prepared according to Example 1, and excipients were added in the ratio of compound crystals to excipients in a mass ratio of 1:3, and granulated and pressed into tablets.

Example 5

Tablet: compound A: 50 mg; starch: 50 mg; corn steep liquor: appropriate amount; magnesium stearate: appropriate amount.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (3)

1. an ergosteroid derivative, ergosta-7, 22-dien-3-ketal thione, its structural formula is

2. a kind of preparation method of the described compound of claim 1, is characterized in that this method comprises the following steps: (1) get ergosta-7,22-dien-3-one, dissolve with petroleum ether; (2) Add thiosemicarbazide and ethanol in the reaction flask, insert a magnetic stirring bar, install a spherical condenser, connect with cooling water, heat to slight boiling under stirring to make thiosemicarbazide completely dissolve, then add ergoster- 7, the petroleum ether solution of 22-dien-3-one, and add a few drops of concentrated hydrochloric acid to make a catalyst, and continue stirring and heating to reflux until the reaction is complete; (3) complete reaction, remove most of the solvent by vacuum rotary evaporation, then add distilled water, extract three times with equal volume of ethyl acetate; (4) Combine the ethyl acetate extracts, wash with saturated sodium carbonate solution and saturated brine solution successively, and finally dry with anhydrous magnesium sulfate, remove the solid by filtration, and recover ethyl acetate from the filtrate by vacuum rotary evaporation; (5) The solid after recovering the ethyl acetate is recrystallized with a petroleum ether-ethanol mixed solvent to obtain ergosta-7,22-dien-3-ketal thizone as colorless crystals. 3. The application of the compound of claim 1 in the preparation of antibacterial drugs.

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