CN112409386B

CN112409386B - Guanine piperazine compound and its preparing method and use

Info

Publication number: CN112409386B
Application number: CN202011229230.7A
Authority: CN
Inventors: 戈惠明; 徐响; 樊瑞; 史净; 焦瑞华
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2021-11-16
Anticipated expiration: 2040-11-06
Also published as: CN112409386A

Abstract

The invention separates and purifies the solid fermentation product of Streptomyces chrysosporium with the preservation number of NRRL F-5755 from Research Laboratory (Northern Regional Laboratory) in Northern America to obtain a piperazine compound, and experimental Research results show that the guanine piperazine has stronger inhibitory activity to saccharomyces cerevisiae and can be further used for preparing saccharomyces cerevisiae inhibitors.

Description

Guanine piperazine compound and its preparing method and use

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a guanine piperazine compound and a preparation method and application thereof.

Background

Abundant microbial resources play an important role in national economy and people's life, and are a resource treasury for potential new drug discovery. Of all natural resources capable of producing drugs, microbial resources have become the last, and largest, field of great development potential. In the discovery research of microbial drug lead compounds in the last half century, a plurality of national research institutions and research groups have carried out considerable systematic natural product chemical and biological activity research on microorganisms in China, a large number of novel framework compounds, high-activity lead compounds with new drug development potential and candidate drugs are discovered, and part of components enter the preclinical research process.

Due to the factors such as difficult separation and purification of microbial resources, although some chemical and biological research reports exist in recent years, most of the microbial resources are still in a state of being researched and developed, and the research on various aspects such as microbial species diversity, distribution diversity, biochemical diversity and biological functions is relatively lagged behind at present, and the current situation becomes the bottleneck of the development and utilization of the microbial resources in China. Deep excavation and systematic research on new microbial resources become one of the main ways of researching and developing new drugs with independent intellectual property rights in China.

The piperazine compounds are widely applied in the field of medicine. Quinolone antibacterial drugs, piperazinrifamycin antibiotics, have been widely used in medical treatment. In recent years, piperazine compounds are also used for treating diseases such as gastrointestinal stromal tumor (avaprinib), duchenne muscular dystrophy (golodisen), thrombocytopenia (avatrompag Maleate), anti-cytomegalovirus infection (Letermovir), and anti-schizophrenia (Cariprazine Hydrochloride), and have a great potential for drug development, and are particularly necessary for the development of piperazine compounds.

Disclosure of Invention

The invention discovers two new active substances from a Streptomyces chrysosporium Streptomyces chrysstilconduit NRRL F-5755 solid fermentation product preserved in a research laboratory in the northern region of the United states for the first time, and the active substances are named guanine piperazine A and guanine piperazine B (guanipiprazine), and an in vitro activity experiment shows that the active substances have stronger inhibitory activity to saccharomyces cerevisiae.

The specific technical scheme of the invention is as follows:

a guanine piperazine compound having the following structural formula:

the invention further provides a preparation method of the compound.

Is obtained by extracting Streptomyces chrysosporium strain by fermentation (preferably solid fermentation). Preferably, the Streptomyces chrysosporium strain deposited at the research laboratory in the northern region of the United states (Beziville, Md., zip code 20704) under accession number NRRL F-5755.

The preparation method comprises the following steps:

(1) culturing Streptomyces brevitamyces strain at 26-30 deg.C for 5-9 days, preferably at 28 deg.C for 7 days;

preferably, culturing Streptomyces coronarius strain (preferably Streptomyces coronarius strain with the biological preservation number of NRRL F-5755) on one or more solid culture media of ISP2, ISP4 and MS at 26-30 ℃ for 7 days;

(2) drying the culture medium obtained in the step (1), re-dissolving the culture medium by using an alcohol solvent, and concentrating the supernatant;

(3) performing gel column chromatography on the concentrate obtained in the step (2), preferably performing Sephadex LH-20 gel column chromatography, and eluting with alcohol solvent to obtain 3 peak-appearing components;

(4) separating and purifying the peak component 2 by HPLC to obtain the compound of the invention.

The alcohol solvents in the steps (2) and (3) are the same or different and are selected from one or more of methanol, ethanol and isopropanol. Preferably, methanol is used in both steps (2) and (3).

Preferably, the gel column in the step (3) is Sephadex LH-20.

The HPLC chromatographic conditions in the step (4) are as follows: using a C18 reverse phase chromatography column, detecting the wavelength of 280nm, mobile phase: methanol: water 3:2, V/V, isocratic elution.

Preferably, in the step (4), the HPLC separation and purification process is as follows: semi-preparative reverse phase high performance liquid chromatography by semihplc: ODS-2Hypersil gum, 5 μm, 250 mm. times.10 mm, mobile phase: methanol-water volume ratio of 3:2, elution is carried out with a flow gradient of 2mL/min for 15 min. The pump may be of the type Hitachi pump L-7100 and the UV lamp may be of the type UV detector L-7400. The retention time of guanine piperazine A is 12min, and the retention time of guanine piperazine B is 13 min.

The Streptomyces chrysosporium chrestomyces NRRL F-5755 used in the preparation method is obtained from research laboratories in the northern region of the United states, and is characterized in that: on ISP4 plate, the hypha in the medium is white at the initial stage of culture, so that single colony is easy to generate, and a large amount of aerial hyphae begin to generate after one week, and the spore amount is small and white.

Has the advantages that:

the invention firstly obtains streptomycin Streptomyces chrysospermus NRRL F-5755 from research laboratories in northern regions of America, and firstly discovers guanine piperazine (guanipiprazine) A and guanine piperazine (guanipiprazine) B from fermentation products of the streptomycin chrysospermus NRRL F-5755.

Drawings

FIG. 1 is an HPLC chromatogram of guanine piperazine of the present invention.

FIG. 2 is a graph showing the results of experiments showing that guanine piperazine of the present invention has a strong inhibitory activity against Saccharomyces cerevisiae.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: activation of Streptomyces chrysogenyticus NRRL F-5755.

The actinomycetes were obtained by coating freeze-stored dry powder of a strain from a research laboratory in northern region of the United states on an ISP2 medium (Yeast Extract 4g, Malt Extract 10g, Glucose 4g, agar 20g, 1L of distilled water, pH7.4 to 7.6) and culturing the dry powder in a thermostat at 28 ℃. On ISP2 plates, the hyphae in the medium are white at the initial stage of culture, single colonies are easy to generate, and a small amount of white spores begin to generate after one week. The strain is identified as Streptomyces chrestomyces of the Streptomyces genus by morphology and 16S rRNA. The strain is named as Streptomyces chrestomyces NRRL F-5755, is deposited in research laboratories in northern America, and has the deposit number: NRRL F-5755.

Example 2: solid fermentation of Streptomyces chrysstilbesticus NRRL F-5755

The strain Streptomyces brestomyces NRRL F-5755 was transferred to a plate ISP4 medium and fermented at 28 ℃ for 7 days.

Example 3: extraction and isolation of guanine piperazines A and B

Drying the fermentation medium obtained in example 2, dissolving the fermentation medium with methanol, and concentrating the supernatant to obtain extract F1. And (3) carrying out gel column Sephadex LH-20 chromatography on the extract F1, and eluting by taking MeOH as a mobile phase to obtain 3 peak-appearing components. And (3) subjecting the second peak component to semi-HPLC (chromatographic column: Allsphere ODS-2.5mm column), and eluting isocratically for 15min at the flow rate of 2mL/min and the volume ratio of methanol to water of 3:2 in a methanol-water system to obtain guanine piperazine A (45mg) and purine piperazine B (55 mg). The pump may be of the type Hitachi pump L-7100 and the UV lamp may be of the type UV detector L-7400.

Example 4: structural identification of guanine piperazine

The structure of the guanine piperazine is determined based on the mass spectrum and the nuclear magnetic resonance spectrum of the guanine piperazine. The results are shown in tables 1 and 2.

Process for preparing guanine piperazine A¹H and¹³the C NMR data are tabulated below:

TABLE 1 guanipiperazine A¹H and ¹³Data attribution of C NMR

The guanine piperazine A has the following structure:

TABLE 2 guanine piperazine B¹H and¹³Data attribution of C NMR

Guanine piperazine B has the following structure:

example 5: activity assay

Experimental materials: saccharomyces cerevisiae, YPD medium, DMSO, guanine piperazine.

The experimental method comprises the following steps:

1) preparation of the culture medium and guanine piperazine solution

Dissolving 10g of Yeast Extract, 20g of polypeptone (peptone) and 20g of glucose in one liter of water, sterilizing at 121 ℃ for half an hour for later use, and adding 2% agar powder if preparing a solid culture medium; guanine piperazine A, B solid was weighed separately and dissolved in DMSO to make up a 10mM solution.

Minimum inhibitory concentration:

the Minimum Inhibitory Concentration (MIC) is the lowest concentration of a drug that inhibits bacteria in a culture medium after culturing the bacteria in vitro for 18 to 24 hours, and is an index for measuring the magnitude of antibacterial activity of an antibacterial drug.

2) Determination of Activity

Yeast were inoculated into 50mL shake flasks containing 5mL YPD medium and cultured overnight at 30 ℃. 1mL of overnight-cultured yeast was mixed with 100mL of YPD medium and dispensed into 96-well plates (100. mu.L per well), and various amounts of guanine piperazine solution were added, and the solutions were incubated overnight at 30 ℃ with a blank of DMSO equivalent to the volume of the solution.

The experimental results are shown in fig. 2: the abscissa is the concentration of guanine piperazine and the ordinate is the difference between the OD of the control and the OD of the inhibition group.

The MIC of guanine piperazine A to Saccharomyces cerevisiae is about 4.5 mug/mL; the MIC of guanine piperazine B for Saccharomyces cerevisiae is about 5.5. mu.g/mL.

In vitro activity experiments show that guanine piperazine has strong inhibitory activity to saccharomyces cerevisiae and can be further used for preparing a yeast inhibitor.

Claims

1. A guanine piperazine compound having the following structural formula:

or

。

2. The process for producing a guanine piperazine compound according to claim 1, wherein the strain Streptomyces coronatines is Streptomyces species (see (1))Streptomyces chrestomyceticusBiological deposit number NRRL F-5755) is obtained by fermentation and extraction.

3. The method of claim 2, comprising the steps of:

(1) mixing a coronatine Streptomyces species (Streptomyces chrestomyceticusBiological deposit number NRRL F-5755) is subjected to solid fermentation by using one or more culture media of ISP2 and ISP 4;

(2) drying the culture medium obtained in the step (1), dissolving the culture medium in an alcohol solvent, and concentrating the supernatant;

(3) performing gel column chromatography on the concentrate obtained in the step (2), and eluting by using an alcohol solvent to obtain 3 peak components;

(4) separating and purifying the peak component 2 by HPLC to obtain the compound of claim 1.

4. The method according to claim 3, wherein the culture conditions in the step (1) are 26 to 30 ℃ for 5 to 9 days.

5. The method according to claim 3, wherein the alcoholic solvents of steps (2) and (3) are the same or different and are selected from one or more of methanol, ethanol, and isopropanol.

6. The method according to claim 3, wherein the gel column in step (3) is Sephadex LH-20.

7. The method according to claim 3, wherein the HPLC chromatographic conditions in the step (4) are as follows: using a C18 reverse phase chromatography column, detection wavelength 280nm, mobile phase: methanol: water = 3:2, V/V, isocratic elution.

8. Use of a guanine piperazine compound according to claim 1 for the preparation of a yeast inhibitor.

9. Use according to claim 8, characterized in that the yeast is Saccharomyces cerevisiae.