CN110330549B

CN110330549B - Cyclic peptide emericellamide G, its preparation method and application in preparation of enzyme inhibitor

Info

Publication number: CN110330549B
Application number: CN201910579614.2A
Authority: CN
Inventors: 漆淑华; 梁潇
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2019-06-28
Filing date: 2019-06-28
Publication date: 2021-05-04
Anticipated expiration: 2039-06-28
Also published as: CN110330549A

Abstract

The invention discloses a cyclic peptide emericellamide G, a preparation method thereof, and application in the preparation of protein tyrosine phosphatase TCPTP inhibitor and SHP1 inhibitor. The structural formula of the cyclic peptide emericellamide G is shown in formula (I). Cyclic peptide emericellamide G is isolated from the fermentation broth of Asprergillus puniceus SCSIO z021. Tests have shown that the cyclic peptide emericellamide G of the present invention has the activity of inhibiting protein tyrosine phosphatase TCPTP and SHP1, and can be used for TCPTP inhibitor and SHP1 Inhibitor lead compound studies.

Description

Cyclic peptide emericella G, preparation method thereof and application thereof in preparation of enzyme inhibitor

Technical Field

The invention belongs to the field of marine organisms, and particularly relates to a cyclic peptide emericellamide G, a preparation method thereof and application thereof in preparation of a protein tyrosine phosphatase TCPTP inhibitor and a SHP1 inhibitor.

Background

At present, the diagnosis and treatment of malignant tumors are still one of the biggest challenges in the medical field, and although the traditional malignant tumor treatment means such as chemotherapy and radiotherapy are continuously developed, certain limitations and disadvantages still exist, such as large adverse reaction, easy induction of drug resistance and the like. Protein Tyrosine Phosphatases (PTPs) are widely present in living bodies, and diseases related to Protein phosphatases include diabetes, cancer, obesity, senile dementia, and the like, and have attracted considerable attention by scientists in recent years. Some PTPs, such as SHP1, SHP2, PTP1B, TCPTP, etc., have become new targets for the development of new drugs, such as antitumor drugs. Finding effective and highly selective inhibitors of PTPs is an important direction for future clinical therapy.

Disclosure of Invention

The first object of the present invention is to provide a novel cyclic peptide emericellamide G having an inhibitory effect on protein tyrosine phosphatase TCPTP and SHP 1.

The structural formula of the novel cyclopeptide emericelle compound G is shown as a formula (I),

a second object of the present invention is to provide a process for the preparation of the compound emericellamide G, which is isolated from the fermentation broth and/or mycelium of the fungus Aspergillus puniceus SCSIO z 021.

Preferably, the specific steps are as follows:

(a) preparing fermentation liquor and mycelium of the fungus Asprergillus puniceus SCSIO z 021;

(b) adsorbing the fermentation liquor obtained in the step (a) by using macroporous resin, washing the macroporous resin by using water to remove culture medium components, washing the macroporous resin by using methanol or ethanol, and concentrating the washing liquid to obtain methanol or ethanol extracts respectively; or extracting the fermentation liquor obtained in the step (a) with ethyl acetate, dichloromethane or chloroform solvent, and concentrating the extract to obtain ethyl acetate extract, dichloromethane extract or chloroform extract; crushing mycelium, soaking in acetone or methanol, concentrating the soaking solution under reduced pressure to remove acetone or methanol, extracting the residual water phase with ethyl acetate, and concentrating to obtain ethyl acetate extract of thallus;

(c) subjecting the methanol extract, the ethanol extract, the ethyl acetate extract of the fermentation broth, the ethyl acetate extract of the thalli, the dichloromethane extract or the chloroform extract obtained in the step (b) to normal-phase silica gel column chromatography, sequentially carrying out gradient elution by using solvent systems with a dichloromethane-methanol volume ratio of 100:0,98:2,95:5,92:8,90:10,80:20 and 70:30, and collecting a sample washed by dichloromethane-methanol volume ratio of 90:10 to obtain a component Fr.6; fr.6 is subjected to normal-phase silica gel column chromatography again, gradient elution is sequentially carried out by using solvent systems with the volume ratio of dichloromethane to acetone being 100:0,95:5,90:10,80:20,70:30 and 50:50, samples washed with the volume ratio of dichloromethane to acetone being 50:50 are collected, and the compound emercellamide G is obtained through recrystallization.

Further preferably, the fermentation broth in step (a) is prepared by: growing fungus Asprergillus puniceus SCSIO z021 in a plate culture medium suitable for the fungus, inoculating the fungus into a fermentation culture medium after the fungus spores, and performing static culture at room temperature for 25 days to obtain fermentation liquor and mycelia, wherein each liter of the fermentation culture medium is prepared by the following method: boiling 200g of potato in 500mL of pure water for 20min, filtering to obtain potato juice, adding 20g of glucose and 30g of sea salt, and supplementing to 1000mL of water.

Further preferably, the concentration in step (b) is performed by concentration under reduced pressure.

The third purpose of the invention is to provide the application of the compound emericellamide G or the medicinal salt thereof in preparing the protein tyrosine phosphatase TCPTP inhibitor and/or SHP1 inhibitor.

It is a fourth object of the present invention to provide a TCPTP inhibitor of protein tyrosine phosphatase or a SHP1 inhibitor, which comprises the compound emericellamide G or a pharmaceutically acceptable salt thereof as an active ingredient.

A fifth object of the present invention is to provide the use of the fungus Aspergillusspuniceus SCSIO z021 in the preparation of the compound emericellamide G.

The invention separates new cyclic peptide emericellamide G with the activity of inhibiting protein tyrosine phosphatase TCPTP and SHP1 from the fermentation liquor of a marine fungus A. penicilleus SCSIO z021, and the cyclic peptide emericellamide G can be used for researching TCPTP inhibitor and SHP1 inhibitor lead compound.

The fungus AspergillusanniceSCSIO z021 of the invention is preserved in Guangdong province microorganism culture Collection (GDMCC) in 2019, 3 and 20 days, and the address is as follows: the Guangzhou city Pieli Zhongluo No. 100 large yard No. 59 building No. 5, the preservation number is GDMCC No: 60611.

drawings

FIG. 1 is a key HMBC, COSY correlation for Compound 1 (Compound emericella G).

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

(1) The fermentation medium is prepared by the following method per liter: boiling 200g of potato in 500mL of pure water for 20min, filtering to obtain potato juice, adding 20g of glucose and 30g of sea salt, adding water to 1000mL, and sterilizing with high-pressure steam at 115 ℃ for 25 min. Fermentation medium 65L was prepared in this manner. About 200 1000mL Erlenmeyer flasks were filled with 65L of fermentation medium, about 300mL per flask.

(2) Preparation of fermentation liquor and mycelium: growing fungus A.penicilleus SCSIO z021 in a plate culture medium suitable for the fungus, after the fungus grows spores, transferring the spores from the plate to a triangular flask containing sterile water by using a bamboo stick to obtain a spore suspension, inoculating the spore suspension of the fungus into a fermentation culture medium (300 mL of the fermentation culture medium is contained in a 1L triangular flask) by using a liquid transfer gun, and after standing and culturing for 25 days at room temperature (26 ℃), collecting fermentation liquid and mycelia.

(3) And (3) separating and purifying a compound: filtering and separating the fermentation liquid and mycelium obtained by culturing the fermentation medium with gauze, wherein 65L of the fermentation liquid is adsorbed by macroporous resin, then washing the macroporous resin with water to remove the components of the culture medium, then washing the macroporous resin with ethanol (or methanol) to obtain an ethanol extract, (the fermentation liquid can also be extracted with ethyl acetate, dichloromethane or chloroform), and concentrating under reduced pressure to obtain an ethanol crude extract; crushing mycelium, soaking in acetone, extracting for three times, mixing extractive solutions, concentrating under reduced pressure to remove acetone, extracting the residual water phase with ethyl acetate, and concentrating to obtain ethyl acetate extract of thallus; mixing the ethanol crude extract and ethyl acetate extract, mixing with normal phase silica gel (100-200 mesh) by dry method, loading into glass chromatography column (H fine silica gel), performing normal temperature pressurized column chromatography, sequentially treating with dichloromethane: performing gradient elution by a dichloromethane-methanol system with methanol volume ratios of 100:0,98:2,95:5,92:8,90:10,80:20 and 70:30 respectively, finally combining eluent by TLC and HPLC detection to obtain 9 components (Fr.1-Fr.9), and collecting a sample washed by dichloromethane-methanol volume ratio of 90:10 to obtain a component Fr.6; fr.6 is subjected to normal phase silica gel column chromatography again, gradient elution is sequentially carried out by using solvent systems with dichloromethane to acetone volume ratio of 100:0,95:5,90:10,80:20,70:30 and 50:50, samples washed with dichloromethane to acetone volume ratio of 50:50 are collected, and the compound erercellamide G is obtained through recrystallization (dichloromethane-methanol volume ratio of 1: 1).

And (3) structure presumption:

compound 1, colorless needle-like crystals, [ M + Na ] given by high resolution Mass Spectrometry (HRESIMS) at M/z 660.4310]⁺Ion peaks, combined with NMR data (Table 1) to infer the formula C₃₃H₅₉N₅O₇The unsaturation degree was 7.¹H and¹³the C NMR data are characteristic of the distinct peptidic compounds,¹five amide NH proton signals (delta) exist in HNMR spectrogram_H8.14,8.03,7.90,7.54,7.36), six alpha-amino protons (delta)_H4.15,4.11,4.08,4.07,3.95,3.76), and a vicinal oxymethylene proton (. delta.) -O_H5.06); in that¹³In the C NMR spectrum, six amide carbonyl or ester carbonyl signals (. delta.) were observed_C171.7,171.3,171.2,170.7,169.4,168.9), one sp linked to oxygen³Hybrid carbon (. delta.)_C74.1) and five alpha-amino carbons (. delta.) -_C60.1,51.5,48.3,47.4,42.4). IR absorption spectra at 1634 and 1755cm^-1The presence of an absorption peak at the wavelength also confirms the presence of both amide and ester functional groups in the structure. Six carbonyl groups contribute 6 unsaturations, and to satisfy 7 unsaturations, compound 1 should be a cyclic peptide.

The 2D NMR data (Table 1) revealed the presence of two alanine (Ala), one valine (Val), one leucine (Leu) and a thirteen-carbon fatty acid fragment in the structure. The spectral data of compound 1 are very similar to those of the known compound emericella B (document: Oh D C, Kauffman C A, et al. journal of Natural Products,2007,70,515-520.), which have the same amino acid fragments, and the main difference is that compound 1 lacks a methyl signal on a long alkyl chain and has more methyl signals on the long alkyl chainOne methylene carbon C-21 (. delta.)_C 38.0)，¹H-¹The continuous correlation between H-21-H-24 was shown in H COSY, suggesting that Compound 1 lacks the methyl group attached to C-21, and the presence of a structural fragment of 3-hydroxy-4, 6-dimethyldodecanoic acid (HDMD) in the structure was confirmed by comparing nuclear magnetic data with the fatty acid fragment of emericellamide B, in combination with the HMBC correlation signal.

2-NH (. delta.) in HMBC spectra (FIG. 1)_H7.36) are associated with the carbonyl carbons of the two Ala fragments, respectively, H-2 is associated with C-4; 8-NH/5-NH/H-5 is related to C-7, H-14 is related to C-18/C-13, 14-NH is related to C-18, H-19/19-NH is related to C-20, the connection sequence of the amino acids is presumed to be Ala1-Ala2-Leu-Val-Gly-HDMD, and the hydroxyl on C-22 and the carboxyl of Ala-1 fragment are dehydrated to form lactone, thereby determining the planar structure of the compound 1. Through Marfey reaction, the amino acids in the structure are determined to be L-Ala, L-Leu and L-Val respectively. The relative configuration of all chiral centers is determined by an X-ray single crystal diffraction experiment, and the absolute configuration of the amino acid fragment is known, so that the C-3/C-4/C-6 of the 3-hydroxy-4, 6-dimethyldodecanecarbonic acid fragment is determined to be S configuration. The identification result of the compound 1 is shown as a formula I and is named as emericellamide G

TABLE 1 preparation of Compound 1¹H and¹³C-NMR data (500,125MHz, DMSO-d)₆,δppm)

EXAMPLE 2 protein tyrosine phosphatase Activity assay of the Compound emericellamide G

Cloning of human protein tyrosine phosphatases PTP1B, SHP1, SHP2, MEG2or TCPTP into Escherichia coli (Esche)richia coli) and purified. The enzyme inhibitory activity was determined using p-nitrophenyl phosphate (pNPP) as substrate in 96-well plates containing 100. mu.L of reaction mixture per plate. Human recombinant PTP1B, SHP1, SHP2, MEG2or TCPTP (0.05. mu.g) was added to 50. mu.L of a reaction buffer (pH 6.5) containing 50mM HEPES, 100mM NaCl, 1mM EDTA and 1mM Dithiothiotriol (DTT), and further samples of the compound to be tested (compound emericelamide G) were added to each 96-well plate, respectively. Na (Na)₃VO₄As a positive control, DMSO was used as a negative control for evaluating this high throughput screening system. After a pre-incubation period of 15 minutes at room temperature, 50. mu.L of buffer containing 50mM pNPP was added and incubation continued at 37 ℃ for 60 minutes. Phosphatase activity was determined by measuring the absorbance of the produced p-nitrophenol at 405 nm. IC (integrated circuit)₅₀Values were calculated using Gen5 software (Synergy2Multi-Mode Microplate Reader, BioTek Instruments, inc., head calibrated in Winooski, VT, USA). Each experiment was repeated 3 times.

The test result shows that: compound emericellamide G selectively inhibits protein tyrosine phosphatase TCPTP and SHP1, IC₅₀Values were 1.40 and 6.57. mu.g/mL, respectively, but were inactive against SHP2, MEG2, and PTP 1B. Positive control Na₃VO₄IC inhibiting TCPTP, SHP1, SHP2, MEG2 and PTP1B₅₀The values are 2.4,4.4, 6.2, 3.2, 1.6. mu.M, respectively; the negative control DMSO was inactive.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. A compound emericella G or a pharmaceutically acceptable salt thereof, having the structural formula shown in formula (I):

2. a process for the preparation of the compound emericella G according to claim 1, which is prepared from the fungus Asprergillus puniceus SCSIO z021 GDMCC No: 60611 and/or mycelium.

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

(a) preparation of the fungus Asprergillus puniceus SCSIO z021 GDMCC No: 60611 fermentation broth and mycelium;

(b) adsorbing the fermentation liquor obtained in the step (a) by using macroporous resin, washing the macroporous resin by using water to remove culture medium components, washing the macroporous resin by using methanol or ethanol, and concentrating the washing liquid to obtain methanol or ethanol extracts respectively; or extracting the fermentation liquor obtained in the step (a) with ethyl acetate, dichloromethane or chloroform solvent, and concentrating the extract to obtain ethyl acetate extract, dichloromethane extract or chloroform extract; crushing mycelium, soaking in acetone or methanol, concentrating the extractive solution under reduced pressure to remove acetone or methanol, extracting the residual water phase with ethyl acetate, and concentrating to obtain ethyl acetate extract of thallus;

(c) subjecting the methanol extract, ethanol extract, ethyl acetate extract, dichloromethane extract, chloroform extract or ethyl acetate extract of the bacteria of the fermentation broth obtained in the step (b) to normal phase silica gel column chromatography, sequentially subjecting to dichloromethane: gradient elution is carried out on a solvent system with the methanol volume ratio of 100:0,98:2,95:5,92:8,90:10,80:20 and 70:30, and a sample washed by dichloromethane and methanol with the volume ratio of 90:10 is collected to obtain a component Fr.6; fr.6 is subjected to normal-phase silica gel column chromatography again, gradient elution is sequentially carried out by using solvent systems with the volume ratio of dichloromethane to acetone being 100:0,95:5,90:10,80:20,70:30 and 50:50, samples washed with the volume ratio of dichloromethane to acetone being 50:50 are collected, and the compound emercellamide G is obtained through recrystallization.

4. The method according to claim 3, wherein the fermentation broth and the mycelia in step (a) are prepared by: the fungus Aspergillus puniceus SCSIO z021 GDMCC No: 60611 growing in a plate culture medium suitable for fungus, inoculating fungus into a fermentation culture medium after fungus spores grow, and standing and culturing at room temperature for 25 days to obtain fermentation liquid and mycelium, wherein the fermentation culture medium is prepared by the following method per liter: boiling 200g of potato in 500mL of pure water for 20min, filtering to obtain potato juice, adding 20g of glucose and 30g of sea salt, and supplementing to 1000mL of water.

5. The method according to claim 3, wherein the concentration in the step (b) is carried out by concentration under reduced pressure.

6. Use of the compound emericella G of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a TCPTP inhibitor of protein tyrosine phosphatase and/or an inhibitor of SHP 1.

7. A protein tyrosine phosphatase TCPTP inhibitor or SHP1 inhibitor, characterized by containing the compound emericellamide G of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

8. Fungus Aspergillus puniceus SCSIO z021 GDMCC No: 60611 for use in the preparation of a compound emericellamide G of formula (I) as claimed in claim 1.