CN109574828B

CN109574828B - Sesquiterpene compound and anti-hepatitis C application thereof

Info

Publication number: CN109574828B
Application number: CN201811588057.2A
Authority: CN
Inventors: 林文翰; 刘�东; 李博; 黄健
Original assignee: CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION; Peking University
Current assignee: CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION; Peking University
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-05-15
Anticipated expiration: 2038-12-25
Also published as: CN109574828A

Abstract

The invention discloses a sesquiterpenoids and application thereof in resisting hepatitis C. The compounds are sesquiterpene compounds with a novel chemical framework, which are found in fermentation extracts of marine fungus Trichoderma harzianum, and the sesquiterpene compounds are found to have anti-HCV activity for the first time. In vitro activity research data shows that the compounds can simultaneously act on the invasion and transcription replication stages of HCV virus infection, have multi-target antiviral efficacy, and are drug mode molecules with great potential for research and development of anti-HCV new substances.

Description

Sesquiterpene compound and anti-hepatitis C application thereof

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to sesquiterpene compounds, a preparation method thereof, and application of the sesquiterpene compounds in the aspect of preventing and treating Hepatitis C Virus (HCV).

Background

Hepatitis C Virus (HCV) infection is a major public health problem worldwide, causing over 35 million deaths each year, affecting approximately 3% of the population worldwide. HCV has a hidden course and high infection chronicity, and patients at the end stage are often accompanied with chronic liver diseases, and even develop liver cancer in severe cases.

The medicine for treating early hepatitis C generally adopts polyethylene glycol interferon plus ribavirin, and the curative effect of the medicine is greatly different among different genotype groups, the treatment time is long, and the adverse reaction is more. In recent years, the advent of targeted drugs against key proteins of the HCV life cycle, namely, directly acting antiviral Drugs (DAAs), has greatly improved HCV treatment and greatly improved cure rates. However, the use of DAA is still limited by problems including the generation of drug-resistant mutants, adverse effects associated with drugs, and high treatment costs. Therefore, the intensive research on the HCV infection process and the development of the multi-target novel medicine aiming at different periods of virus infection have important social and economic significance for guaranteeing the life safety of the people and improving the life quality of the people.

Disclosure of Invention

In the research process of exploring active chemical components in marine microorganisms, two sesquiterpene compounds with novel chemical frameworks are discovered from fermentation extracts of marine fungus Trichoderma harzianum, and the novel sesquiterpenes are discovered to have anti-HCV activity for the first time. In vitro activity research data shows that the compounds can simultaneously act on the invasion and transcription replication stages of HCV virus infection, have multi-target antiviral efficacy, and are drug mode molecules with great potential for research and development of anti-HCV new substances.

In a first aspect of the invention, there is provided a compound of formula I and formula II:

in a second aspect of the present invention, there is provided a process for the preparation of the above compound, comprising the steps of:

1) taking marine filamentous fungus Trichoderma harzianum as an engineering bacterium, and performing fermentation culture to obtain a fermentation product;

2) extracting and concentrating the fermentation product, and preparing the compounds of the formula I and the formula II from the bacterial strain fermentation product extract through chromatographic separation and purification.

Specifically, in the step 1), Trichoderma harzianum (Trichoderma harzianum) is inoculated on a solid culture medium for culture, preferably on a rice solid culture medium, and fermentation culture is carried out for 30-40 days at room temperature.

Smashing the mycelium and the culture medium collected in the step 2), ultrasonically extracting by ethyl acetate, standing overnight, filtering to remove filter residues, concentrating, and extracting for multiple times to obtain a fermented substance extract; and then sequentially carrying out silica gel column chromatography, reversed phase column chromatography separation and HPLC purification on the fermentation extract, and preparing the compounds of the formula I and the formula II from the fermentation extract.

The silica gel column chromatography is Vacuum Liquid Chromatography (VLC), 160-200-mesh silica gel particles are adopted, and a system with a petroleum ether/acetone volume ratio of 50: 1-1: 1 is used as an elution solvent for gradient elution. The chromatographic separation of the reverse phase column adopts a C-18ODS reverse phase column and gradient elution with 20-100% methanol/water. The HPLC purification was performed using semi-preparative HPLC, 35% acetonitrile/water isocratic elution.

In a third aspect of the invention, there is provided the use of a compound of formula I and formula II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of hepatitis c.

The anti-HCV activity of the compounds shown in the formula I and the formula II is evaluated by constructing an experimental model for inhibiting virus invasion and replication in vitro. The activity evaluation of an in vitro pseudovirion model, an SPR experiment and the docking analysis of small molecules and target protein binding molecules prove that the compound can block the combination of HCV and host cells by acting on the binding site of an HCV membrane protein E2 on an LEL (extracellular large loop region) of a cell host factor CD81 protein, so that the HCV is prevented from invading the host cells. In addition, the anti-HCV effect of the above compounds in huh7.5 cells acutely infected with HCV was verified by in vitro experiments. Molecular docking analysis proves that the compound acts on the Palm I region on HCV virus NS5B to inhibit the transcription and replication of HCV. Finally, the compounds shown in the formula I and the formula II are proved to be multi-target HCV inhibitors with a brand new structure type.

Drawings

FIG. 1 shows the inhibition effect of compound (A) of formula I and compound (B) of formula II on the virus invasion stage by pseudovirion model, wherein the left panel shows the VSV pseudovirion test results, and the right panel shows the VSV pseudovirion test results.

FIG. 2 is a data chart of SPR experiments on the binding of compounds (A) and (B) of formula I and II to CD 81.

FIG. 3 is a graph of data from SPR experiments in which compounds of formula I (A) and formula II (B) blocked the binding of CD81 to E2 protein.

FIG. 4 is a molecular docking analysis of the binding of a compound of formula I to CD81, wherein: a is a molecular docking analysis chart of the compound of the formula I and CD81 in a whole body, and B is a molecular docking analysis chart of the compound of the formula I and a binding site of CD 81.

FIG. 5 molecular docking analysis of the binding of a compound of formula I to NS5B, wherein: a shows the three pharmaceutically active binding sites of NS5B protein, B shows the binding of the compound of formula I to site 1, C shows the binding of the compound of formula I to site 2, and D shows the binding of the compound of formula I to site 3.

Detailed Description

The essence of the present invention will be better understood by those skilled in the art from the disclosure of the present invention, and the following embodiments are merely examples.

Example 1: preparation and structural identification of compounds

(1) Extraction and separation of compounds

The marine fungus Trichoderma harzianum is inoculated on 100 bottles of 500mL sterilized rice solid culture medium, and is statically cultured for 30 days at 25 ℃, and the mycelium is collected after the culture medium is full of mycelium.

Mashing the rice culture medium with mycelium, adding appropriate amount of ethyl acetate (just by soaking in the culture medium), ultrasonic extracting for 30min, and standing overnight. Filtering to remove residue, and concentrating ethyl acetate under pressure to obtain crude extract. The total extraction is carried out for 3 times, and 15.8g of total extract is obtained after combination.

Separating the total extract by using VLC silica gel column chromatography, mixing the total extract with 160-200 mesh silica gel, performing gradient elution (50: 1-1: 1, v/v) by using a petroleum ether/acetone system as an elution solvent, and combining to obtain 8 fractions. The fraction (3.4g) of fraction 5 was subjected to C-18ODS reverse phase column chromatography, and methanol/water gradient elution was carried out at 20% to 100% (by volume) to obtain a total of 5 fractions. Semi-preparative HPLC purification of fraction 3 (202.3mg) was performed with 35% acetonitrile/water isocratic elution to afford the compound of formula I (5.2mg) and the compound of formula II (2.1 mg).

(2) Structural identification of compounds

Samples were dried using 0.55mL DMSO-d₆The solution was dissolved and loaded into a nuclear magnetic tube for 1D and 2D NMR measurements. Then, the planar structures of the compounds are analyzed according to NMR data, and the relative stereo configurations of the two compounds are determined according to NOE coupling correlation. On this basis, the absolute configuration of the compound was determined by ECD calculation. The physical and chemical parameters are as follows:

a compound of formula I, a colorless powder, [ α ]]²⁵ _D-5.3(c 0.05,MeOH)；IR(KBr)νmax 3420,2925,2854,1739,1679,1460,1376,1248,1123,1042cm^-1；HRESIMS m/z 267.1234[M-H]^－(calcd forC₁₄H₁₉O₅267.1232); the nuclear magnetic data are shown in table 1.

A compound of the formula II [ α ] as a colorless powder]²⁵ _D10.0(c 0.05,MeOH)；IR(KBr)νmax 3366,2922,2851,1714,1681,1451,1374,1205,1138,1023cm^-1；HRESIMS m/z 313.1289[M-H]^－(calcdfor C₁₅H₂₁O₇313.1287); the nuclear magnetic data are shown in table 1.

TABLE 1 preparation of the compounds of formula I and II¹H and¹³c NMR data attribution

Example 2: in vitro anti-HCV transcriptional replication Activity evaluation of Compounds

(1) Cell lines

Huh7.5 cells, a human liver cell line, was gifted by Vertex pharmaceuticals, USA, using a culture Medium of high-sugar Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% inactivated Fetal Bovine Serum (FBS) and 100 units of Penicillin/Streptomycin per ml (Penicillin-Streptomycin, Pen-Strep). Cells in 5% CO₂And culturing in a 37 ℃ cell culture box with saturated humidity.

(2) Virus

The infectious Hepatitis C Virus (HCV) strain is J6/JFH-1/JC. pFL-J6/JFH/JC1 plasmid (gift from Vertex Pharmaceuticals Inc. of USA) containing recombinant full-length HCV cDNA is digested and purified to obtain HCV cDNA, HCV RNA is obtained by in vitro transcription, and normal Huh7.5 cells are transfected to obtain infectious virus liquid.

(3) Control drug

VX-950: is known under the generic name Telaprevir and is an HCV NS3/4A serine protease inhibitor.

PSI-7977: under the generic name Sofosbuvir, is an HCV NS5B polymerase inhibitor.

Both drugs are available from MedChemExpress.

(4) Test method

Designing and preparing the concentration of a compound solution:

compounds of formula I and II were prepared in 10mM stock solution in DMSO, and both Telaprevir and Sofosbuvir were prepared in 20mM stock solution in DMSO, diluted to the desired concentration in DMEM complete medium at the time of use. The compound concentration design was formulated as follows:

in a cytotoxicity experiment and an anti-HCV efficacy experiment of the compound in Huh7.5 cells acutely infected by HCV, the final concentration range of the compound is 0-100 mu M, and the compound is diluted by 3 times; the final concentration range of the Telaprevir and the Sofosbuvir is 0-200 mu M, and the final concentration range is diluted by 5 times.

anti-HCV effects of compounds in acutely HCV infected huh7.5 cells:

1) huh7.5 cells at 3X 10⁴Per cm²The density of (2) is planted into a 96-well plate;

2) after 24h of incubation, the old culture medium was discarded, and HCV infection was performed while adding compound of the corresponding concentration or an equivalent solvent control (MOI 0.1);

3) after 72h incubation, intracellular total RNA was extracted using Qiagen RNeasy Mini Kit, and intracellular HCV RNA levels were measured using qRT-PCR, and half-maximal effective concentrations were calculated using the Reed & Muench method.

Cytotoxicity experiments:

2) after 24h of culture, the old culture medium is discarded, and the cells are treated with 100 μ L of compound solutions with different concentrations;

3) after the drug treatment for 72h, 10 μ L of MTT solution (concentration of 5 μ g/mL) is added to each well and then culture is continued;

4) after 4h of culture, the culture solution was carefully discarded, 150 μ L of DMSO was added to each well, and the mixture was shaken for 15min to sufficiently dissolve formazan crystals; the absorbance at 570nm was then measured at a reference wavelength of 630nm and the half-cytotoxic concentration was calculated using the Reed & Muench method.

Real-time qRT-PCR：

TaqMan probe method real-time fluorescent quantitative PCR, kit AgPath-ID TM One-Step RT-PCRKit was used to quantify housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HCV RNA.

TABLE 2 primer and Probe sequences used in Real time qRT-PCR

2) And a result processing method comprises the following steps:

using GAPDH as internal reference, normalizing CT value of target gene, adopting 2^-△△CTThe method calculates the expression difference of the target gene.

The test result shows that: the anti-HCV activity of the compounds of formula I and II and the positive controls Telaprevir, Sofosbuvir on Huh7.5 cells is shown in the following table:

TABLE 3 anti-HCV Activity of Compounds on Huh7.5 cells

SI is a selection index of CC₅₀And EC₅₀The ratio of (a) to (b). CC (challenge collapsar)₅₀And EC₅₀Are expressed as mean ± standard deviation.

Example 3: evaluation of Activity of Compounds for inhibiting HCV invading cells

(1) Pseudovirion assay

Pseudoviral packaging

Packaging of HCV pseudoviruses: 293T cells in six-well plates were transfected with pCMV-HCV GP at 0.7. mu.g per well; HIV-Luc1 μ g; pAdvantage 0.3. mu.g. Transfection reagent Megatrans1.08. mu.L. After transfection, the solution is changed for 4-6h, and then changed into 3% FBS and DMEM containing TPCK-Trypsin and neuraminidase. After culturing for 72h, the supernatant was harvested and filtered through a 0.45 μm filter, and the supernatant was stored frozen at-80 ℃ for further use. VSV pseudovirus packaging procedures were as above, except that the pCMV-HCV GP was replaced with a plasmid expressing the VSVG protein.

Experiment of pseudovirus infection

The established pseudovirus model can be used for screening antiviral drugs taking virus entering cells as targets. The specific experimental steps are as follows:

1) 1 day in advance, 293T cells or A549 cells were passaged to 96-well plates at 5X 10 per well³Culturing the cells at 37 ℃;

2) mixing a compound with a certain concentration with viruses, adding the mixture into cells, and culturing the cells at 37 ℃ for 72 hours;

3) measurement of luciferase Activity: the Bright-Glo detection kit is used for detecting the activity of luciferase, and the operation method is shown in the product instruction book. Calculating the inhibition ratio and IC of the compound according to the measured signal value₅₀. Percent inhibition ═ percent (blank fluorescence reading-compound added fluorescence reading)/blank fluorescence reading.

The results of the experiments (see FIG. 1) show that the compounds of formula I and formula II inhibit HCV invasion of host cells.

SPR (surface plasmon resonance) experiment

In order to further determine the action target of the compounds shown in the formula I and the formula II for inhibiting HCV invasion of host cells, the binding capacity of the small molecule compounds with an HCV membrane protein E2 receptor and a host factor CD81 is tested by an SPR experiment.

The experimental results are shown in fig. 2 and fig. 3, which show that the compounds of formula i and formula ii can act on the binding site of E2 on the LEL (extracellular large loop region) of the host factor CD81 protein, and further block the binding of the receptor of the HCV membrane protein E2 with the host factor CD81, and further exert the function of resisting virus invasion.

Example 4: molecular docking experiments confirm the action target of the compound

The anti-HCV target of the compound is confirmed by utilizing a molecular docking experiment. According to the two-dimensional conformation of the compound to be detected in the formula I, a LIG molecular structure is drawn by using ChemOffice software, and then the LIG molecular structure is converted into a mol2 format by using Open Babel software and is subjected to structure optimization for later use.

Small molecule compounds were subjected to mock docking analysis with HCV critical target proteins NS3/4A, NS5A, NS5B, E2, and host factor CD 81. The results are shown in fig. 4 and fig. 5, the compound of formula i can be well combined with the drug action targets of CD81 and NS5B, the results of docking analysis are consistent with the data of in vitro activity tests in examples 2 and 3, and the results of analysis of the compound of formula II are similar, further confirming that the compounds of formula i and formula II are a multi-target anti-HCV active molecule which can act on both host factor CD81 and virus NS 5B.

SEQUENCE LISTING

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Claims

1. A compound of formula I and formula II:

2. a process for the preparation of a compound according to claim 1, comprising the steps of:

1) taking Trichoderma harzianum as an engineering bacterium, and obtaining a fermentation product through fermentation culture;

2) extracting and concentrating the fermentation product, and separating and purifying by chromatography to obtain the compounds of formula I and formula II.

3. The method according to claim 2, wherein the Trichoderma harzianum (Trichoderma harzianum) is inoculated to the solid medium in the step 1) and cultured.

4. The method according to claim 3, wherein the solid medium is a rice solid medium, and the solid medium is inoculated and then subjected to fermentation culture at room temperature for 30 to 40 days.

5. The preparation method according to claim 2, wherein step 2) is that mycelium and culture medium are crushed, then extracted by ethyl acetate ultrasound, kept stand overnight, filtered to remove filter residue, concentrated and extracted for many times to obtain fermented extract; and then sequentially carrying out silica gel column chromatography, reversed phase column chromatography separation and HPLC purification on the fermentation extract, and preparing the compounds of the formula I and the formula II from the fermentation extract.

6. The preparation method according to claim 5, wherein the silica gel column chromatography is vacuum liquid chromatography, and a system of 160-200 mesh silica gel particles and a petroleum ether/acetone volume ratio of 50: 1-1: 1 is used as an elution solvent for gradient elution.

7. The method according to claim 5, wherein the reverse phase column chromatography is performed using a C-18ODS reverse phase column,

gradient elution with 20-100% methanol/water.

8. The method of claim 5, wherein the HPLC purification is performed using semi-preparative HPLC, with 35% acetonitrile/water isocratic elution.

9. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of hepatitis c.