CN112294806A

CN112294806A - Application of 1-formyl-beta-carboline derivative in preparation of anti-newcastle disease virus drugs

Info

Publication number: CN112294806A
Application number: CN202011154489.XA
Authority: CN
Inventors: 王俊儒; 王重阳; 杨增岐; 李娜; 代江坤; 王婷; 安治远; 胡若辰
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-02-02
Anticipated expiration: 2040-10-26
Also published as: CN112294806B

Abstract

The 1-formyl-beta-carboline derivative is applied to preparing the anti-newcastle disease virus medicine. The molecular structure of the related 1-formyl-beta-carboline derivative is shown as formulas (I), (II) and (III); in-vitro experimental research proves that the derivative can play an obvious inhibiting role in the early and later stages of the proliferation of the Newcastle disease virus, can play an antiviral role in related cell lines, can obviously inhibit the proliferation of 3 NDV with different genotypes, and has higher inhibiting rate along with the increase of concentration; meanwhile, the compound has low cytotoxicity to DF-1, and has the potential of being applied to resisting Newcastle disease virus alone or in combination with other medicines.

Description

Application of 1-formyl-beta-carboline derivative in preparation of anti-newcastle disease virus drugs

Technical Field

The invention belongs to a new antiviral application of 3 1-formyl-beta-carboline derivatives, and particularly relates to an application of the derivatives in preparation of a medicament for resisting Newcastle disease viruses.

Background

Newcastle Disease Virus (NDV), a member of the family paramyxoviridae, a enveloped, non-segmented, single-stranded negative-strand RNA Virus that infects a variety of hosts, including humans. Among them, hendra virus (HeV) and Nipah virus (NiV) cause extremely high mortality after infecting humans, posing a great threat to human health.

Newcastle Disease virus can infect poultry and over 200 birds, and Newcastle Disease (ND) caused by the Newcastle Disease virus is one of the most important diseases endangering the poultry industry and is listed as an epidemic Disease which must be reported by the world animal health Organization (OIE). China is the country with the largest number of chickens in the world, and the development of poultry industry in China is seriously harmed by Newcastle disease.

The beta-carboline alkaloid is widely present in plants, animals and microorganisms, and has good biological activity. Deyan Chen et al (2015) reported that harmine inhibits enterovirus type 71 activity; veranica m.quintana et al (2016) reported the activity of harmine and its derivatives in inhibiting dengue virus; paromita Bag et al (2014) reported that beta-carboline alkaloids inhibit replication of herpes simplex virus type I.

Disclosure of Invention

Based on the new research findings of the inventor, the invention provides the application of the 1-formyl-beta-carboline derivatives in preparing the anti-newcastle disease virus drugs, wherein the structures of the 1-formyl-beta-carboline derivatives are shown as formulas (I), (II) and (III):

preferably, the concentration of the 1-formyl-beta-carboline derivatives in the medicine is 5-30 mu M.

Further, the 1-formyl-beta-carboline derivatives can be used alone or in combination with other drugs.

Furthermore, the 1-formyl-beta-carboline derivatives can resist the proliferation of the Newcastle disease virus by directly inhibiting the adsorption of the Newcastle disease virus.

On the other hand, the invention also provides a medicament for resisting the Newcastle disease virus, and the effective component of the medicament provided by the invention comprises one or more than two compositions of the compounds shown in the structural formulas (I), (II) and (III).

The 1-formyl-beta-carboline derivatives have the highest inhibition rate of over 95 percent on the Newcastle disease virus, and have no cell specificity in antiviral action; after the 3 derivatives are treated, the virus titer in the cell supernatant and the virus mRNA in the cells are obviously reduced; and the compound can be added in the early and later stages of the proliferation of the Newcastle disease virus to play an inhibiting effect.

The 1-formyl-beta-carboline derivatives can be used as NDV adsorption inhibitors to directly inhibit the adsorption of Newcastle disease viruses, so that the virus proliferation is inhibited at an early stage, and the inhibition rate is 33-45%.

Drawings

Figure 1 shows that the derivative can significantly inhibit the expression of NDV P mRNA after being treated.

FIG. 2 shows that NDV proliferation can be significantly inhibited by adding the derivative to different cell lines.

FIG. 3 shows that the derivatives can be added at different times after NDV vaccination to significantly inhibit the proliferation of Newcastle disease virus, and FIGS. 3A, 3B, and 3C show the antiviral effects of compounds I, II, and III, respectively.

FIG. 4 shows that the derivatives inhibit the adsorption of Newcastle disease virus.

FIG. 5 is a graph showing the results associated with comparative example compounds 1-9 failing to inhibit NDV proliferation (F48E 9).

FIG. 6 is a graph showing the results associated with the inability of comparative compounds 1-9 to inhibit NDV proliferation (PPMV-1/SX-01/Ch/15).

Detailed Description

The technical solution of the present invention is further explained by the following description, and the examples provided are only illustrative of the method of the present invention and do not limit the remaining relevant contents of the present disclosure in any way.

The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby. Unless otherwise specified, the examples follow conventional experimental conditions.

The compounds I to III and the compounds 1 to 9 used in the following examples were synthesized by the inventors themselves according to the methods which have been disclosed in the prior art. Other experimental materials were purchased from commercial products.

Example 1

This example demonstrates in vitro NDV-resistant EC for compounds (I) - (III)₅₀The values were determined:

four strains of Newcastle disease viruses (provided by animal medical college at northwest university of agriculture and forestry) with different virulence and different genotypes (Blackbird/China/08, PPMV-1/SX-01/Ch/15, La Sota and F48E9) are respectively used for testing EC by a plaque assay₅₀The value is obtained.

Inoculating DF-1 cells (purchased from American ATCC) into a 12-well plate, infecting the cells with four strains of Newcastle disease virus (MOI is 0.01) after the cells grow to 70-80%, adding DMEM culture solutions (purchased from Gibco company in America) containing compounds I, II and III with different concentrations after virus infection, and continuing incubation and culture for 24 h;

collecting cell supernatant, diluting in gradient, and performing plaque test: BHK-21 cells (purchased from ATCC in USA) were cultured in 12-well plates, and after 70-80% of the cells were infected with the diluted sample at 37 ℃ for 1h, the supernatant was discarded, and a fresh medium containing methylcellulose (1%) was added, and the results were observed after 72 h.

Calculating EC according to the statistical result by applying GraphPad Prism software₅₀The values, results are shown in Table 1. EC of said compound₅₀The values are all lower than 16 mu M, the antiviral effect can be better exerted under the concentration without cytotoxicity, and the inhibition effect on 3 genotypes of strains with different virulence is better.

TABLE 1 EC of Compounds (I) to (III) for 4 strains of NDV₅₀Value (μ M)

Comparative example:

this example performed an in vitro anti-NDV activity assay on compounds 1-9, and the results showed that none of compounds 1-9 had anti-NDV activity:

the specific method comprises the following steps: inoculating DF-1 cells into a 12-well plate, infecting the cells with F48E9 or PPMV-1/SX-01/Ch/15 (MOI is 0.01) after the cells grow to 70-80%, adding culture solution containing different compounds 1-9 with different concentrations after virus infection, and continuing incubation and culture for 24 h; collecting cell supernatant, diluting in gradient, and performing plaque test: BHK-21 cells were cultured in 12-well plates, and after 70-80% growth, the cells were infected with the diluted sample at 37 ℃ for 1h, the supernatant was discarded, and a fresh medium containing methylcellulose (1%) was added, and the results were observed after 72 h. The results are shown in FIG. 5(F48E9) and FIG. 6 (PPMV-1/SX-01/Ch/15).

Example 2:

this example is a related assay for cytotoxicity of compounds (i) - (iii):

the cytotoxic effect of the compound on DF-1 cells was tested using a CCK-8 kit (purchased from Shanghai ceramic Biotechnology Co., Ltd.); DF-1 cells were cultured in a 96-well plate, and after the cells grew to 70-80%, 100. mu.L of DMEM medium containing compounds I, II, III (5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, 150. mu.M and 200. mu.M) at different concentrations was added, and 5 wells were repeated at each concentration, and after culturing was continued for 48 hours, 10. mu.L of CCK-8 reagent was added to each well. At 37 ℃ 5% CO₂After culturing for 1h in the dark, the absorbance at 450nm was measured. And calculating the inhibition rate according to the absorbance value. The inhibition rate calculation formula is as follows:

cell inhibition ratio (%) ═ 1- [ a (dosed) -a (blank) ]/[ a (0 dosed) -a (blank) ] × 100%

Calculation according to inhibition ratio by GraphPad Prism softwareCC₅₀The values and results are shown in Table 2. Shows the Compound CC₅₀The values are all higher than 50 mu M, and the toxicity is lower.

TABLE 2 CC of Compounds (I) - (III) in DF-1 cells₅₀Value (μ M)

Example 3

This example demonstrates that compounds (i) - (iii) significantly inhibit NDV P mRNA expression:

inoculating DF-1 cells into a 12-well plate, infecting the cells with F48E9 (MOI is 0.01) after the cells grow to 70-80%, adding culture solutions containing compounds I, II and III with different concentrations (5 mu M, 10 mu M and 20 mu M) after virus infection, and further incubating and culturing for 24 h;

adding TRIzol (purchased from Takara Bio), extracting RNA according to the instruction, and reverse-transcribing the RNA into cDNA using StarScript II First-strand cDNA Synthesis Kit-II (purchased from Genstar);

finally, the NDV P mRNA expression level was measured by real-time fluorescent quantitative PCR (qPCR) using a 2 × RealStar green power mix kit (available from Genstar) according to the instructions.

The results of figure 1 show that the compounds I-III can obviously inhibit NDV P mRNA expression, and the inhibition effect is better with the increase of the compound concentration, and can reach more than 95 percent at most.

Example 4:

this example demonstrates that compounds (i) - (iii) significantly inhibit NDV P mRNA expression on different cell lines:

inoculating Hela, Vero or BHK-21 cells (purchased from ATCC in USA) into 12-well plate, infecting with F48E9 (MOI 0.01) after the cells grow to 70-80%, adding culture solution containing compounds I, II and III with concentration of 20 μ M after virus infection, incubating and culturing for 24h, and collecting cell sample; after RNA extraction and reverse transcription, NDV P mRNA expression was detected using qPCR. The results in fig. 2 show that the compound can significantly inhibit the expression of NDV P mRNA in different cell lines, and the inhibition rate of 3 compounds is more than 90% at the concentration.

Example 5:

this example demonstrates that compounds (i) - (iii) inhibit NDV proliferation when added at various stages of NDV proliferation:

DF-1 cells are inoculated on a 12-well plate, after the cells grow to 70-80%, compounds (I) - (III) are respectively added at 6h and 2h before inoculation of the virus or at 2h, 4h, 8h and 12h after inoculation of the virus (F48E 9; 0.01MOI), cell supernatants are collected 24h after inoculation of the virus, after gradient dilution, the virus titer is detected by using a plaque test, and the result is shown in 3.

FIGS. 3A, 3B, and 3C show the antiviral effects of compounds (I), (II), (III), respectively, and the results show that the addition of the compounds in both early and late stages of Newcastle disease Virus propagation exerts an inhibitory effect, with the best effect of adding the derivatives as soon as possible after Newcastle disease Virus infection.

Example 6:

this example demonstrates that compounds (i) - (iii) significantly inhibit virus-adsorbed cells:

BHK-21 cells were inoculated into a 12-well plate, after 70-80% of the cells had grown, they were placed at 4 ℃ for 1h with a pre-cool, then infected with F48E9 (200PFU) and compounds I, II, III (20. mu.M) were added at the same time; after incubation at 4 ℃ for 1h, the supernatant was discarded, and washed three times with precooled PBS, followed by addition of fresh medium containing methylcellulose (1%), and the results were observed after 72 h.

The results are shown in FIG. 4, which demonstrates that the compound can directly inhibit the adsorption of the newcastle disease virus to BHK-21 cells, and the inhibition rates of the compounds (I) to (III) are 43.5%, 44.5% and 33%, respectively.

Claims

The application of the 1-formyl-beta-carboline derivatives in preparing the anti-newcastle disease virus drugs is disclosed, wherein the 1-formyl-beta-carboline derivatives have the structures shown as formulas (I), (II) and (III):
2. the use of claim 1, wherein the concentration of the 1-formyl- β -carboline derivative in the medicament is 5-30 μ M.
3. The use of claim 1, wherein the 1-formyl- β -carboline derivative is used alone or in combination with other drugs.
4. The use according to claim 1, wherein the 1-formyl- β -carboline derivative is resistant to the proliferation of newcastle disease virus by directly inhibiting the adsorption of newcastle disease virus.
5. The medicament for resisting the Newcastle disease virus is characterized in that the effective component of the medicament comprises one or more than two compositions of compounds shown in structural formulas (I), (II) and (III).