CN106580980B

CN106580980B - Application of the aromatic ester compound in preparing anti-enterovirns type 71 drug

Info

Publication number: CN106580980B
Application number: CN201611192748.1A
Authority: CN
Inventors: 魏艳红; 奚彩丽; 胡康洪; 张谦; 李栋; 尧晨光
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2016-12-21
Filing date: 2016-12-21
Publication date: 2018-11-13
Anticipated expiration: 2036-12-21
Also published as: CN106580980A

Abstract

The invention belongs to antiviral drugs fields.It provides Novel series aromatic ester compound and is preparing anti-enterovirns type 71（EV71）Application in drug.The aromatic ester compound is formula WY59, WY112 compound represented.By Novel series pyridine aromatic ester compound for EV71 inhibitory activity research experiments, WY59, WY112 inhibit the cytopathic effect that EV71 is generated on host cell RD（CPE）, enhance cell survival rate, reduce progeny virus production, and can inhibit Apoptosis of Host Cells caused by EV71 infection, mainly act on duplication multiplicative stages of the EV71 in host cell.Show that these novel aromatic ester type compounds there are the potentiality for preparing anti-EV71 virus drugs, these compound synthesis technologies are simple, economical quickly to be easy to mass produce, and have potential applicability in clinical practice.

Description

Application of the aromatic ester compound in preparing anti-enterovirns type 71 drug

Technical field

The invention belongs to antiviral drugs fields, and in particular to aromatic ester compound is preparing anti-enterovirns type 71 Application in drug.

Background technology

Enterovirns type 71 (EV71) is Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus) member is to cause one of main pathogen of Children, sometimes with serious maincenter god Through systematic complication, including aseptic meningitis, encephalitis, polio sample is benumbed, and nerve cardiopulmonary failure etc. even results in It is dead.Since 1974 report for the first time, EV71 infectious diseases has worldwide repeatedly been broken out and prevalence, in Asia-Pacific The situation is tense for especially China of area.The very big danger brought to our people's life and health in view of the spread and epidemic of hand-foot-and-mouth disease Evil, hand-foot-and-mouth disease was classified as Class C infectious disease in 2008 and is included in management by the Chinese government, and formulated a series of correlation method laws Rule, the popular of hand-foot-and-mouth disease of keeping under strict control are propagated.There is no specific medicament, relevant vaccine for the disease treatment infected by EV71 at present It was just listed in 2015, preventive effect need further to investigate.Therefore special effective anti-EV71 drugs gesture is developed must Row.

Ester type compound is a kind of important fine chemical product, is widely used in drug, material, food, plasticizer, molten The chemical industries such as agent, aromatic ester can be widely used for fragrance, preservative, solvent, drug, dyestuff, coating and materials synthesis etc.. Project team where the applicant, which has independently synthesized six kinds, has new structural fragrant ester compounds, and in 2015 in periodical Tetrahedron Letters disclose the preparation method of these types of fragrant ester compounds.Its biological activity is not commented Valence.

Invention content

The object of the present invention is to provide application of the aromatic ester compound in the drug for preparing anti-enterovirns type 71, institutes Aromatic ester compound WY59, WY112 stated, structural formula such as following formula：

The present invention cellular level find WY59, WY112 can with cytopathic effect caused by strong inhibition EV71 viruses, Enhance the survival rate of infection cell, reduce progeny virus production, and Apoptosis caused by virus infection can be inhibited, mainly Act on duplication multiplicative stages of the EV71 in host cell.

Further, the aromatic ester compound is preferably formula WY112.

Second object of the present invention is to provide a kind of drug of anti-enterovirns type 71, including the conduct of effective dose is lived The compound WY59 or WY112 or their salt of sexual element, and pharmaceutically acceptable carrier.

Further, the pharmaceutical preparation is granule, tablet, pill, capsule, injection or dispersant.

Being indicated above compound WY59, WY112 has the potentiality for the specific therapy drug for preparing anti-EV71 infection, has clinic Application prospect.

The preparation of the Novel series aromatic ester compound of the present invention, reference literature Tetrahedron Letters 2015,56,6136-6141 method, specifically using transition metal palladium as catalyst, under the ortho position inducing action of pyridine, in virtue The ortho position of ring is acted on high price iodobenzene, is carried out aryl acyloxy, is obtained final esterification products.

The present invention has the following advantages：

1, these compound synthesis low in raw material price, are easy to buy；Synthesis technology is simple, economical quick, is easy to advise greatly Mould production is promoted.

2, anti-EV71 drugs are found from the similar compound of structure, are easy to confirm that its acts on target by structure-activity relationship discussion Point provides valuable guiding role for further drug development.

Description of the drawings

Fig. 1 is influence of six kinds of novel aromatic ester type compounds for the EV71 RD cell survival rates acted on.

Fig. 2 is the depression effect of WY59 and WY112 for RD cells CPE caused by EV71.

Fig. 3 is inhibiting effect of the WY112 for EV71 progeny virus productions.

Fig. 4 is inhibiting effect of the WY112 for RD Apoptosis caused by EV71.

Fig. 5 is the inhibitory activity for EV71 under the processing of WY112 difference dosing methods.

Specific implementation mode

By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.

Hereinafter, if not specified, material therefor of the present invention and operating method are well known in the art.

【Embodiment 1】The synthesis of novel aromatic ester type compound

The present invention has synthesized following six kinds of novel aromatic ester type compounds, reference literature Tetrahedron Letters 2015,56,6136-6141 method, specifically using transition metal palladium as catalyst, under the ortho position inducing action of pyridine, in virtue The ortho position of ring is acted on high price iodobenzene, is carried out aryl acyloxy, is obtained final esterification products.

【Embodiment 2】Six kinds of anti-EV71 activity of pyridine aromatic ester compound are assessed

1, test method：

Toxicity of 1.1 compounds for host's RD cells

By 96 orifice plate of RD plating cells, at 37 DEG C, 5%CO₂After single layer is covered in incubator culture, cell culture fluid is discarded, The cell maintenance medium for testing compound respectively plus containing various concentration continues to cultivate, microscopic visual measurement and to record it respectively thin after 48h Cellular toxicity, mtt assay measure cell survival rate.11.5 softwares of SPSS calculate median toxic concentration (Median of the drug for cell Cyctoxic concentration, CC50).Cell survival rate=(medicine group is averaged OD₄₉₂Value/cell controls group is averaged OD₄₉₂ Value) × 100%.

Inhibitory activity of 1.2 compounds for EV71

By 96 orifice plate of RD plating cells, at 37 DEG C, 5%CO₂After single layer is covered in incubator culture, culture solution is discarded, The EV71 virus liquid infection cell 1h of 100TCID50, the test compound of various concentration is added, and (Ribavirin is as positive control Drug) incubated cell.It waits continuing to cultivate about 48h, when 90% or so CPE lesions occur in virus control wells, microscopically observation Cytopathic effect (CPE).CPE's observes and records method：Acellular lesion is denoted as-, 25% or less cytopathy is denoted as+, 25%-50% cytopathies are denoted as ++, 50%-75% cytopathies are denoted as +++, 75% or more cytopathy is denoted as ++++.

After CPE is observed, inhibiting rate of the drug to EV71 is detected using MTT methods.The specific steps are：It is added per hole MTT 50μL(5mg·mL^-1), remove supernatant after being incubated 3-4h, isometric DMSO dissolving precipitations are added.Existed with microplate reader Corresponding absorbance (OD is read at 492nm₄₉₂Value).Inhibiting rate of the drug to EV71 is calculated using following formula.Use SPSS 11.5 softwares calculate the medium effective concentration (Concentration for 50%of maximal effect, EC50) of drug.

The therapeutic index (SI) of 1.3 drugs

SI=CC50/EC50.Therapeutic index is higher, illustrates that antiviral potentiality are bigger.

2, test result

Table 1 has new structural aromatic ester Compound Cytotoxicity and anti-EV71 activity

a"-"：Inhibition or invalid less than 50%

The results are shown in Table 1 for Compound Cytotoxicity and anti-EV71 active testings.The compound of concentration dependant is for EV71 The influence of the RD cell survival rates of effect is as shown in Figure 1.The invention detects that WY59 and WY112 has strong inhibition to live EV71 Property, it is better than positive reference compound Ribavirin.Wherein WY112 has better inhibition (IC50=7.56), higher to control Index is treated, shows big application potential.Other compounds do not detect anti-EV71 activity or inhibition less than 50%.Change It is as shown in Figure 2 to close RD cell CPE effects caused by object WY59 and WY112 inhibit EV71.The RD cell roundings aggregation of EV71 infection, It is detached from from cell wooden partition, WY59 and WY112 (40 μ g/mL) processing have certain inhibiting effect, WY112 can its lesion effect RD cytopathic effects caused by completely inhibit EV71, WY59 have 67% inhibitory activity.

【Embodiment 3】Inhibiting effect of the WY112 for EV71 progeny virus productions

1, test method

24 orifice plate of RD plating cells of exponential phase, covers with 100TCID after single layer₅₀EV71 infection cells, 37 DEG C of incubations Virus liquid is removed after 1.5h, PBS is washed three times, and the cell maintenance medium containing 40 μ g/mL WY112 is added.After 48h collect cell and Supernatant culture solution, -20 DEG C and 37 DEG C three times after freezing-thawing and cracking, TCID₅₀Method measures EV71 virus titers.

2, test result

As shown in figure 3, the RD cells of 40 μ g/mL WY112 processing are relative to virus control group, under virus titer is notable Drop, illustrates compound for the strong inhibiting effect of EV71 progeny virus productions.

【Embodiment 4】Inhibiting effect of the WY112 for RD Apoptosis caused by EV71

1, test method

24 orifice plate of RD plating cells of exponential phase covers with 100TCID50EV71 infection cells after single layer, 37 DEG C of incubations Virus liquid is removed after 1.5h, and the cell maintenance medium containing 40 μ g/mL WY112 is added.After about 48h, cell is collected, is used Annexin V-FITC/PI apoptosis detection kits carry out the detection of Apoptosis on flow cytometer.

2, test result

Experimental result such as Fig. 4 shows that WY112 can effectively inhibit Apoptosis caused by EV71.It is thin in virus control group Born of the same parents' apoptosis rate is 95.62%, in the case of normal untreated cell apoptosis rate 8.02%, the cell of 40 μ g/mL WY112 processing Apoptosis rate only has 8.85%.It can be seen that WY112 can be with Apoptosis caused by effective protection EV71.

【Embodiment 5】The anti-EV71 active pharmacologicals researchs of compound WY112

1, test method

1.1 WY112 analyze the direct killing effect of EV71

10⁴TCID₅₀EV71 suspensions and 100 μ g/mL WY112 of compound be incubated for 24 hours in 4 DEG C of refrigerators, viral suspension dilution 100 multiples, the effective concentration for making its be less than compound for HIV suppression, titration adherent cover with single layer in ready in advance RD cells in, pass through Reed and Muench methods calculate viral suspension TCID₅₀Value.

Analyses of 1.2 WY112 for the EV71 modes of action

Pass through three kinds of different dosing methods (before EV71 infection, test compound is added between infection, after infection) point It Ce Ding not influence of the compound for EV71.

(1) prevention effect：Compound effects are added before EV71 infection.

After RD cells cover with single layer in 96 orifice plates, culture solution is abandoned in suction, and WY112 containing various concentration is added and is incubated 2h, discards After drug culture solution, 100TCID₅₀EV71 suspensions adsorb 1h, remove viral suspension, cell culture maintenance medium added to continue to train It supports.Cell CPE is observed when 48h, measures cell survival rate.

(2) inhibit suction-operated:Compound is added during EV71 infects.

By EV71 suspensions and various concentration WY112 mixings, direct titration is in the RD cells for having covered with 96 orifice plate of single layer, and 37 DEG C, 5%CO₂After incubator adsorbs 1h, infection liquid is discarded, cell maintenance medium is added to continue to cultivate.Cell survival rate is measured when 48h.

(3) therapeutic effect：Compound is added after EV71 infection.

After RD cells cover with single layer in 96 orifice plates, culture solution, 100TCID are abandoned in suction₅₀EV71 suspensions adsorb 1h, discard disease Venom is added WY112 containing various concentration and continues to cultivate, cell CPE and survival rate are detected when 48h.

2, test result

The external direct killing EV71 effects of 2.1 compound WY112

The processed EV71 suspensions of compound WY112 infect RD cells, and 48h detects its virus titer.As a result, it has been found that drug Processing group compares virus control group, and virus titer illustrates that WY112 does not have and directly kill in vitro without significant change (data are not shown) Hinder EV71 effects.

2.2 compound WY112 inhibit the mode of action of EV71

The results show that WY112 pretreatment cells infect without apparent CPE depression effects (Fig. 5) subsequent EV71, WY112 and EV71 is incubated this dosing method of infection cell and obtains similar as a result, illustrating that WY112 cannot be by making in advance simultaneously Inhibit EV71 for cell, i.e., for virus infection without prevention effect, and WY112 for EV71 adsorptive internalization without apparent shadow It rings.Compound WY112 is added after EV71 infection, as a result shows for inhibitory activity strong EV71, can almost press down completely Thus cytopathic effect (Fig. 5) caused by EV71 processed illustrates that the duplication after WY112 main functions EV71 infects in the cell increases Grow process, such duplication proliferation that can be used for preparing with new structural ester type compound after anti-EV71 infects in the cell The drug of process.

In conclusion it is active with stronger inhibition EV71 with new structural ester type compound WY59 and WY112, Middle WY112 has more preferably inhibition.WY112 mainly acts on duplication breedings of the EV71 in RD cells, potential Prepare a kind of drug clinically effectively antagonizing EV71 infection.

Claims

1. application of the aromatic ester compound in the drug for preparing anti-enterovirns type 71, which is characterized in that the aromatic ester Class compound is WY59, WY112, structural formula such as following formula：

2. application according to claim 1, which is characterized in that the aromatic ester compound is WY112.

3. a kind of drug of anti-enterovirns type 71, which is characterized in that include the compound as active ingredient of effective dose WY59 or WY112 or their salt, and pharmaceutically acceptable carrier.

4. the drug of anti-enterovirns type 71 according to claim 3, which is characterized in that the pharmaceutical preparation is particle Agent, tablet, pill, capsule, injection.