CN105748474A - Gramine analogue with heterocyclic structure and application of gramine analogue with heterocyclic structure to preparation of anti-CVB3 virus medicines - Google Patents

Gramine analogue with heterocyclic structure and application of gramine analogue with heterocyclic structure to preparation of anti-CVB3 virus medicines Download PDF

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CN105748474A
CN105748474A CN201510954297.XA CN201510954297A CN105748474A CN 105748474 A CN105748474 A CN 105748474A CN 201510954297 A CN201510954297 A CN 201510954297A CN 105748474 A CN105748474 A CN 105748474A
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cvb3
gramine
analog
virus
analogue
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魏艳红
柯少勇
杨自文
石丽桥
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Hubei University of Technology
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Hubei University of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone

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  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

The invention relates to a gramine analogue with a heterocyclic structure and application of the gramine analogue with the heterocyclic structure to preparation of anti-CVB3 virus medicines.A chemical formula of the gramine analogue with the heterocyclic structure is as shown in the specification.According to anti-CVB3 activity research experiments of the gramine analogue with the heterocyclic structure, anti-CVB3 pharmacological researches and experiments of CVB3 replication inhibition effect of an analogue G19, the gramine analogue with the heterocyclic structure inhibits CPE (cytopathic effect) of CVB3 on a host cell Hep-2, increases cell survival rate, inhibits progeny virus production and viral nucleic acid generation, mainly inhibits an early replication stage of CVB3 viruses in the host cell and can be applied to preparation of the anti-CVB3 virus medicines.

Description

There is the application in preparing anti-CVB3 virus drugs of the Gramine analog of heterocycle structure
Technical field
Antiviral drugs technical field of the present invention, relates to the application in preparing anti-CVB3 virus drugs of a kind of Gramine analog with heterocycle structure.
Background technology
Coxsackie virus (Coxsaekievirus) is Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus) member, its infection can cause multiple disease, such as hand-foot-mouth disease, aseptic meningitis, encephalitis, myocarditis, epidemic myositis pain, herpangina etc..The CV reported has 29 serotypes, according to its pathogenic characteristic to neonatal rat and the difference to cellular sensitivity, can be classified as A and B two groups, i.e. CVA (CVA1-22,24) and CVB (CVB1-6).Infection with CVBs is most commonly seen, and wherein CVB3 the strongest pathogenic type in being 6 serotypes of CVB, is the topmost pathogenesis of viral myocarditis.According to disease prevention and control center of the U.S. (CDC) statistical number it has been found that CVB (1-6 type) may result in about 5,000,000 people every year suffers from intestinal tract disease, wherein 10%-20% is the acute myocarditis caused by CVB3.CVB3 causes the trend of hand-foot-mouth disease also rising in recent years, also has the diseases induced popular report of a lot of CVB3 in China.At present for Coxsackie virus infection without specific medicament, clinic there is no treatment means targetedly.Many researchers have found numerous compound in vivo and in vitro with suppression CVB3 activity, but these are substantially also in the primary stage of laboratory test, and distance actual application clinically is the remotest.Therefore special effective anti-CVB3 medicine is developed imperative.
From the chemical compound lot of different Source of Drug Plants, the antiviral activity strong with it and hypotoxicity are widely studied.Gramine is as natural indoles alkaloid, separated from various plant materials and coal tar, present pharmaceutically active widely, such as relax bronchial smooth muscle, vasodilation, alleviate bronchitis and bronchial asthma, but find no the report of antiviral activity so far.Up to the present, Gramine is widely used as the leading support containing indoles structural compounds, and synthesis has the compound of various biologic activity.Many is had the analog of splendid biologic activity and is synthesized by different approach.The applicant discloses series in periodical European Journal of Medicinal Chemistry in the research work of early stage and has the Gramine analog of heterocycle structure, and it was found that this compounds has certain antitumor action.
Summary of the invention
It is an object of the invention to for above-mentioned present situation, it is desirable to provide there is the application in preparing anti-CVB3 virus drugs of the Gramine analog of heterocycle structure.
The implementation of the object of the invention is, has the application in preparing anti-CVB3 virus drugs of the Gramine analog of heterocycle structure.
The Gramine analog with heterocycle structure has following chemical structural formula
In formula:
R1For the one in hydrogen, methyl, ethyl, benzyl or substituted benzyl；
R2For the one in hydrogen, phenyl, 2-chlorphenyl, 4-chlorphenyl, 2-fluorophenyl, 4-fluorophenyl, 4-trifluoromethyl, 2-trifluoromethyl, pyridine-2-base, pyridin-4-yl, 2-hydroxy phenyl, 4-hydroxy phenyl；
R3For the one in hydrogen, methyl；
R4For the one in methyl, pyridine-2-base, benzothiazole-2-base；
Or R3And R4Collectively form
The applicant passes through substantial amounts of biological experiment, finds to have a lot of compounds in the Gramine analog of heterocycle structure and has the activity of anti-CVB3 virus.It is embodied in the cytopathic effect that can cause with strong inhibition CVB3 virus, strengthens the survival rate of infection cell, suppression progeny virus production and the synthesis of viral nucleic acid.And mainly act on the early stage duplicate stage after CVB3 infection cell.It is indicated above the potential specific therapy medicine preparing anti-CVB3 infection of Gramine analog with heterocycle structure, there is big potential applicability in clinical practice.
The invention have the advantages that
The Gramine analog 1, with heterocycle structure is cheap, it is easy to buy；Synthesis technique is simple, economical quick, it is easy to large-scale production is promoted.
2, numerous Gramine analog with heterocycle structure have the inhibitory activity for CVB3, it is easy to therefrom find optimum medicine efficacy, are suitable for the compound of clinical practice；And its action target spot can be searched out by structure activity study, provide valuable guide effect for preparing drug development further；
The Gramine analog 3, with heterocycle structure has antitumor and antiviral activity concurrently, and this can lower the risk of later development application significantly, and antitumor and the using value of antiviral activity medicine and the market competitiveness are prepared in raising.
Accompanying drawing explanation
Fig. 1 is the depression effect of the Hep-2 cell CPE that Gramine and the like causes for CVB3,
Fig. 2 is the inhibitory activity under the different dosing method of Gramine and the like processes for CVB3,
Fig. 3 is that different time points adds the Gramine and the like the depression effect for CVB3,
Fig. 4 is the impact that Gramine analog G19 replicates in Hep-2 cell for CVB3.
Detailed description of the invention
The preparation of the Gramine analog with heterocycle structure of the present invention, reference literature European Journal of Medicinal Chemistry, 2012,54, the method of 248, specifically with Gramine as initiation material, uses and can facilitate preparation without catalyst one kettle way.
The present invention relates to the application in preparing anti-CVB3 virus drugs of the Gramine analog with heterocycle structure.
Described application refers to that the Gramine analog with heterocycle structure adds the acceptable adjuvant of pharmaceutics and carrier, for preparing the preparation of anti-CVB3 virus.
Described preparation is granule, tablet, pill, capsule, injection or dispersant.
The present invention has the Gramine analog of heterocycle structure and has following chemical structural formula
In Formulas I:
R1For the one in hydrogen, methyl, ethyl, benzyl or substituted benzyl；
R2For the one in hydrogen, phenyl, 2-chlorphenyl, 4-chlorphenyl, 2-fluorophenyl, 4-fluorophenyl, 4-trifluoromethyl, 2-trifluoromethyl, pyridine-2-base, pyridin-4-yl, 2-hydroxy phenyl, 4-hydroxy phenyl；
R3For the one in hydrogen, methyl；
R4For the one in methyl, pyridine-2-base, benzothiazole-2-base；
Or R3And R4Collectively form
The Gramine analog with anti-CVB3 activity has G1, G2, G3, G4, G5, G7, G9, G10, G11, G13, G15, G16, G17, G18, G19, G20, G21；The chemical structural formula of G1, G2, G3, G4, G5, G7, G9, G10, G11, G13, G15, G16, G17, G18, G19, G20, G21 is
There is the Gramine analog application as anti-CVB3 virus of heterocycle structure, the cytopathic effect (CPE) produced on host cell Hep-2 including this compounds suppression CVB3, strengthen cell survival rate.Parent Gra mine suppresses CVB3 weak activity, and maximal percentage inhibition is about 50%.A few derivant (G6, G8, G12, G14) is entirely without inhibitory activity, and remaining derivant presents stronger anti-CVB3 effect.In these derivants, G18 and G19 has the highest therapeutic index, demonstrates the strongest application potential.
Having the application in preparing anti-CVB3 virus drugs of the Gramine analog of heterocycle structure, it is optimal for having in the Gramine analog of heterocycle structure with analog G18, G19.
Preliminary mechanism research shows, the commitment replicated after these analog target CVB3 infection cells.G19 effect is added after virus infects, can be with the progeny virus production of strong inhibition CVB3 and the synthesis of viral nucleic acid.When 40 μ g/mL G19 process, its progeny virus titre presents the minimizing of about 5.0log, and the nucleic acid synthesis suppression for CVB3 reaches 103Times.
These results indicate that such Gramine analog (with G19 as representative) is potential develops into the medicine that effectively treatment CVB3 infects.Such Gramine analog designs synthesis for antiviral drugs and provides preferable leading support and valuable enlightenment effect, and the basic research of its anti-CVB3 effect is also prepared as clinical treatment medicine further for it and provides theory support.
The applicant has carried out the experiment of anti-CVB3 activity research to the above-mentioned Gramine analog with heterocycle structure, and experimental conditions is as follows: hereinafter, if not specified, material therefor of the present invention and operational approach are well known in the art.
1, content of the test:
Compound anti-CVB3 activity analysis: the present invention will measure cell survival rate detection method in conjunction with cytopathic effect analysis and MTT, be estimated the anti-CVB3 activity of Gramine and derivant thereof.
2, test method:
2.1.1 analog is for the toxicity of host's Hep-2 cell
By Hep-2 plating cells 96 orifice plate, at 37 DEG C, 5%CO2Incubator is cultivated after covering with monolayer, discards cell culture fluid, adds the cell maintenance medium containing variable concentrations test compound respectively and continues to cultivate, microscopic visual measurement record its cytotoxicity respectively after 48h, mtt assay mensuration cell survival rate.SPSS 11.5 computed in software medicine is for the median toxic concentration (Median cyctoxic concentration, CC50) of cell.Cell survival rate=(average OD of medicine group492The average OD of value/cell controls group492Value) × 100%.
2.1.2 compound is for the inhibitory activity of CVB3
By Hep-2 plating cells 96 orifice plate, at 37 DEG C, 5%CO2Incubator is cultivated after covering with monolayer, discarding culture fluid, the CVB3 virus liquid infection cell 1h of 100TCID50, before virus infects, during infection, after infection, all add test compound (ribavirin is as the positive control medicine) incubated cell of variable concentrations.Treat that about 48h is cultivated in continuation, when there is the CPE pathological changes of about 90% in virus control wells, basis of microscopic observation cytopathic effect (CPE).The observed and recorded method of CPE: acellular pathological changes is denoted as-, less than 25% cytopathy is denoted as+, 25%-50% cytopathy is denoted as ++, 50%-75% cytopathy is denoted as +++, more than 75% cytopathy is designated as ++++.
After CPE observes, MTT method is utilized to detect the medicine suppression ratio to CVB3.Concretely comprise the following steps: every hole adds MTT 50 μ L (5mg mL-1), remove supernatant after hatching 3-4h, add isopyknic DMSO dissolution precipitation.With microplate reader read at 492nm corresponding to absorbance (OD492Value).Equation below is utilized to calculate the medicine suppression ratio to CVB3.With the medium effective concentration (Concentration for 50%of maximal effect, EC50) of SPSS 11.5 computed in software medicine.
2.1.3 the therapeutic index (SI) of medicine
SI=CC50/EC50.Therapeutic index is the highest, illustrates that antiviral potentiality are the biggest.
3, result of the test
Gramine and the like cytotoxicity and anti-CVB3 active testing result are as shown in table 2.
Table 2
As seen from Table 2, parent Gramine of the present invention only detects the inhibitory activity weak for CVB3, except G6 in analog, G8, G12, G14 entirely without inhibitory activity beyond, most compounds all presents preferable anti-CVB3 effect.On Gramine parent heterocyclic units be added in the inhibitory activity improving compound to a certain extent for CVB3, and its anti-CVB3 activity is all had a certain impact by the change of substituent group in heterocyclic units.Derivant G2-G5 has the anti-CVB3 effect similar with G1, but toxicity is stronger.Introducing methyl (G6-G9) at parent Gramine unit and improve the cytotoxicity of these compounds, but the reduction of G6 and G8 antiviral activity is even lost, G7 and G9 then maintains antiviral activity in various degree.In general, parent Gramine unit with methyl group can reduce cellulotoxic effect (cytotoxicity: G2 > G6, G3 > G7, G4 > G8, G5 > G9, G10 > G11, G17 > G18).-CF on benzene unit group3Existence (G10, G11) reduce anti-CVB3 activity and enhance cytotoxicity, there is the lowest therapeutic index.Carrying the analog (G12-G15) of pyridine 4 substituent group and pyridine 2 substituent group, cytotoxicity reduces, and antiviral activity is lost or very poor, G13 and G15 has the depression effect weak for CVB3.But, by introducing benzothiazole group, the anti-CVB3 activity of analog G16-G19 has had and has been greatly improved.G16 and G17 presents sizable cytotoxicity, and analog G18 and G19, and its stronger cytotoxicity active and low for anti-CVB3 result in high therapeutic index, and positive control agents Ribavirin close to or quite.Analog G20 comprises pyrazoles unit and G21 carries 1,2,4-triazole unit, presents strong cytotoxicity and good antiviral activity.
In conjunction with analog construction features and anti-CVB3 activity thereof, these similar are classified, represent G1, G2, G7, G10, G19, G20, G21 of each class analog respectively and Hep-2 cell CPE effect that parent Gramine suppression CVB3 causes as shown in Figure 1.The Hep-2 cell rounding that CVB3 infects, departs from from cell wooden partition, and different compound treatment result in for cytopathy inhibitory action in various degree.Parent Gramine (40 μ g/mL) depression effect is inconspicuous, not as good as 50%.Analog G1 (10 μ g/mL), G2 (10 μ g/mL), G10 (2.5 μ g/mL), G19 (40 μ g/mL), G20 (2.5 μ g/mL), G21 (2.5 μ g/mL) all present stronger anti-CVB3 activity, wherein compound G10, G19, G21 depression effect is the strongest, can completely inhibit the CPE of Hep-2 cell.G7 (20 μ g/mL) inhibitory activity is taken second place, about 70% suppression ratio.
The applicant has carried out the research of anti-CVB3 active pharmacological to the above-mentioned Gramine analog with heterocycle structure.
1, content of the test
The pharmaceutical research of 1.1 analog suppression CVB3
1.1.1 analog is for the direct killing effect analysis of CVB3
Analog is for the effect of virus, it may be possible to certain link in suppression virus replication, causes the suppression of virus multiplication, it is also possible to be lethal effect direct to virus, causes virus to lose the ability continuing propagation.Therefore the applicant first confirms that whether analog can be with direct killing virus.
1.1.2 analog is for the analysis of CVB3 model of action
Design three kinds of different dosing methods (respectively before CVB3 infects, between infection, test analog is added after infection) measure its impact for CVB3, thereby determine that medicine is to play preventive effect for virus, suppression adsorptive internalization effect or its biosynthesis effect, thus judge the analog model of action for virus.
1.1.3 different time points adds the medicine function analysis for viral yield
CVB3 proliferating cycle, about 10h, completed following steps: virus adhere to, enters, shelled, polyprotein translation and cutting, viral RNA duplication, assemble and discharge in a replicative cycle.These committed steps all can be as the Effective target site of antiviral drugs effect.Therefore present invention design different time points in one replicative cycle of virus adds medicine, measures its progeny virus infectivity respectively when an end cycle, determines the medicine active phase for CVB3.
2, test method
2.1 analog are for the direct killing effect analysis of CVB3
The CVB3 suspension of high titre hatches 24h with Gramine and derivant (selecting to suppress to greatest extent the concentration of cell CPE) thereof at 4 DEG C of refrigerators, viral suspension dilution certain multiple, it is made to be much smaller than the compound valid density for HIV suppression, titrate in the most adherent the most ready Hep-2 cell covering with monolayer, calculated the TCID50 value of viral suspension by Reed and Muench method.
2.2 analog are for the analysis of CVB3 model of action
The compound impact for CVB3 is measured respectively by three kinds of different dosing methods (before CVB3 infects, between infection, adding test compound after infection).
(1) preventive effect: added analog effect before CVB3 infects.
After Hep-2 cell covers with monolayer in 96 orifice plates, inhale and abandon culture fluid, add and test compound incubation 2h containing variable concentrations, after discarding medicine culture fluid, the CVB3 suspension absorption 1h of 100TCID50, removes viral suspension, adds cell culture maintenance medium and continue to cultivate.Observation of cell CPE during 48h, measures cell survival rate.
(2) suppression adsorption: add analog effect during CVB3 infects.
By CVB3 suspension and test compound mixing, direct titration in the Hep-2 cell covering with monolayer 96 orifice plate, 37 DEG C, 5%CO2After incubator absorption 1h, discard infection liquid, add cell maintenance medium and continue to cultivate.Cell survival rate is measured during 48h.
(3) therapeutical effect: add analog effect after CVB3 infects.
After Hep-2 cell covers with monolayer in 96 orifice plates, inhale and abandon culture fluid, the CVB3 suspension absorption 1h of 100TCID50, discard virus liquid, add the test compound containing variable concentrations and continue to cultivate, during 48h, detect cell CPE and survival rate.
In order to verify the different dosing method inhibitory action for CVB3 further, selection represents analog G19, set concentration as 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, cultivated by above-mentioned three kinds of dosing methods respectively, observation of cell CPE during 48h, TCID50 method measures progeny virus production.
2.3 different time points add the medicine function analysis for viral yield
100TCID50CVB3 infects Hep-2 cell, 37 DEG C of cell culture incubator absorption 1h, discard virus liquid, respectively at-3-(-1) h ,-1-0h, 0-10h, 2-10h, 4-10h, 6-10h, 8-10h and-3-10h (-3-(-1) h: virus infects the last stage；-1-0h: virus infective stage；0-10h: virus duplicate stage in cell) each time period adds Gramine, G1, G2, G7, G10, G19, G20, G21 of 2 times of EC50 concentration.After each time period drug incubation terminates, wash three thorough removing residual liquors with PBS, add the DMEM cell maintenance medium containing 2%FBS and continue to cultivate.After infection, 10h collects cell and supernatant,-20 DEG C and 37 DEG C of three alternate freezing and thawings, titrate on Hep-2 cell 96 orifice plate being already prepared in advance after diluting 10 times again, treats that virus control group cell CPE reaches more than 90%, observing drug treating group cytopathy situation, mtt assay measures cell survival rate.
In order to verify the active phase of analog further, selection represents analog G19, set concentration as 40 μ g/mL, during different phase in-3-(-1) h ,-1-0h, 0-10h, 2-10h, 4-10h, 6-10h, 8-10h ,-3-10h and 0-2h, 2-4h, 0-4h, 4-6h virus infection adds Hep-2 cell respectively, after virus infects, 10h collects cell and supernatant, after freezing-thawing and cracking, pass through TCID50Assay method detection progeny virus titre, determines that different time sections adds the medicine impact for progeny virus production.
3, result of the test
3.1Gramine and the like external direct killing CVB3 effect
The CVB3 suspension that Gramine and the like processed infects Hep-2 cell, and 48h detects its virus titer.It was found that drug treating group compares virus control group, virus titer, without significant change (data do not show), illustrates that these analog the most do not have external direct killing CVB3 effect.
The model of action of 3.2Gramine and the like suppression CVB3
All analog pretreatment cells infect without obvious CPE depression effect (Fig. 2 A) for CVB3 subsequently, the highest suppression only about 50%.And the cell that analog G19 processes is compared with the virus control group cell not processed, virus titer is without significant difference (see Fig. 2 D), illustrate that these analog all can not suppress CVB3, Gramine and the like to infect without preventive effect for virus by acting on cell in advance.
Gramine and the like and CVB3 is hatched infection Hep-2 cell simultaneously, adds cell maintenance medium and continue to cultivate 48h, observation of cell CPE.Result shows, this processing mode of all analog all can not completely inhibit cell CPE (see Fig. 2 B), and G19 process group and virus control papova titre are almost as good as, illustrate that the Gramine and the like the adsorptive internalization for CVB3 is without obvious effect (see Fig. 2 D).
CVB3 adds Gramine and the like effect after infecting, find that these analog show the inhibitory activity strong for CVB3, its cell survival rate maintains a high level mostly, some analog can almost completely inhibit the cytopathic effect (Fig. 2 C) that CVB3 causes, the virus titer of G19 process group is significantly reduced relative to virus control, virus titer can be made when concentration is 40 μ g/mL to reduce about 5.0log (Fig. 2 D), thus illustrate these analog Main Function CVB3 infect after in intracellular duplication breeding, there is the Gramine analog of heterocycle structure for preparing after anti-CVB3 infects the medicine in intracellular duplication breeding.
The active phase of 3.3Gramine and the like suppression CVB3
As shown in Figure 3A, when these eight kinds of analog of Gramine, G1, G2, G7, G10, G19, G20, G21 be present in CVB3 duplicate stage whole during (-3-10h) time, cell survival rate is the highest, and this effect is similar to add analog in 0-10h, 2-10h stage.And in 4-10h, adding analog, cell survival rate has a certain degree of decline, drug inhibition substantially to weaken.Adding analog in 6-10h, 8-10h ,-3-(-1) h and-1-0h, cell survival rate is the lowest, and the especially 8-10h stage acts on, and cell survival rate is almost nil.These results indicate that these analog Main Function CVB3 is at the intracellular commitment replicating propagation of Hep-2.Result above is passed through G19 different phase in virus infection and is added in Hep-2 cell, detects its viral yield respectively and verify further during 10h.Result is as shown in Figure 3 B, analog is added in 0-10h, 2-10h ,-3-10h, 0-2h, 2-4h, 0-4h stage that virus infects, progeny virus titre is kept at a relatively low level, 4-10h, the 4-6h stage acts on, virus titer is significantly increased, and other times point virus titer is all close to virus control group.Illustrate that these analog mainly work in 4 hours after virus infected cell, i.e. suppress the commitment of virus replication.
The applicant implements the inhibitory action test that the Gramine analog G19 with excellent anti-CVB3 activity replicates for CVB3, and test situation is as follows:
1, content of the test detection is after CVB3 infects Hep-2 cell, the inhibitory action that Gramine derivant G19 synthesizes for CVB3 progeny virus production and viral nucleic acid.
2, test method
Hep-2 plating cells 24 orifice plate of exponential phase, covers with 100TCID after monolayer50CVB3 infection cell, 37 DEG C hatch 1.5h after remove virus liquid, PBS washs three times, adds the cell maintenance medium containing G19.Process detection CVB3 virus titer and rna expression situation respectively in such a way.
(1) cell and supernatant culture fluid are collected at 4,8,16,24 or 36h (pi) respectively, after-20 DEG C and 37 DEG C of three freezing-thawing and crackings, TCID50 method mensuration CVB3 virus titer.
(2) respectively at 4,8,16,24h (pi) collection cell and supernatant culture fluid, pass through-20 DEG C and 37 DEG C of three freezing-thawing and crackings, measure the rna expression situation of CVB3.Use TaKaRa (RNAisoTMPlus) Total RNA extracts test kit and extracts RNA, carries out quantitative fluorescent PCR after reverse transcription, and after detection drug treating, CVB3RNA relative amount in cell, calculates the suppression multiple that medicine synthesizes for CVB3RNA.The virus control group not adding drug treating is set.
3, result of the test
(1) G19 is for the inhibitory action of CVB3 progeny virus production
As shown in Figure 4 A, in virus control cell, virus titer presents from 4h to 36h and is significantly increased, and illustrates the duplication state that in cell, virus presents.And until 36h in the cell that 40 μ g/mL G19 process, virus titer is all without substantially increasing.In the cell that 20 μ g/mL G19 process, CVB3 yield has certain increase, but still presents obvious depression effect relative to virus control.
(2) inhibitory action that G19 synthesizes for CVB3RNA
The inhibitory action that G19 synthesizes is determined for CVB3RNA by fluorescence quantifying PCR method.The RNA of all samples distinguishes relative to 4h virus control cell RNA Production rate multiple.Fig. 4 B shows, the rna level of virus control group can be detected in the 4h moment, and passage presents and strongly increases over time.And G19 concentration is when being 40 μ g/mL, CVB3 progeny virus production in Hep-2 cell and nucleic acid synthesis can be suppressed to greatest extent, present the strongest depression effect at 24h, suppression be synthesized for the nucleic acid of CVB3 and reaches 103Times.
Result above further illustrates Gramine and the like mainly preparation suppression virus at the medicine of intracellular duplicate stage.
In sum, there is heterocycle structure Gramine and the like there is stronger suppression CVB3 effect, the commitment replicated after Gramine and the like target CVB3 infection cell.The compound G19 wherein comprising pyridine and benzothiazole unit shows the strongest application potential, is hopeful to prepare the medicine that a kind of the most effectively treatment CVB3 infects.Gramine and the like designs for antiviral drugs, synthesis provides preferable leading support and valuable guide effect.

Claims (7)

1. there is the application in preparing anti-CVB3 virus drugs of the Gramine analog of heterocycle structure, its feature It is: the Gramine analog with heterocycle structure has following chemical structural formula
In formula:
R1For the one in hydrogen, methyl, ethyl, benzyl or substituted benzyl；
R2For hydrogen, phenyl, 2-chlorphenyl, 4-chlorphenyl, 2-fluorophenyl, 4-fluorophenyl, 4-trifluoromethyl, One in 2-trifluoromethyl, pyridine-2-base, pyridin-4-yl, 2-hydroxy phenyl, 4-hydroxy phenyl；
R3For the one in hydrogen, methyl；
R4For the one in methyl, pyridine-2-base, benzothiazole-2-base；
Or R3And R4Collectively form
The Gramine analog with heterocycle structure the most according to claim 1 is preparing anti-CVB3 virus Application in medicine, it is characterised in that:
The Gramine analog with anti-CVB3 activity has G1, G2, G3, G4, G5, G7, G9, G10, G 11, G13, G15, G16, G17, G18, G19, G20, G2；G1, G2, G3, G4, G5, G7, G9, G The chemical structural formula of 10, G11, G13, G15, G16, G17, G18, G19, G20, G21 is
The Gramine analog with heterocycle structure the most according to claim 1 is preparing anti-CVB3 virus Application in medicine, it is characterised in that:
Have in the Gramine analog of heterocycle structure and have analog G18, G19.
The Gramine analog with heterocycle structure the most according to claim 1 is preparing anti-CVB3 virus Application in medicine, it is characterised in that: when G19 concentration is 40 μ g/mL, CVB3 can be suppressed to greatest extent at H Progeny virus production in ep-2 cell and nucleic acid synthesis.
The Gramine analog with heterocycle structure the most according to claim 1 is preparing anti-CVB3 virus Application in medicine, it is characterised in that: the Gramine analog with heterocycle structure infects for preparing anti-CVB3 After at the medicine of intracellular duplication breeding.
Application in preparing anti-CVB3 virus drugs the most according to claim 1, it is characterised in that: institute State application and refer to have the Gramine analog interpolation acceptable adjuvant of pharmaceutics and the carrier of heterocycle structure, be used for Prepare the preparation of anti-CVB3 virus.
7. according to the application in preparing anti-CVB3 virus drugs described in claim 1 or 6, it is characterised in that: Described preparation is granule, tablet, pill, capsule, injection or dispersant.
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CN106668002A (en) * 2016-12-20 2017-05-17 湖北工业大学 Applications of Gramine and derivatives thereof to preparation of medicaments for resisting adenovirus Type 7
CN106905300A (en) * 2017-02-21 2017-06-30 中国科学院昆明植物研究所 Giantreed alkali derivant and its pharmaceutical composition and its application in pharmacy
CN109516978A (en) * 2019-01-28 2019-03-26 河北工业大学 Giantreed alkali derivant and its preparation method and application
CN109645008A (en) * 2019-01-28 2019-04-19 河北工业大学 The application of donaxine and its derivative in Antiphytoviral and germ

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106668002A (en) * 2016-12-20 2017-05-17 湖北工业大学 Applications of Gramine and derivatives thereof to preparation of medicaments for resisting adenovirus Type 7
CN106905300A (en) * 2017-02-21 2017-06-30 中国科学院昆明植物研究所 Giantreed alkali derivant and its pharmaceutical composition and its application in pharmacy
CN109516978A (en) * 2019-01-28 2019-03-26 河北工业大学 Giantreed alkali derivant and its preparation method and application
CN109645008A (en) * 2019-01-28 2019-04-19 河北工业大学 The application of donaxine and its derivative in Antiphytoviral and germ
CN109516978B (en) * 2019-01-28 2020-04-14 河北工业大学 Giantreed alkali derivative and preparation method and application thereof
CN109645008B (en) * 2019-01-28 2020-12-15 河北工业大学 Application of arundoin and derivatives thereof in resisting plant viruses and germs

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