CN103735546A - Novel application of 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate - Google Patents
Novel application of 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate Download PDFInfo
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- CN103735546A CN103735546A CN201410048957.3A CN201410048957A CN103735546A CN 103735546 A CN103735546 A CN 103735546A CN 201410048957 A CN201410048957 A CN 201410048957A CN 103735546 A CN103735546 A CN 103735546A
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Abstract
The invention relates to new application of 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate which is a post-market drug. 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate is a selective leukotrienes D4-receptor antagonist and mainly used for asthma treatment clinically. The project is based on the known drug prediction of a drug repositioning strategy and includes the steps of screening and activity verification for an anti-dengue virus drug through experiments. 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate is found out to have the significant anti-dengue virus activity and possible to be developed into a novel anti-dengue virus drug.
Description
Technical field
The present invention relates to small-molecule drug 4-oxo-8-[4-(4-phenyl butoxy) benzamido] the new purposes of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate, 4-oxo-8-[4-(4-phenyl butoxy) benzamido specifically]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate preparation treatment and prevention dengue virus (dengue virus, DENV) infect and diseases related medicine in new purposes.
Background technology
DENV belong to flaviviridae (Flaviviridae) Flavivirus (member of (Flavivirus), spherical in shape, diameter 37-50nm, contains the structures such as lipid envelope, nucleocapsid, peplos has the thrusts such as surface protein outward.DENV can cause large-scale outbreak of epidemic, and dengue fever spread speed is fast, and sickness rate is high, very easily causes eruption and prevalence; Dengue hemorrhagic fever and the dengue shock syndrome state of an illness are violent, and case fatality rate is high.It is very serious that DENV infects harm, become a serious public health problem.At present, the disease causing for DENV, there is no the vaccine and the effective medicine that can be used for human body.Therefore, research and development effectively prevent to have become the task of top priority with the method for controlling DENV.
Develop effective anti-DENV medicine costly, and the R&D cycle very long (over 12 years); The effective means that reduces anti-DENV medicament research and development input and cost is medicine reorientation strategy.Recently, medicine method for relocating has been applied to the prevention of acquired immune deficiency syndrome (AIDS) and has treated research suffered.But also about the medicine reorientation of anti-DENV, do not report so far.This laboratory is found 4-oxo-8-[4-(4-phenyl butoxy) benzamido by applying biological informatics first in conjunction with the strategy of experimental verification research]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can suppress copying of DENV, and find that first it all has the activity of significant anti-DENV in vitro.
Summary of the invention
The object of the invention is to study 4-oxo-8-[4-(4-phenyl butoxy) benzamido] activity of the anti-DENV Infective of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate, prevention and the treatment of for DENV, infecting relevant disease provide medicine.
The present invention relates to a kind of medicine 4-oxo-8-[4-that gone on the market (4-phenyl butoxy) benzamido] the new purposes of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate is a kind of selectivity leukotriene D-receptor antagonist, is clinically mainly used in treating asthma.This project is carried out reorientation prediction based on medicine reorientation strategy to known drug early stage, and carry out by experiment anti-DENV drug screening and verify with active, find 4-oxo-8-[4-(4-phenyl butoxy) benzamido] to have significant anti-DENV active for the-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate, may develop into anti-DENV drug candidate.
4-oxo-8-[4-(4-phenyl butoxy) benzamido] the structure of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate shows below:
This research is to find micromolecule 4-oxo-8-[4-(4-phenyl butoxy) benzamidos by a series of extracorporeal antivirus effect active appraisal experiments]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate has the object of obvious DENV Infective.BHK21 cell (hamster nephrocyte cell) be and Vero (African green time nephrocyte) in evaluated 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses DENV and copies and cytopathogenic characteristic, result shows 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can suppress copying and cytopathic effect of DENV2 and DENV3 in dose dependent ground, further by plaque experimental evaluation 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate anti-DENV2 Infective is in vitro active, result shows half effective inhibition concentration (50%inhibiting concentration, IC50) be 5.56 ± 0.58 μ M.In addition, by series of experiments, verified 4-oxo-8-[4-(4-phenyl butoxy) benzamido under different time points]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate effectiveness suppressed the virus titer of DENV2; The mRNA that has suppressed DENV copies significance; And in BHK21 cell, suppressed the expression of DENV2E envelope glycoprotein.Prompting 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can develop into the medicine that prevention and treatment DENV infect relevant disease.
According to the present invention, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can effectively suppress the infection duplication of DENV, likely becomes treatment and the medicine that DENV infects relevant disease is stepped in prevention.
The treatment that enforcement of the present invention is infected the DENV of serious harm human health has important Social benefit and economic benefit.
Accompanying drawing explanation:
Fig. 1 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses DENV2 cytopathic effect in BHK21 cell dose dependent.
Fig. 2 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses DENV3 cytopathic effect in Vero cell dose dependent.
Fig. 3 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses the virus titer of DENV2 in BHK21 cell dose dependent.
Fig. 4 4-oxo-8-[4-(4-phenyl butoxy) benzamido] the toxic action of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate to BHK21 cell.
4-oxo-8-[4-under Fig. 5 different time points (4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate effectiveness suppressed the virus titer of DENV2.
Fig. 6 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate mRNA of significantly having suppressed DENV copies.
Fig. 7 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the expression of the E envelope glycoprotein of DENV2 in BHK21 cell.
The specific embodiment
Embodiment:
The present embodiment mainly illustrates 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate is in the activity of BHK21 cell and the anti-DENV Infective of Vero cell.
Materials and methods
1. cell, virus and medicine preparation
DENV2 strain is international standard strain New Guinea strain (DENV2-NGC), by infecting the breeding of increasing of BHK21 cell; DENV3 breeds by Vero cell amplification, then respectively to two-strain mix, subpackage and be stored in-70 ℃ standby.BHK21 cell and Vero cell culture fluid are DMEM (GIBCO) culture fluid containing 10% hyclone (FBS).When measuring, viral infection and cytopathy use the maintenance medium containing 2%FBS.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate is dissolved as 20mM with DMSO, after subpackage, be stored in-20 ℃ standby.2.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the cytopathogenic dosage dependence of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate inhibition DENV2 effect detection
BHK21 cell is in containing the DMEM culture medium (GIBCO) of 10%FBS, in 37 ℃, 5%CO
2in incubator, cultivate.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, with 8000～10000 cells/well, cell is seeded in 96 porocyte culture plates, and cover with next day 75～80%.With DMEM dilution 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate to 2000 μ M.Change 96 orifice plates that cover with the DMEM culture medium (GIBCO) containing 10%FBS of 75～80%BHK21 cell into DENV2 virus liquid (infection multiplicity MOI=0.1) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and control wells supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively five Concentraton gradient of 5,10,20,40,80 μ M (containing the maintenance medium of 2%FBS) to add in 96 orifice plates, each concentration is established three multiple holes, cultivates 4d for 37 ℃.Utilize Cell Counting Kit-8 (DoJinDo product) method at microplate reader (BIO-RAD product, model: Model680) above measure cytopathy degree (representing with OD450), and calculate the viral suppression ratio of medicine with following formula: [(OD test-OD virus)/(OD cell-OD virus)] * 100.Repeated trials twice, calculating mean value.
3.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the cytopathogenic dosage dependence of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate inhibition DENV3 effect detection
Vero cell is in containing the DMEM culture medium (GIBCO) of 10%FBS, in 37 ℃, 5%CO
2in incubator, cultivate.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, with 8000～10000 cells/well, cell is seeded in 96 porocyte culture plates, and cover with next day 75～80%.With DMEM dilution 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate to 2000 μ M.Change 96 orifice plates that cover with the DMEM culture medium (GIBCO) containing 10%FBS of 75～80%BHK21 cell into DENV3 virus liquid (infection multiplicity MOI=0.01) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and contrast supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively tetra-Concentraton gradient of 10,20,40,80 μ M (containing the maintenance medium of 2%FBS) to add in 96 orifice plates, each concentration is established three multiple holes, cultivates 4d for 37 ℃.Utilize Cell Counting Kit-8 (DoJinDo product) method at microplate reader (BIO-RAD product, model: Model680) above measure cytopathy degree (representing with OD450), and calculate the viral suppression ratio of medicine with following formula: [(OD test-OD virus)/(OD cell-OD virus)] * 100.Repeated trials twice, calculating mean value.
4.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the dosage dependence effect that the suppresses DENV2 titre of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate detects
With the DMEM culture fluid containing 10%FBS, cultivate BHK21 cell to exponential phase, by 8000 left and right, an every hole cell (200 μ l), be inoculated in 96 porocyte culture plates, after adherent 16 hours, cover with next day 75～80%, abandons culture fluid.Add the DENV2 virus liquid (infection multiplicity MOI=0.1) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and control wells supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively tetra-Concentraton gradient of 2.5,5,10,20 μ M (containing the maintenance medium of 2%FBS) to add in 96 orifice plates, each concentration is established three multiple holes, cultivates 4d for 37 ℃.Get respectively normal group, virus control group and variable concentrations 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate well supernatant ,-70 ℃ save backup.Next, the supernatant of preserving is carried out to plaque experiment.With BHK21 cell, spread 6 porocyte culture plates, treat that Growth of Cells monolayer is fine and close, abandon supernatant.The supernatant of preservation is carried out to gradient dilution, and add culture plate.37 ℃, 5% CO
2hatch 1hr, balance on a plank is once gently in centre; Every hole adds 3ml to contain the DMEM of 2%FBS and the mixed liquor of 2% low melting-point agarose gel, makes mixeding liquid temperature be maintained 30 ℃ of left and right; Room temperature is standing, until agarose solidifies; 37 ℃, 5% CO
2incubator, cultivates 5 days.Add 3ml fixative, 37 ℃ of fixing 1h, then abandon fixative and nutrition Qiong Gai, and by washed with de-ionized water 2-3 time; 1.5% crystal violet solution that added 1m, 37 ℃ of dyeing 1h, abandon crystal violet solution, and by washed with de-ionized water 2-3 time, count speckle number.Plaque reduction ratio (%)=(virus control group plaque number-drug treating group plaque number)/(virus control group plaque number) * 100%, logarithm value with drug level is done abscissa, with Plaque reduction ratio, do vertical coordinate mapping, according to Plaque reduction ratio result, make the sensitivity curves of virus to medicine, draw thus the IC of medicine
50, experiment repeats 3 times.
5.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the toxic action detection of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate to BHK21 cell
Totally 3 of 96 orifice plates that BHK21 cell spread respectively to different cell densities, cell density is followed successively by 6000 cells/well, 8000 cells/well and 10000 cells/well, and cover with next day 75～80%.The BHK21 cell that covers with 75～80% is changed into containing the maintenance medium of 2%FBS standby.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively five Concentraton gradient of 10,20,80,160,320 μ M, each concentration is set up three multiple holes, and set up BHK21 cell negative control group, cultivate 4d, Microscopic observation 4-oxo-8-[4-(4-phenyl butoxy) benzamido for 37 ℃] the impact of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate on cellular morphology.Utilize Cell Counting Kit-8 (DoJinDo product) method in microplate reader (BIO-RAD product, model: Model680) the upper cytopathy degree of measuring is (with OD
450represent), and calculate median toxic dose CC
50.
6. 4-oxo-8-[4-(4-phenyl butoxy) benzamido under different time points]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses the detection of DENV2 virus titer effectively
With the DMEM culture fluid containing 10%FBS, cultivate BHK21 cell to exponential phase, by 8000 left and right, an every hole cell (200 μ l), be inoculated in 96 porocyte culture plates, after adherent 16 hours, cover with next day 75～80%, abandons culture fluid.Add the DENV2 virus liquid (infection multiplicity MOI=0.1) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and control wells supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively tetra-Concentraton gradient of 10,20,40,80 μ M (containing the maintenance medium of 2%FBS) to add in 96 orifice plates, each concentration is established three multiple holes, cultivates respectively 2d, 3d and 4d for 37 ℃.Get respectively virus control group and variable concentrations 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate well supernatant ,-70 ℃ save backup.Next, the supernatant of preserving is carried out to plaque experiment.Specific operation process refers to discussion above.Plaque number to every group is added up, and carries out significance test analysis.*, represent P < 0.05; *, represents P < 0.01.
7.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the RT-PCR that DENV mRNA copies and detected
With the DMEM culture fluid containing 10%FBS, cultivate BHK21 cell to exponential phase, by every hole 100,000 left and right cell (2mL), be inoculated in 6 porocyte culture plates, after adherent 16 hours, cover with next day 75～80%, abandons culture fluid.Add the DENV2 virus liquid (infection multiplicity MOI=0.1) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and control wells supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively tetra-Concentraton gradient of 10,20,40,80 μ M (containing the maintenance medium of 2%FBS) to add in 6 orifice plates, each concentration is established three multiple holes, cultivates respectively 3d for 37 ℃.Abandon supernatant, by Trizol method extraction method, extract respectively total RNA of cell.With the RNA of above extraction, as template, carry out Quant one-step method RT-PCR amplification.Concrete grammar is with reference to TIIANGEN " Quant One Step RT-PCR Kit " explanation.DENV2 primer is: DENV2F5 '-AATATGCTGAAACGCGAGAGA-3 '; DENV2R5 '-
GGGATTGTTAGGAAACGAAGG-3 '; Internal reference primer is: GAPDH F5 '-
GAGTCAACGGATTTGGTCGT-3’；GAPDH?R5’-TTGATTTTGGAGGGATCTCG-3’。And amplification is carried out to significance analysis.*, represent P < 0.05.
8.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the expression of the E envelope glycoprotein of DENV2
With the DMEM culture fluid containing 10%FBS, cultivate BHK21 cell to exponential phase, by every hole 10,000 left and right cell (2mL), be inoculated in the burnt culture plate of copolymerization, after adherent 16 hours, cover with next day 75～80%, abandons culture fluid.Add the DENV2 virus liquid (infection multiplicity MOI=0.1) containing the maintenance medium of 2%FBS, and set up viral infection matched group and normal cell matched group, 37 ℃, 5%CO
2hatch 1hr.Abandon virus and control wells supernatant, 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively tri-Concentraton gradient of 20,40,80 μ M (containing the maintenance medium of 2%FBS) to add in the burnt culture plate of copolymerization, each concentration is established three multiple holes, cultivates respectively 2d for 37 ℃.Abandon supernatant; PBS washes plate 2 times, 5min/ time; Fixing: the paraformaldehyde room temperature with 4% is placed 20min; PBS washes plate 2 times; Wear film: with the PBS room temperature that comprises 0.25%TritonX-100, hatch 10min, PBS washes plate 2 times; Sealing: 2% sheep blood serum (containing 0.4%Triton X-100) incubated at room 30min; Add primary antibodie (1: 10 dilution) incubated at room 1h or 4 ℃ to spend the night, PBS washes three times, 5min/ time; Add two anti-(lucifuge, dilution in 1: 100) incubated at room 1h, PBS washes three times, 5min/ time; Set up simultaneously and do not add primary antibodie, only add the anti-negative control of fluorescent labeling two; Dyeing: with DAPI, carry out nuclear staining, static 3-5min, PBS washes three times, 5min/ time; Finally, under Laser Scanning Confocal Microscope, observe.
Result:
1.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the cytopathogenic dosage dependence of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate inhibition DENV2 effect
As shown in Figure 1,4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can dose dependent within the scope of 0-80 μ M the cytopathic effect of inhibition DENV2.
2.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the cytopathogenic dosage dependence of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate inhibition DENV2 effect
As shown in Figure 2,4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can dose dependent within the scope of 0-80 μ M the cytopathic effect of inhibition DENV3.
3.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppresses the dosage dependence effect of DENV2 titre
As shown in Figure 3,4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate can dose dependent within the scope of 0-80 μ M the titre of inhibition DENV, plaque result shows that it suppresses the IC of DENV2
50be 5.56 ± 0.58 μ M.
4.4-oxo-8-[4-(4-phenyl butoxy) benzamido] the toxic action of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate to BHK21 cell
4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate sets up respectively five Concentraton gradient of 10,20,80,160,320 μ M to carry out toxicity trial, CCK8 testing result shows 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate concentration while reaching 160 μ M on cell proliferation do not make significant difference (Fig. 4), its median toxic dose CC
50be 320 μ M.
5. 4-oxo-8-[4-(4-phenyl butoxy) benzamido under different time points]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the virus titer of DENV2 effectively
After DENV2 infects, add 4-oxo-8-[4-(4-phenyl butoxy) benzamido] after-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate 48hr to 96hr, along with the prolongation cell conditioned medium liquid virus titer of time reduces (Fig. 5) gradually.During 48hr, 40 μ M and 80 μ M papova titres are zero, and relatively viral matched group utmost point significance ground has suppressed viral release (P < 0.01).During 72hr, be similar to 48hr, and the also significance reduction (P < 0.05) of 20 μ M group titre.During 96hr, 20 μ M, 40 μ M and 80 μ M group titre are reduced to zero, and relatively viral matched group utmost point significance ground has suppressed viral titre (P < 0.01).
6.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate mRNA of significantly having suppressed DENV copies
After DENV2 infects, add 4-oxo-8-[4-(4-phenyl butoxy) benzamido] hatch to 72hr after the-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate, RT-PCR amplification shows with respect to virus control group, and what variable concentrations dosing group significance had reduced virus mRNA copies (P < 0.05) (Fig. 6).
7.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the expression of the E envelope glycoprotein of DENV2
After DENV2 infects, add 4-oxo-8-[4-(4-phenyl butoxy) benzamido] hatch to 48hr after the-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate, immunofluorescence result shows the increase along with dosing group concentration, the expression intensity of peplos E albumen lowers gradually, shows 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate suppressed the expression (Fig. 7) of virus E protein effectively.
Conclusion
4-oxo-8-[4-(4-phenyl butoxy) benzamido] the external effect with the significant anti-DENV Infective of-2-tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate.
Claims (4)
1.4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate or its can medication salt in the purposes for the preparation of in treatment dengue virus infection and diseases related medicine thereof.
2. according to claim 1, wherein said 4-oxo-8-[4-(4-phenyl butoxy) benzamido]-2-the tetrazole-4H-1-.alpha.-5:6-benzopyran-semihydrate is also named as pranlukast, its English Pranlukast by name.
3. according to claim 1, wherein said medicine contains one or more pharmacy acceptable carriers.
4. purposes according to claim 1, wherein said medicine can be made injection, freeze-dried powder, microsphere, powder, powder spray, aerosol, enteric coating, nanoparticles, microemulsion or emulsion.
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| CN201410048957.3A Pending CN103735546A (en) | 2014-02-11 | 2014-02-11 | Novel application of 4-oxo-8-[4-(4-phenyl butoxyl) benzamido]-2-tetrazol-4H-1-benzopyran-semihydrate |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178647A (en) * | 2011-04-28 | 2011-09-14 | 重庆圣华曦药物研究开发有限公司 | Pranlukast injection preparation |
| WO2013148366A1 (en) * | 2012-03-27 | 2013-10-03 | Duke Unversity | Compositions and methods for the prevention and treatment of mast cell-induced vascular leakage |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178647A (en) * | 2011-04-28 | 2011-09-14 | 重庆圣华曦药物研究开发有限公司 | Pranlukast injection preparation |
| WO2013148366A1 (en) * | 2012-03-27 | 2013-10-03 | Duke Unversity | Compositions and methods for the prevention and treatment of mast cell-induced vascular leakage |
Non-Patent Citations (1)
| Title |
|---|
| 佘文煜等: "普仑司特对实验性变应性鼻炎鼻黏膜组织重塑的影响", 《中华耳鼻咽喉头颈外科杂志》 * |
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