CN112694513A - Dehydroepiandrosterone derivatives, medicaments with anti-flavivirus activity and use thereof - Google Patents
Dehydroepiandrosterone derivatives, medicaments with anti-flavivirus activity and use thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
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- C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a dehydroepiandrosterone derivative, a medicament with anti-flavivirus activity and application thereof. The dehydroepiandrosterone derivative has excellent effect on inhibiting flavivirus, and can remarkably improve normal cell lesion caused by flavivirus infection at a concentration without toxic effect on cells. The dehydroepiandrosterone derivative or the medicine containing the dehydroepiandrosterone derivative and having anti-flavivirus activity can be used for preparing a flavivirus inhibitor or a flavivirus treatment medicine independently or after being combined with other anti-flavivirus medicines, and has huge application prospect.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a dehydroepiandrosterone derivative, a medicament with anti-flavivirus activity and application thereof.
Background
Flaviviridae (Flaviviridae) flaviviruses (flaviviruses) are enveloped single-stranded positive-strand RNA viruses that cause infections by transmission from blood-sucking arthropods (mosquitoes, ticks, sand flies, etc.). The flavivirus virus includes about 70 viruses, most of which are pathogens of zoonotic infectious diseases, such as Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Yellow fever virus (Ylow river virus, YFV), Dengue virus type 1-4 (DENGue virus, DENV), Zika virus (ZIKV), and the like.
Flavivirus infection can cause fever, headache, joint pain, conjunctivitis, and rash itching, and in severe cases, can also cause death of the infected person. In recent years, the distribution of flaviviruses has been gradually expanded in the world with the change of global climate, and at the same time, the infection of flaviviruses shows a tendency to spread further due to the adaptation of the virus gene to a new transmission medium by mutation at certain sites. And the lack of vaccines against flaviviruses currently, the need to develop drugs against flaviviruses infections is increasingly pressing.
On the basis of analyzing the chemical structure of the known drug, designing derivatives of the synthetic drug is the starting point of modern new drug research. The method has the advantages of less investment, short period and high benefit for modifying natural compounds, is favorable for constructing a large amount of compound libraries for activity screening, and lays a foundation for the research and development of antiviral drugs. Dehydroepiandrosterone (DHEA) is an intermediate in testosterone biosynthesis, a steroid and sex hormone precursor. Dehydroepiandrosterone can affect fat formation, regulate NADPH, regulate interleukin and interferon production, bind to endothelial cell membrane receptors, and antagonize various viruses.
The invention patent with application number CN99813658.1 discloses the application of a 17-ketosteroid compound and derivatives, metabolites and precursors thereof in the treatment of hepatitis C virus and other togaviruses. The invention patent with the application number of CN201711426066.7 discloses a dehydroepiandrosterone-6-glucoside derivative and application thereof as an anti-hepatitis C virus medicament.
However, the above compounds or derivatives have limited efficacy against flavivirus activity and may also have the technical drawbacks of high cytotoxicity and poor water solubility, which further limits the function of antiviral activity. In view of the above, there is a need to design an improved dehydroepiandrosterone derivative, a drug with anti-flavivirus activity and its application to solve the above problems.
Disclosure of Invention
The invention aims to provide a dehydroepiandrosterone derivative, a medicament with anti-flavivirus activity and application thereof.
In order to achieve the above object, the present invention provides a dehydroepiandrosterone derivative having an anti-flaviviridae activity, which has the following structural formula:
wherein R1 is phenyl; r2 is a hydrogen atom.
As a further improvement of the invention, the structural formula of the dehydroepiandrosterone derivative is as follows:
in order to achieve the above objects, the present invention also provides a pharmaceutical composition having anti-flaviviridae activity, which comprises the dehydroepiandrosterone derivative as an active ingredient and has anti-flaviviridae activity.
As a further improvement of the invention, the medicament with anti-flavivirus activity further comprises a pharmaceutically acceptable carrier, diluent or excipient.
As a further improvement of the invention, the dosage form of the drug with anti-flavivirus activity is a gastrointestinal administration dosage form; the gastrointestinal administration dosage form comprises but is not limited to one of powder, tablet, granule, capsule, solution, emulsion and suspension.
As a further improvement of the present invention, the dosage form of the drug having anti-flavivirus activity is a parenteral dosage form; the parenteral administration dosage form comprises but is not limited to one of injection dosage form, respiratory administration dosage form, nasal drops, skin administration dosage form, mucous membrane administration dosage form and cavity administration dosage form.
As a further improvement of the present invention, the flaviviridae virus includes a flavivirus virus.
As a further improvement of the invention, the flavivirus virus includes but is not limited to one of Japanese encephalitis virus, Zika virus and dengue virus.
In order to achieve the above object, the present invention also provides the use of the dehydroepiandrosterone derivative or the anti-flavivirus activity of the above drug in the preparation of a flavivirus inhibitor or a flavivirus treatment drug.
To achieve the above objects, the present invention also provides a method for inhibiting flaviviridae virus activity. The dehydroepiandrosterone derivative is used for inhibiting the translation stage of the flaviviridae virus in host cells and remarkably improving normal cytopathy caused by flaviviridae infection by contacting a chemical drug containing the anti-flaviviridae active drug with the host infected with the flaviviridae virus.
The invention has the beneficial effects that:
1. the dehydroepiandrosterone derivative provided by the invention has excellent effect of anti-flavivirus activity. In vitro experiment tests show that the compound can obviously improve normal cytopathy caused by flavivirus infection at a concentration which has no toxic effect on cells. In addition, the dehydroepiandrosterone derivative inhibits Japanese encephalitis virus IC50Inhibition IC of Zika virus with a value of 3.056. mu.M50Value of 5.436. mu.M, inhibition IC of dengue virus50The value was 2.603. mu.M. The in vivo experiment results show that the medicine is prepared byThe mouse animal model infected with the Japanese B encephalitis virus analyzes the survival rate and clinical symptoms of a mouse treated by medication, and meanwhile, a healthy mouse and a mouse not treated by the medication after being infected with the Japanese B encephalitis virus are used as references, and through data statistics analysis, in the aspect of survival rate, the dehydroepiandrosterone derivative has a certain treatment effect on the infection of flaviviridae viruses such as the Japanese B encephalitis virus and the like, so that a new choice is provided for clinical medication; the dehydroepiandrosterone derivative is introduced into the application of inhibiting flavivirus, has great practical significance, and is expected to be used for preparing novel anti-flavivirus medicaments.
2. The dehydroepiandrosterone derivative provided by the invention has excellent effect on inhibiting flavivirus, can be used for preparing a flavivirus inhibitor or a flavivirus treatment drug independently or after being combined with other anti-flavivirus drugs, and has huge application prospect.
3. The dehydroepiandrosterone derivative provided by the invention takes dehydroepiandrosterone as a matrix, is modified at 17 sites of the dehydroepiandrosterone matrix, introduces different groups from the angle of structure-activity relationship, and has the advantages of novel structure, good water solubility and low cytotoxicity by virtue of the modified physicochemical property and the newly introduced inert electronegative functional group (constructed by connecting a carbon-nitrogen double bond with electronegative phenyl) compared with the unmodified dehydroepiandrosterone or the dehydroepiandrosterone derivative modified at other sites, so that the anti-flavivirus activity effect of the modified dehydroepiandrosterone derivative is remarkably improved, and the anti-flavivirus activity effect of the derivative is more excellent compared with the unmodified dehydroepiandrosterone or the dehydroepiandrosterone derivative modified at other conservative sites; the mechanism of action of which exerts anti-flavivirus activity is: after the flavivirus enters the host cell, the derivative exerts an antiviral effect either before or during translation of the virus, but not during viral replication, indicating that the derivative exerts an antiviral effect after the flavivirus enters the host cell.
Drawings
FIG. 1 shows the results of comparing the in vitro inhibition of Japanese encephalitis virus by dehydroepiandrosterone and derivatives according to the present invention.
FIG. 2 is CC of the cytotoxic effect of dehydroepiandrosterone derivatives provided by the present invention50The measurement of (1).
FIG. 3 shows the in vitro inhibitory effect of dehydroepiandrosterone derivatives against Japanese B encephalitis virus (A in FIG. 3 is an inhibitory experiment of different drug concentrations; B in FIG. 3 is a drug-to-Japanese B encephalitis virus IC calculated according to a dose-dependent experiment)50Numerical values).
FIG. 4 shows the effect of dehydroepiandrosterone derivatives of the present invention on the in vitro inhibition of Zika virus (A in FIG. 4 is an inhibition experiment with different drug concentrations; B in FIG. 4 is the IC of Zika virus with drug calculated by a dose-dependent experiment50Numerical values).
FIG. 5 shows the effect of dehydroepiandrosterone derivatives of the present invention on in vitro inhibition of dengue virus (A in FIG. 5 is an inhibition experiment with different drug concentrations; B in FIG. 5 is a dengue virus IC calculated from a dose-dependent experiment for drugs50Numerical values).
FIG. 6 shows that the dehydroepiandrosterone derivative provided by the present invention reduces the mortality caused by JEV.
FIG. 7 shows the virus adsorption experiment of dehydroepiandrosterone derivatives according to the present invention.
FIG. 8 shows the virus invasion test of dehydroepiandrosterone derivatives according to the present invention.
FIG. 9 shows replicon experiments of dehydroepiandrosterone derivatives provided by the present invention.
FIG. 10 shows CHX experiments of dehydroepiandrosterone derivatives provided by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not closely related to the present invention are omitted.
In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Experimental materials: vero cells (Vero monkey kidney cells); BHK-21 cells (milk hamster kidney cells); hela cells (human cervical cancer cells); dehydroepiandrosterone derivatives; japanese B encephalitis virus (GenBank: U47032.1); zika virus (GenBank: KJ 776791); dengue virus (DENV type 2, GenBank: AF 038403.1).
Example 1
The embodiment 1 of the invention provides a dehydroepiandrosterone derivative, which is recorded as AV1004, and the preparation route is as follows: the 17 site of the dehydroepiandrosterone parent is modified, a new group is introduced from the structure-activity relationship, and the modified site and structural formula change are shown as the following formula:
comparative example 1
Comparative example 1 provides an unmodified Dehydroepiandrosterone (DHEA).
Comparative example 2
Comparative example 2 is a blank comparative example with addition of dimethyl sulfoxide (DMSO).
Functional tests for anti-flavivirus activity were performed:
firstly, the dehydroepiandrosterone derivative (AV1004) provided in example 1, the Dehydroepiandrosterone (DHEA) provided in comparative example 1 and the dimethyl sulfoxide (DMSO) blank control group provided in comparative example 2 were tested for in vitro inhibition of Japanese encephalitis virus, and the experimental method was as follows:
s1, taking Vero cells with good growth state for digestion and passage, and adjusting the cell density to 2 x 10 by using cell growth liquid5Perml, inoculated in a 24-well plate at 500. mu.L/well, incubated at 37 ℃ with 5% CO2The incubator is used for 14 h.
S2, after 14h of culture, the supernatant was aspirated, and lower concentrations of 10. mu.M dehydroepiandrosterone derivative AV1004 of example 1, dehydroepiandrosterone of comparative example 1, and DMSO of comparative example 2 were divided into three groups, added to the corresponding cell wells, respectively, and treated in a 37 ℃ cell incubator for 1 hour.
S3 adding Japanese encephalitis virus (JEV virus) (MOI 1) to each well, standing at 37 deg.C and 5% CO2Incubators were infected for 1 hour.
S4, the supernatant was aspirated and washed 3 times with serum-free DMEM, and a maintenance medium containing various compounds was added to the cell wells and the culture was continued for 24 hours.
S5, culturing for 24 hours, collecting cell supernatant, and freezing in-80 deg.C refrigerator.
S6, detecting the virus titer of the cell supernatant after 24 hours of the culture by a plaque experiment.
As shown in FIG. 1, the titer of progeny virus in the cell supernatant was significantly reduced compared to the dehydroepiandrosterone-treated group of comparative example 1 after Vero cells were treated with the dehydroepiandrosterone derivative AV1004 of example 1 for 24 hours, indicating that the dehydroepiandrosterone derivative AV1004 of example 1 had a stronger anti-flavivirus function than the dehydroepiandrosterone of comparative example 1 at a lower concentration of the compound. It can also be seen from figure 1 that the progeny viral titer of dehydroepiandrosterone derivative AV1004 of comparative example 1 was lower than the DMSO blank of comparative example 2, indicating that the unmodified dehydroepiandrosterone had a weak function against flavivirus.
Secondly, the dehydroepiandrosterone derivative (AV1004) provided in example 1 was tested for cell activity according to the following experimental method:
s1, taking Vero cells with good growth state for digestion and passage, and adjusting the cell density to 3 x 10 by using cell growth liquid4Perml, inoculated in a 96-well plate at 100. mu.L/well, incubated at 37 ℃ with 5% CO2Culturing for 14h in an incubator;
s2, after 14h, taking out a 96-well plate, discarding the culture medium in the well, and washing with sterile PBS for three times;
s3, the dehydroepiandrosterone derivative (AV1004) is diluted to 200. mu.M, 100. mu.M, 50. mu.M, 10. mu.M, 5. mu.M, 2. mu.M, 1. mu.M and 0.1. mu.M with the cell maintenance medium, and then added to Vero cells in sequence.
After S4, 48h, CellTiter-
And (3) carrying out cell viability detection by using the One Solution Assay kit. The 96-well plate was allowed to stand at room temperature for 30min, 100. mu.L of CellTiter-Glo reagent was added to each well and stirred on a shaker for 2 min, and then the cells were incubated at room temperature for 10 min in the absence of light. Finally, the luminescence signal is recorded by a multi-lane fluorescence reader.
And (3) detection results:
the cell viability was measured in response to the toxic effect of the compound on the cells and the mean cell viability (%) was calculated from the formula as the mean luminescence value of the compound-treated cells/the mean luminescence value of the control cells.
As shown in FIG. 2, the dehydroepiandrosterone derivative AV1004 provided in example 1 has less toxic effect on Vero cells, and CC thereof50Greater than 100. mu.M.
Thirdly, detecting the effect of the dehydroepiandrosterone derivative (AV1004) on inhibiting virus infection by a plaque reduction experiment
1. Confirming the inhibition effect of dehydroepiandrosterone derivative AV1004 on JEV virus, which comprises the following steps:
p11, Vero cells were trypsinized and counted, cells were added at 1.0X 10/well in 24-well plates5And (4) respectively.
P12, after 14h of incubation, the supernatant was aspirated off, and diluted AV1004 of example 1 and DMSO of comparative example 2 (20. mu.M, 10. mu.M, 5. mu.M, 2. mu.M, and 1. mu.M) were added to the corresponding cell wells, respectively, and treated in a 37 ℃ cell incubator for 1 hour.
P13, JEV Virus (MOI ═ 1) was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Incubators were infected for 1 hour.
P14, aspirate the supernatant, wash 3 times with PBS, add maintenance medium containing the drug to the cell wells, and continue culturing for 24 hours.
P15, after 24 hours of culture, cell supernatants were harvested and frozen in a-80 ℃ freezer.
P16, cell supernatant virus titer was measured by plaque assay after 24 hours of the above culture.
Results as shown in a in fig. 3, the progeny virus titer in the cell supernatant in example 1 was significantly reduced compared to comparative example 2 after 24h treatment of Vero cells with the concentration gradient compound.
2. The method confirms the inhibition effect of the dehydroepiandrosterone derivative AV1004 on the ZIKV virus, and comprises the following specific steps:
p21, Vero cells were trypsinized and counted, cells were added at 1.0X 10/well in 24-well plates5And (4) respectively.
P22, cultured for 14h, the supernatant was aspirated off, and diluted AV1004 of example 1 and DMSO of comparative example 2 (20. mu.M, 10. mu.M, 5. mu.M, 2. mu.M, and 1. mu.M) were added to the corresponding cell wells and treated in a 37 ℃ cell incubator for 1 hour.
P23, ZIKV virus (MOI 1) was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Incubators were infected for 1 hour.
P24, aspirate the supernatant, wash 3 times with PBS, add maintenance medium containing the drug to the cell wells, and continue culturing for 24 hours.
P25, after 24 hours of culture, cell supernatants were harvested and frozen in a-80 ℃ freezer.
P26, detecting the virus titer of the cell supernatant after 24 hours of culture by a plaque experiment; results as shown in a in fig. 4, the progeny virus titer in the cell supernatant in example 1 was significantly reduced compared to comparative example 2 after 24h treatment of Vero cells with the concentration gradient compound.
3. The method confirms the inhibition effect of dehydroepiandrosterone derivative AV1004 on DENV virus, and comprises the following specific steps:
p31, Vero cells were trypsinized and counted, cells were added at 1.0X 10/well in 24-well plates5And (4) respectively.
P32, cultured for 14h, the supernatant was aspirated off, and diluted AV1004 of example 1 and DMSO of comparative example 2 (20. mu.M, 10. mu.M, 5. mu.M, 2. mu.M, and 1. mu.M) were added to the corresponding cell wells and treated in a 37 ℃ cell incubator for 1 hour.
P33, DENV virus (MOI 1) was added to each well and incubated at 37 ℃ with 5% CO2Incubators were infected for 1 hour.
P34, aspirate the supernatant, wash 3 times with PBS, add maintenance medium containing the drug to the cell wells, and continue culturing for 24 hours.
P35, after 24 hours of culture, cell supernatants were harvested and frozen in a-80 ℃ freezer.
P36, detecting the virus titer of the cell supernatant after 24 hours of culture by a plaque experiment; results as shown in a in fig. 5, the progeny virus titer in the cell supernatant in example 1 was significantly reduced compared to comparative example 2 after 24h treatment of Vero cells with the concentration gradient compound.
The results of the three treatment groups show that the dehydroepiandrosterone derivative AV1004 provided in example 1 is effective in inhibiting flavivirus infection.
Half maximal effect concentration (IC)50) Experimental results of the tests:
half maximal effect concentration (IC)50) Can reflect the inhibition effect of the dehydroepiandrosterone derivative AV1004 on viruses, namely can reach an effective concentration corresponding to 50 percent of the maximum biological effect (inhibiting viruses). IC can be calculated according to the experimental data of drug dose dependence50Numerical values.
IC from B in FIG. 3, B in FIG. 4 and B in FIG. 550As can be seen from the graph, the dehydroepiandrosterone derivative AV1004 provided in example 1 inhibits Japanese encephalitis virus IC50Inhibition IC of Zika virus with a value of 3.056. mu.M50Value of 5.436. mu.M, inhibition IC of dengue virus50The value was 2.603. mu.M.
Note that: in the invention, the plaque experiment specifically comprises the following steps:
r1, BHK-21 cells were dispensed into 24-well plates at a density of 2.0X 105one/mL.
After 14h, R2, plaque experiments were prepared. Washing with serum-free DMEM twice, diluting the sample by 10 times gradient, adding 200 μ L of sample per well for infection, repeating each concentration step in two wells, and standing at 37 deg.C、5％CO2Incubators were infected for 1 hour.
R3, after completion of infection, the supernatant was aspirated and washed once with serum-free DMEM, and 1mL of a medium containing 1.0% methylcellulose and 2% FBS was added to each well, and incubated at 37 ℃ without movement.
R4, culturing for 3 days, taking out the cell plate, sucking away the cell cover, adding formaldehyde solution, and fixing the cells for 12 h.
R5, the formaldehyde solution is removed by suction, and the cells are treated for 4h by adding a crystal violet staining solution containing 0.1 percent.
R6, recovering the crystal violet staining solution, rinsing the cell plate with tap water, and drying in an oven. Pictures were then taken and plaque counts were counted.
Fourthly, evaluating the treatment effect of the dehydroepiandrosterone derivative AV1004 on mice infected with Japanese B encephalitis virus, and specifically comprising the following experimental steps:
a1, six week old C57BL/6 female mice were divided into four groups: DMSO group (comparative example 2, 10), dehydroepiandrosterone derivative group (example 1, 10), JEV + DMSO group (P3 strain infection group, 10), and JEV + dehydroepiandrosterone derivative group (P3 strain infection treatment group, 10).
A2, injecting 105PFU encephalitis B virus into abdominal cavity of mouse, injecting DMEM culture medium with the same volume into abdominal cavity of control group mouse. Mice were gavaged with 35mg/kg of the derivative 1h after infection with the virus, followed by gavage once a day in six weeks old mice. Animal experiments were continued for 23 days, and surviving mice were weighed daily and their behavioral manifestations and death were recorded.
The experimental results are as follows:
detecting the influence of the dehydroepiandrosterone derivative AV1004 on the survival rate of JEV-infected mice: the challenge experiment continued until 23 days post-infection, and the surviving mice had essentially no apparent neurological symptoms. As can be seen from FIG. 6, the number of deaths in the JEV + DMSO group increased sharply between 6 and 13 days of infection, and no dead mice appeared until day 14, with a final mortality of 90%. The death number of mice in the JEV + dehydroepiandrosterone derivative group is obviously less than that of mice in the JEV group, and the final death rate is 60 percent, which shows that the dehydroepiandrosterone derivative can effectively reduce the death of the mice caused by JEV infection. The DMSO group of comparative example 2 did not show any death with the dehydroepiandrosterone derivative group of example 1, indicating that the derivative had no effect on the health of the mice.
Fifthly, the verification and process analysis of the specific mechanism of the dehydroepiandrosterone derivative (AV1004) against the flavivirus provided by the invention are as follows:
p51, carrying out virus adsorption experiment, the steps are as follows:
1) when the growth of the monolayer Vero cells reaches 80% in the cell plate, the cells are washed with DMEM, the medium containing 20 mu M of the derivative or the medium containing the same volume of DMSO is added to the solution, and the mixture is incubated at 37 ℃ and 5% CO2Incubate for 1 hour.
2) JEV virus (MOI ═ 5) was added to each well and incubated in a refrigerator at 4 ℃ for 1 hour. The cells were washed three times with DMEM and cell samples were collected for subsequent detection.
The data of the virus adsorption experiment shown in figure 7 show that the dehydroepiandrosterone derivative has no inhibition effect in the virus adsorption stage.
P52, carrying out virus invasion experiment, and the steps are as follows:
1) when the growth of the monolayer Vero cells reaches 80% in the cell plate, the cells are washed with DMEM, the medium containing 20 mu M of the derivative or the medium containing the same volume of DMSO is added to the solution, and the mixture is incubated at 37 ℃ and 5% CO2Incubate for 1 hour.
2) JEV virus (MOI ═ 5) was added to each well and incubated in a refrigerator at 4 ℃ for 1 hour. Cells were washed three times with DMEM.
3) The new medium was replaced and the cell plates were placed at 37 ℃ in 5% CO2And incubating the culture box for 1 hour, and collecting a cell sample for subsequent detection.
The data of the virus invasion experiment shown in figure 8 show that the dehydroepiandrosterone derivative has no inhibition effect in the virus invasion stage.
P53, performing replicon experiments, the procedure was as follows:
1) to confirm whether the derivatives were effective at the replication stage of JEV, we used replicons to quantitatively assess their inhibitory effects. In vitro transcripts were synthesized from linearized JEV replicons using the T7 mMessage mMachine kit (Invitrogen).
2) When Hela cells grew to 80%, the cells were transfected and 20. mu.M of the derivative was added to the medium and cultured for 24 h. Cells were harvested and luciferase activity was measured using the Rluc assay system (Promega).
The replicon experimental data shown in fig. 9 indicate that the dehydroepiandrosterone derivative has no inhibitory effect in the virus replication stage.
P54, CHX experiment was performed, the procedure was as follows:
1) vero cells were digested with pancreatin and cell counts were performed, adding cells 1.0X 10 per well of 24-well plate5And (4) respectively.
2) After 14h incubation, the supernatants were aspirated, JEV virus (MOI ═ 1) was added to each well, and the wells were incubated at 37 ℃ with 5% CO2Incubators were infected for 1 hour.
3) The supernatant was aspirated, washed 3 times with PBS, and maintenance medium containing drug and CHX was added to the cell wells and incubation continued for 9 hours.
4) After 9 hours of culture, cell samples were harvested and the expression levels of viral mRNA in the different treatment groups were determined by RT-PCR.
The CHX experimental data shown in FIG. 10 indicate that the dehydroepiandrosterone derivative plays an antiviral role before or during translation of the virus after the virus enters the cell.
The results of the above experiments are combined to prove that the dehydroepiandrosterone derivative plays an antiviral role after viruses enter host cells.
Example 2
Example 2 of the present invention provides a drug having anti-flavivirus activity comprising the dehydroepiandrosterone derivative (AV1004) as described above, which has excellent anti-flavivirus activity by using the dehydroepiandrosterone derivative (AV1004) as an active ingredient. The dosage form of the medicament can be adjusted according to the actual application requirement, and is not limited herein.
It should be noted that, as will be understood by those skilled in the art, the dehydroepiandrosterone derivative with anti-flavivirus activity provided by the present invention is not limited to the derivative AV1004 provided in example 1, and can be derivatives with other configurations, all of which have good anti-flavivirus activity efficacy, and is not exhaustive.
In conclusion, the invention provides a dehydroepiandrosterone derivative, a medicament with anti-flavivirus activity and application thereof. The dehydroepiandrosterone derivative has excellent effect on inhibiting flavivirus, and can remarkably improve normal cell lesion caused by flavivirus infection at a concentration without toxic effect on cells. The dehydroepiandrosterone derivative or the medicine containing the dehydroepiandrosterone derivative and having anti-flavivirus activity can be used for preparing a flavivirus inhibitor or a flavivirus treatment medicine independently or after being combined with other anti-flavivirus medicines, and has huge application prospect.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. A dehydroepiandrosterone derivative characterized by: the dehydroepiandrosterone derivative has the function of resisting the activity of flaviviridae viruses, and the structural formula of the derivative is shown as the formula I:
wherein R1 is phenyl; r2 is a hydrogen atom.
2. The dehydroepiandrosterone derivative according to claim 1 wherein: the structural formula of the dehydroepiandrosterone derivative is shown as a formula II:
3. a medicament having anti-flavivirus activity, characterized by: the drug having anti-flavivirus activity comprises the dehydroepiandrosterone derivative according to claim 1 or 2 as an active ingredient and has anti-flavivirus activity.
4. The agent with anti-flavivirus activity of claim 3, wherein: the medicament having anti-flavivirus activity further comprises a pharmaceutically acceptable carrier, diluent or excipient.
5. The agent with anti-flavivirus activity of claim 3, wherein: the dosage form of the drug with anti-flavivirus activity is a gastrointestinal administration dosage form; the gastrointestinal administration dosage form comprises but is not limited to one of powder, tablet, granule, capsule, solution, emulsion and suspension.
6. The agent with anti-flavivirus activity of claim 3, wherein: the dosage form of the drug with anti-flavivirus activity is a parenteral dosage form; the parenteral administration dosage form comprises but is not limited to one of injection dosage form, respiratory administration dosage form, nasal drops, skin administration dosage form, mucous membrane administration dosage form and cavity administration dosage form.
7. The agent with anti-flavivirus activity of claim 3, wherein: the flaviviridae viruses include flaviviridae viruses.
8. The agent with anti-flavivirus activity of claim 7, wherein: the flavivirus virus includes but is not limited to one of Japanese encephalitis virus, Zika virus and dengue virus.
9. Use of a dehydroepiandrosterone derivative according to claim 1 or 2 or a medicament according to any one of claims 3-8 having anti-flavivirus activity for the preparation of a flavivirus inhibitor or flavivirus treatment.
10. A method of inhibiting flaviviridae virus activity, comprising: contacting a chemical agent comprising an effective amount of the dehydroepiandrosterone derivative of claim 1 or 2 or the agent having anti-flavivirus activity of any one of claims 3 to 8 with a host infected with a flaviviridae virus to inhibit the translation stage of the flaviviridae virus in the host cell and significantly ameliorate normal cytopathia resulting from the flavivirus infection.
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| CN103768073A (en) * | 2014-02-18 | 2014-05-07 | 湖北省生物农药工程研究中心 | Application of steroid derivative with dihydrazone structure as antiviral drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US3419549A (en) * | 1966-08-16 | 1968-12-31 | Searle & Co | 3beta-hydroxyandrost-5-en-17-one azines |
| CN103768073A (en) * | 2014-02-18 | 2014-05-07 | 湖北省生物农药工程研究中心 | Application of steroid derivative with dihydrazone structure as antiviral drug |
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