CN109223780A - Using influenza A virus RNA polymerase as the counterfeit Ka Weiding of the drug molecule of the target spot and counterfeit Ka Weiding of dehydrogenation and preparation method - Google Patents
Using influenza A virus RNA polymerase as the counterfeit Ka Weiding of the drug molecule of the target spot and counterfeit Ka Weiding of dehydrogenation and preparation method Download PDFInfo
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- CN109223780A CN109223780A CN201810877367.XA CN201810877367A CN109223780A CN 109223780 A CN109223780 A CN 109223780A CN 201810877367 A CN201810877367 A CN 201810877367A CN 109223780 A CN109223780 A CN 109223780A
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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
- A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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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
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
Abstract
The present invention provides a kinds using influenza A virus RNA polymerase as the antiviral drugs of target spot, the antiviral drugs includes at least one of structure as shown in following formula 1, following formula 2, or the antiviral drugs includes the compound using at least one of structure as shown in following formula 1, following formula 2 as prodrug
Description
Technical field
The invention belongs to field of medicinal chemistry more particularly to a kind of resisting using influenza A virus RNA polymerase as target spot
Virus drugs molecule and preparation method thereof, specifically, being related to counterfeit using influenza A virus RNA polymerase as the drug molecule of target spot
The Ka Weiding and counterfeit Ka Weiding of dehydrogenation and preparation method.
Background technique
Bird flu is communicable disease caused by a kind of dye by avian influenza virus, and avian influenza virus has a variety of hypotypes, and
Easily morph.At present common avian influenza virus subtype mainly include H5N1, H1N1, H5N2, H7N3, H7N7, H9N2 and
H7N9 hypotype.Wherein, H7N9 virus is a kind of influenza subtype of latest find.Since 2013, the H7N9 disease of high lethality rate
Poison is repeatedly popular in the U.S. and China.It is up to 40% lethality, causes the highest attention of global range, and researcher opens
Begin actively drug of the research about such disease.
The Tamiflu reported at present can be divided into four major class according to its action target spot.The first kind is amantadine and second
Amine, such drug can inhibit the M2 albumen of virus, cause virus that cannot shell into born of the same parents, to prevent the release of influenza virus.So
And such target drug is only effective to Flu-A, and it was found that influenza virus produces patience to such drug.Second class is
Neuraminidase inhibitor, action target spot are the neuraminidases on peplos, which can inhibit the neural ammonia of virus
Sour enzyme prevents progeny virus from discharging out of infection cell, to play the effect for killing virus.Widely used drug is at present
Oseltamivir, zanamivir and Peramivir.Wherein, zanamivir is unfavorable for bio-absorbable, and utilization rate is low；Peramivir is
One neuraminidase inhibitor that can be injected intravenously.Oseltamivir is developed on the basis of zanamivir, Oseltamivir
Flu-A and influenza B can effectively be treated.The medicine has obtained the approval of FDA, is approved within 2002 to enter China.As controlling
Treat the active drug of severe influenza.However, there are medicine feelings hard to find in Oseltamivir under the background of national influenza pandemic
Condition.In addition, Oseltamivir has side effect, including abnormal behavior, there is illusion and dysopia etc., safety is by scholar
Query.Third class drug is Favipiravir, and also referred to as T-705, structure is similar to pyrimidine.It, can be thin after T-705 enters cell
The enzyme of born of the same parents carries out triphosphoric acid, becomes its active constituent, because its structure is similar to nucleoside triphosphate GTP, and then can influence viral RNA
Duplication and transcription.4th class drug is the novel Tamiflu Xofluza of Japan's listing in 2018.It is different from the past
Drug, the medicine effect possibility target spot be RNA polymerase in PA restriction endonuclease.PA restriction endonuclease can be from host cell precursor
That shears acquisition mRNA in mRNA by way of " CAP-snatching " emits shape structure, the synthesis for influenza virus itself.
" CAP-snatching " is the key link in influenza virus replicative cycle, blocks this link, by the blocking for the property of can choose
The transcription of influenza virus.Granted also turn out of this drug can be using the RNA polymerase of influenza virus as new drug target.
The drug for obtaining Ministry of Public Health's recommendation at present is neuraminidase inhibitor, but its side effect includes that illusion and row are abnormal
Cause people's attention.2017 days reported 3 plants of H1N1 and reduce to neuraminidase inhibitor sensibility height, and drug resistance occurs
It is exposed to the mankind again in the threat of influenza virus.For a variety of hypotypes and drug resistance, more efficiently object target is needed
The newtype drug selected and designed for novel targets.Therefore, effective anti-influenza virus medicament is developed, it appears especially urgent.
Summary of the invention
The purpose of the present invention is to provide a kind of using influenza A virus RNA polymerase as the antiviral drugs molecule of target spot
And preparation method thereof, it is intended to it is limited to solve existing influenza virus drug, the obvious problem of side effect.
For achieving the above object, The technical solution adopted by the invention is as follows:
One aspect of the present invention provide it is a kind of using influenza A virus RNA polymerase as the antiviral drugs of target spot, it is described anti-
Virus drugs include at least one of structure as shown in following formula 1, following formula 2 or the antiviral drugs comprising with such as following formula 1,
Compound of at least one of the structure shown in following formula 2 as prodrug,
Another object of the present invention is to provide a kind of using influenza A virus RNA polymerase as the antiviral drugs of target spot
The preparation method of molecule, the drug molecular structure is as shown in following formula 2, and the preparation method comprises the following steps:
Benzaldehyde substrate shown in tetrahydroisoquinoline substrate, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene, in potassium carbonate
Or in the solution of sodium carbonate, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
Under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, formula 2 is prepared
Shown drug molecule；
Or
The drug molecular structure is as shown in following formula 1, and the preparation method comprises the following steps:
Benzaldehyde substrate shown in tetrahydroisoquinoline substrate, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene, in potassium carbonate
Or in the solution of sodium carbonate, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
Under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, formula 2 is prepared
Shown compound；
Drug molecule shown in formula 1 is prepared through catalytic hydrogenation in compound shown in formula 2；
Another object of the present invention is to provide a kind of drug molecules to prepare the application in influenza A virus medicament, institute
Stating drug molecule includes at least one of structure as shown in following formula 1, following formula 2 or the drug molecule with such as following formula 1, following formula 2
At least one of shown structure is used as prodrug,
Antiviral drugs provided by the invention contains at least one of molecular structure shown in equation 1 above, formula 2 or described anti-
Virus drugs include the compound using at least one of structure as shown in following formula 1, following formula 2 as prodrug.Formula 1,2 institute of formula
Show molecular structure using influenza A virus RNA polymerase as target spot, by inhibit influenza virus RNA polymerase activity, directly
Connect the transcription and replication process for blocking influenza virus.The In vitro cell experiments such as luciferase prove, organic small point shown in formula 1, formula 2
The activity of RNA polymerase can be effectively suppressed in son, and small organic molecule shown in formula 1, formula 2 is for the RNA polymerase of influenza virus
Target spot has high efficiency and highly selective.Molecular structure provided by the invention, compared to commodity drug Favipiravir T-705, tool
There are stronger inhibitory effect and lower cytotoxicity, having exploitation is the potentiality of anti-influenza A virus medicament molecule.
It is provided by the invention using influenza A virus RNA polymerase as the preparation method of the antiviral drugs molecule of target spot,
Using benzaldehyde shown in tetrahydroisoquinoline shown in formula 3, formula 4 and trimethyl silicane ethyl-acetylene as raw material, drawn by removing trimethyl silicon substrate
Enter alkynyl chain extension, then carries out drug molecule shown in ring-closure reaction preparation formula 2, molecule shown in formula 2 is further subjected to catalysis hydrogen
Change reaction, obtains drug molecule shown in formula 1.This method is simple and easy to control, and obtained product yield is higher.
Detailed description of the invention
Fig. 1 is compound shown in formula 1 provided in an embodiment of the present invention1H-NMR map；
Fig. 2 is compound shown in formula 1 provided in an embodiment of the present invention13C-NMR map；
Fig. 3 is compound shown in formula 2 provided in an embodiment of the present invention1H-NMR map；
Fig. 4 is compound shown in formula 2 provided in an embodiment of the present invention13C-NMR map；
Fig. 5 is formula 1, formula 2 and influenza virus RNA polymerase binding site schematic diagram provided in an embodiment of the present invention.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
The RNA polymerase of influenza virus is responsible for the transcription and duplication of RNA, is the core ingredient of influenza activity.Effectively
Inhibit the activity of RNA polymerase that can block the duplication of influenza virus.Different from people intracorporal DNA transcription and reproduction process, for
The drug of RNA polymerase will not generate injury to human body.Existing research person has carried out the drug for RNA polymerase and has set at present
Meter, due to the genetic sequence conservative of the RNA polymerase height of A type influenza B virus, the especially sequence in critical active position
Column similitude, viral RNA polymerase are referred to as " the super target " of drug.Neuraminidase different from the past is target spot,
When viral rna polymerase is as drug target, since polymerase has very strong hereditary conservation, it is not easy virus to drug
It develops drug resistance, and such drug will show wide spectrum inhibition to a series of avian influenza virus with such RNA polymerase；
Meanwhile because viral rna polymerase Active Site Specific, the drug functioned will not affect that the normal of human body cell
Function has lower cytotoxicity.Currently, being modified and being transformed for the structure of natural products to enhance the work of drug
Property, dissolubility and targeting cause the interest and concern of pharmaceutical chemists, and are successfully applied to the research and development of new drug and set
Meter.
On this basis, the embodiment of the invention provides a kind of using influenza A virus RNA polymerase as the disease-resistant of target spot
Cytotoxic drug, the antiviral drugs include at least one of structure as shown in following formula 1, following formula 2 or the antiviral drugs packet
Containing with such as following formula 1 (counterfeit Ka Weiding, pseudocavidine), following formula 2 (13- dehydrogenation counterfeit Ka Weiding, 13-
Dehydropseudocavidine compound of at least one of the structure shown in) as prodrug,
Antiviral drugs provided in an embodiment of the present invention, containing at least one of molecular structure shown in equation 1 above, formula 2, or
The antiviral drugs includes the compound using at least one of structure as shown in following formula 1, following formula 2 as prodrug.Formula
1, molecular structure shown in formula 2 is using influenza A virus RNA polymerase as target spot, by the RNA polymerase for inhibiting influenza virus
Activity directly blocks the transcription and replication process of influenza virus.The In vitro cell experiments such as luciferase prove, shown in formula 1, formula 2
The activity of RNA polymerase can be effectively suppressed in small organic molecule, and small organic molecule shown in formula 1, formula 2 is directed to the RNA of influenza virus
Polymerase is that target spot has high efficiency and highly selective.Molecular structure provided in an embodiment of the present invention, compared to commodity drug method
Draw Wei T-705, have more efficiently inhibitory effect and lower cytotoxicity, have exploitation be anti-influenza A virus medicine
The potentiality of object molecule.
It should be appreciated that in the embodiment of the present invention, using influenza A virus RNA polymerase as in the antiviral drugs of target spot,
Small organic molecule shown in formula 1 can be contained or using small organic molecule shown in formula 1 as medicine precursor compound, can also be contained
Small organic molecule shown in formula 2 or using small organic molecule shown in formula 2 as medicine precursor compound, it is of course also possible to contain simultaneously
There is small organic molecule shown in small organic molecule shown in formula 1 and formula 2, or is shown with simultaneously with small organic molecule shown in formula 1 and formula 2
Machine small molecule is as medicine precursor compound.
As a kind of specific implementation situation, the structure of the active drug molecule of the antiviral drugs as shown in following formula 1,
Small organic molecule shown in formula 1 can effectively inhibit the activity of the RNA polymerase of influenza virus, directly blocking influenza disease
The transcription and replication process of poison, and the RNA polymerase of infected by influenza is that target spot has high efficiency and highly selective.
As another kind be embodied situation, the structure of the active drug molecule of the antiviral drugs as shown in following formula 2,
Small organic molecule shown in formula 2 can effectively inhibit the activity of the RNA polymerase of influenza virus, directly blocking influenza disease
The transcription and replication process of poison, and the RNA polymerase of infected by influenza is that target spot has high efficiency and highly selective.
Small organic molecule shown in formula 1 provided in an embodiment of the present invention, small organic molecule shown in formula 2, can pass through following sides
Method synthesis, it is of course also possible to by the way that acquisition is extracted from plants.
Correspondingly, the embodiment of the invention provides a kind of using influenza A virus RNA polymerase as the antiviral agent of target spot
The preparation method of object molecule, the drug molecular structure is as shown in following formula 2, and the preparation method comprises the following steps:
S01. tetrahydroisoquinoline substrate shown in formula 3, benzaldehyde substrate and trimethyl silicane ethyl-acetylene shown in formula 4 are provided, in carbon
In the solution of sour potassium or sodium carbonate, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
S02. under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, be prepared into
To drug molecule shown in formula 2；
Or
The drug molecular structure is as shown in following formula 1, and the preparation method comprises the following steps:
S01. tetrahydroisoquinoline substrate shown in formula 3, benzaldehyde substrate and trimethyl silicane ethyl-acetylene shown in formula 4 are provided, in carbon
In the solution of sour potassium or sodium carbonate, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
S02. under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, be prepared into
To compound shown in formula 2；
S03. drug molecule shown in formula 1 is prepared through catalytic hydrogenation in compound shown in formula 2；
It is provided in an embodiment of the present invention using influenza A virus RNA polymerase as the preparation of the antiviral drugs molecule of target spot
Method, using benzaldehyde shown in tetrahydroisoquinoline shown in formula 3, formula 4 and trimethyl silicane ethyl-acetylene as raw material, by removing trimethyl silicane
Base introduces alkynyl chain extension, then carries out drug molecule shown in ring-closure reaction preparation formula 2, further urges molecule shown in formula 2
Change hydrogenation, obtains drug molecule shown in formula 1.This method is simple and easy to control, and obtained product yield is higher.
Specifically, in above-mentioned steps S01, with benzaldehyde shown in tetrahydroisoquinoline shown in formula 3, formula 4 and trimethyl silicon substrate second
Alkynes is as raw material, under the effect of the catalyst, reacts in the solution of potassium carbonate or sodium carbonate, which is conducive to front three
The removing of trimethyl silicon substrate in base silicon substrate acetylene.
Preferably, in the step of tetrahydroisoquinoline shown in catalysis reaction preparation formula 5, catalyst is that benzoic acid and iodate are sub-
Copper.With benzoic acid and cuprous iodide collectively as catalyst, catalysis reaction yield can effectively improve.Specifically, compared to list
Solely using cuprous iodide collectively as catalyst, with benzoic acid and cuprous iodide collectively as catalyst after, tetrahydro shown in formula 5 is different
The yield of quinoline is promoted by 5% to 69%.The dosage of the benzoic acid and the cuprous iodide is respectively tetrahydroisoquinoline mole
The 10% of amount, to obtain optimal reaction effect, and yield highest.
It is specific preferred, in above-mentioned steps S01, with benzaldehyde and trimethyl silicane shown in tetrahydroisoquinoline shown in formula 3, formula 4
Ethyl-acetylene is as raw material, and under the effect of the catalyst, in the solution of potassium carbonate or sodium carbonate, reaction obtains tetrahydro shown in formula 5
The reaction equation of isoquinolin is as follows:
In above-mentioned steps S02, it is anti-that tetrahydroisoquinoline shown in formula 5 under the action of palladium catalyst and reducing agent to that closed loop occur
It answers, drug molecule shown in formula 2 is prepared.Preferably, the palladium catalyst is Pd (PPh3)4, the reducing agent is HCO2Na,
It is hereby achieved that drug molecule shown in the formula 2 of high yield (72%).Preferably, the temperature of ring-closure reaction is 95 DEG C -105 DEG C,
More preferably 100 DEG C.In the reaction, it is preferred to use DMF and H2The mixed solvent of O is as reaction medium, to be conducive to closed loop
The generation of reaction, and miscellaneous side reaction can be reduced.
It is specific preferred, in above-mentioned steps S02, by tetrahydroisoquinoline shown in formula 5 palladium catalyst and reducing agent effect
Lower generation ring-closure reaction, the reaction equation that drug molecule shown in formula 2 is prepared are as follows:
In above-mentioned steps S03, drug molecule shown in formula 1 is prepared through catalytic hydrogenation in compound shown in formula 2, it is excellent
Choosing, the catalyst of the catalytic hydrogenation is PtO2, reaction dissolvent is acetic acid, obtains the product that yield is greater than 99%.Instead
It should be carried out under reducing atmosphere such as hydrogen atmosphere.
The reaction equation that drug molecule shown in formula 1 is prepared through catalytic hydrogenation in compound shown in formula 2 is as follows:
Another object of the present invention is to provide a kind of drug molecules to prepare the application in influenza A virus medicament, institute
Stating drug molecule includes at least one of structure as shown in following formula 1, following formula 2 or the drug molecule with such as following formula 1, following formula 2
At least one of shown structure is used as prodrug,
As a kind of specific implementation situation, the drug molecular structure as shown in following formula 1,
As a kind of specific implementation situation, the drug molecular structure as shown in following formula 2,
It is illustrated below with reference to being embodied.
Embodiment 1
It is a kind of using influenza A virus RNA polymerase as the preparation method of the antiviral drugs molecule of target spot, the drug
Molecular structure is as shown in following formula 2, and the preparation method comprises the following steps:
Benzaldehyde 229mg shown in tetrahydroisoquinoline 212mg, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene 0.26mL,
12mg benzoic acid is added, 19mg CuI reacts 12 hours at 80 DEG C, and filtering and concentrating obtains crude product, and crude product is dissolved in
In 10ml methanol, potassium carbonate 276mg is added, tetrahydroisoquinoline shown in formula 5 is obtained by column chromatography for separation after reaction
297mg；
Tetrahydroisoquinoline 215mg shown in modus ponens 5 is placed in flask, is added DMF (9mL), H2O (3mL), adds HCO2Na
(68mg), nitrogen are saturated 15 minutes, and Pd (PPh is then added3)4(29mg) is placed in 100 DEG C of oil bath and reacts 1h.Reaction terminates
DCM extraction, concentration are added afterwards, column chromatography for separation obtains compound 126mg shown in product formula 2.
Compound shown in formula 2 is subjected to nuclear-magnetism detection, is obtained1H-NMR map as shown in figure 3,13C-NMR map such as Fig. 4
It is shown.
Embodiment 2
It is a kind of using influenza A virus RNA polymerase as the preparation method of the antiviral drugs molecule of target spot, the drug
Molecular structure is as shown in following formula 1, and the preparation method comprises the following steps:
Benzaldehyde 229mg shown in tetrahydroisoquinoline 212mg, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene 0.26mL,
12mg benzoic acid is added, 19mg CuI reacts 12 hours at 80 DEG C, and filtering and concentrating obtains crude product, and crude product is dissolved in
In 10ml methanol, potassium carbonate 276mg is added, Tetrahydroisoquinoli- 297mg shown in formula 5 is obtained by column chromatography for separation after reaction；
Tetrahydroisoquinoline 215mg shown in modus ponens 5 is placed in flask, is added DMF (9mL), H2O (3mL), adds HCO2Na
(68mg), nitrogen are saturated 15 minutes, and Pd (PPh is then added3)4(29mg) is placed in 100 DEG C of oil bath and reacts 1h.Reaction terminates
DCM extraction, concentration are added afterwards, column chromatography for separation obtains compound 126mg shown in product formula 2；
Compound 35mg shown in modus ponens 2 is dissolved in HOAc (2.0mL), and catalyst Pt O is added2(2.3mg), it is big at one
It is reacted under the hydrogen atmosphere of air pressure 12 hours, DCM extraction is added after reaction, column chromatography for separation obtains natural products formula 1,
Compound shown in formula 1 is subjected to nuclear-magnetism detection, is obtained1H-NMR map as shown in Figure 1,13C-NMR map such as Fig. 2
It is shown.
The cell transfecting of 3 HEK-293T of embodiment and the toxicity test of drug
Culture medium and solution needed for preparing: prepare TransIT-1L transfection agents and by 5% FBS and 1%P/S
Culture medium composed by Opti-MEM medium.Prepare and cultivate HEK-293T cell.Prepare the 0.2 every hole μ g in 96 orifice plates
DNA solution.
Cell culture: in T-75cm2Flask in cultivate the cell of HEK-293T and reach 70%- until cell density
80%, and continuous passage 3 times.Test first 24 hours collection cells, each 96 orifice plate inoculation about 2 × 106A cell, and be incubated for
Cell pellet overnight.
Cellulation compound: prepare medium of the 1.43mL without serum and prepare to transfect and be added the DNA plasmid of 22 μ g, add
Enter the TransIT-1L of 66 μ l, is uniformly mixed.Hatching 15-30 minutes at room temperature makes to form transfection composite, on 96 orifice plates
The premixed liquid of 13 μ L is added in each hole.
By the cell trypsinized in standard medium, appropriate culture medium is added, is uniformly mixed, uses haemocytometer
Determine the cell quantity of every milliliter of culture medium.Ensure that every milliliter of cell quantity in every hole is no less than 2-4 × 104.In 96 holes
The 87 diluted cell solutions of μ L are added in each hole of plate.
By the plasmid of normal cell transfection influenza A virus polymerase, then the cell of transfection is divided with drug respectively
1,2 and T-705 of son contacts with each other.Drug molecule is added in cell and is cultivated, the shadow that measurement drug molecule grows cell
It rings.Cell is harvested after waiting 40 hours.Lactic dehydrogenase experiment is carried out, corresponding half lethal dose concentration is quantitatively obtained.
Since lactic dehydrogenase exists only in cell interior, if small organic molecule kills cell, lactic dehydrogenase can quilt
It is discharged into solution, by measuring the quantity of lactic dehydrogenase in the solution, and then quantitatively can measure organic molecule to cell
Toxicity.In 40 hours chronic tests, measured by lactic dehydrogenase (Lactate dehydrogenase) test, shown in formula 1
Compound shown in compound, formula 2, T-705 toxicity numerical value be respectively as follows: 2862.83 μM, 1371.17 μM, 3359.64 μM.
It can be seen that drug molecule provided by the invention does not have the ability of inducing cell cytolysis, show as lower thin
Cellular toxicity shows that the molecule safety of drug is good.Using commodity Tamiflu T-705 as reference, the embodiment of the present invention is provided
Organic molecule show lower toxicity.
Embodiment 4 measures the IC50 experiment of the inhibitory effect of drug molecule
Respectively prepare concentration gradient be 1000 μM, 200 μM, 40 μM, 8 μM, drug molecule, structure shown in 1.6 μM of structure 1
The solution of drug molecule shown in 2.
Normal cell in embodiment 3 is transfected into influenza virus polymerase DNA, and is separately added into drug molecule solution and is formed altogether
The influenza polymerase plasmid system of transfection.Cell with and drug molecule contact with each other under the concentration gradient solution prepared culture
After 40 hours, the 503nhibiting concentration IC50 that is measured by luciferase assay (luciferase assay).Compound shown in formula 1,
Compound shown in formula 2, T-705 IC50 be respectively as follows: 44.13 μM, 31.13 μM, 21.63 μM.
From experimental result as can be seen that antiviral compound, which all has, inhibits the active work of influenza virus polymerase complex
With.
This example demonstrates that drug molecule shown in structure 1, drug molecule shown in structure 3 are able to suppress answering for influenza virus
Synthase activity.
The selectivity factor (selectivity index) of 5 drug of embodiment
The effective component T-705 of Favipiravir is chosen as reference, by embodiment 3 and embodiment 4, this kind of structure can be calculated
Drug molecule shown in 1, drug molecule shown in structure 2 selectivity factor be 83.88 and 37.97, be significantly better than the selection of T-705
Property coefficient 149.65.Both Medicine small molecules show as better choice with having more preferably drug selectivity than T-705
Property, the RNA polymerase of influenza virus can be effectively suppressed without damaging normal cell, can needle can be carried out for the inhibitor molecules
MOLECULE DESIGN is to prepare Tamiflu.
This example demonstrates that drug molecule shown in structure 1, drug molecule shown in structure 2 have drug more better than T-705
Selectivity.
The molecular simulation analysis of 6 drug molecule combination target spot of embodiment
Using molecular docking (molecular docking) technique study formula 1,2 formula 2 of formula and influenza A virus
The energy in site and combination that RNA polymerase combines.Formula 1, formula 2 and influenza virus RNA polymerase binding site schematic diagram are shown in figure
5, formula 1 is similar to 2 structure of formula, can behave as binding site similar with RNA polymerase.By molecular docking as a result, we
Molecule shown in discoverable type 1, formula 2 can be combined effectively two positions.First, formula 1,2 compound represented of formula may be combined
The substrate of RNA polymerase enters the position in channel, and the Conjugated free energy being calculated is respectively -9.0kcal/mol, -
8.3kcal/mol, numerical value is close to the Conjugated free energy -9.0kcal/mol of the T-705 of phosphorylation.Chemical combination shown in 1 formula 2 of formula
Object can effectively combine the channel position in RNA polymerase, pass through resistance by interacting with Arg658, Lys659 and His1363
The substrate of disconnected RNA polymerase enters and then inhibits the transcription and replication process of RNA polymerase.Secondly being to change shown in 1 formula 2 of formula
Closing object may be incorporated in the position of PA restriction endonuclease of influenza A virus, and calculating resulting Conjugated free energy is -9.1kcal/mol, -
8.5kcal/mol.As shown, formula 1, formula 2 may be incorporated in the bag structure of RNA polymerase, and His41, Arg84,
Lys196 forms advantageous combination, blocks RNA polymerase and the combination of its substrate.1 formula 2 of formula as the result is shown of molecular docking can
To be effectively integrated to the inside of RNA polymerase, blocking substrate to enter channel i.e. can be effectively suppressed the transcription and replication process of RNA.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of using influenza A virus RNA polymerase as the antiviral drugs of target spot, which is characterized in that the antiviral drugs
It include as shown in following formula 1, following formula 2 comprising at least one of structure as shown in following formula 1, following formula 2 or the antiviral drugs
Compound of at least one of the structure as prodrug,
2. as described in claim 1 using influenza A virus RNA polymerase as the antiviral drugs of target spot, which is characterized in that
The structure of the active drug molecule of the antiviral drugs as shown in following formula 1,
3. as described in claim 1 using influenza A virus RNA polymerase as the antiviral drugs of target spot, which is characterized in that
The structure of the active drug molecule of the antiviral drugs as shown in following formula 2,
4. a kind of using influenza A virus RNA polymerase as the preparation method of the antiviral drugs molecule of target spot, which is characterized in that
The drug molecular structure is as shown in following formula 2, and the preparation method comprises the following steps:
Benzaldehyde substrate shown in tetrahydroisoquinoline substrate, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene, in potassium carbonate or carbon
In the solution of sour sodium, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
Under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, is prepared shown in formula 2
Drug molecule；
Or
The drug molecular structure is as shown in following formula 1, and the preparation method comprises the following steps:
Benzaldehyde substrate shown in tetrahydroisoquinoline substrate, formula 4 shown in offer formula 3 and trimethyl silicane ethyl-acetylene, in potassium carbonate or carbon
In the solution of sour sodium, tetrahydroisoquinoline shown in catalysis reaction preparation formula 5；
Under the action of palladium catalyst and reducing agent ring-closure reaction is occurred into for tetrahydroisoquinoline shown in formula 5, is prepared shown in formula 2
Compound；
Drug molecule shown in formula 1 is prepared through catalytic hydrogenation in compound shown in formula 2；
5. as claimed in claim 4 using influenza A virus RNA polymerase as the preparation side of the antiviral drugs molecule of target spot
Method, which is characterized in that catalysis was reacted in the step of tetrahydroisoquinoline shown in preparation formula 5, and catalyst is benzoic acid and cuprous iodide.
6. as claimed in claim 4 using influenza A virus RNA polymerase as the preparation side of the antiviral drugs molecule of target spot
Method, which is characterized in that the palladium catalyst is Pd (PPh3)4, the reducing agent is HCO2Na；And/or
The catalyst of the catalytic hydrogenation is PtO2, reaction dissolvent is acetic acid.
7. as claim 4-6 is described in any item using influenza A virus RNA polymerase as the antiviral drugs molecule of target spot
Preparation method, which is characterized in that the solution of the potassium carbonate or sodium carbonate is the methanol solution of potassium carbonate or sodium carbonate.
8. a kind of application of drug molecule in preparation treatment influenza A virus medicament, which is characterized in that the drug molecule
Comprising at least one of structure as shown in following formula 1, following formula 2 or the drug molecule in the structure as shown in following formula 1, following formula 2
At least one as prodrug,
9. application of the drug molecule as claimed in claim 8 in preparation treatment influenza A virus medicament, which is characterized in that
The drug molecular structure as shown in following formula 1,
10. application of the drug molecule as claimed in claim 8 in preparation treatment influenza A virus medicament, feature exist
In, the drug molecular structure as shown in following formula 2,
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660850A (en) * | 2004-12-29 | 2005-08-31 | 中国人民解放军第二军医大学 | Method for preparing dehydrogenated cavidine, dehydrogenated apocavidine and combination |
| CN105287539A (en) * | 2015-11-12 | 2016-02-03 | 江苏康缘药业股份有限公司 | Novel application of corydaline |
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2018
- 2018-08-03 CN CN201810877367.XA patent/CN109223780B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660850A (en) * | 2004-12-29 | 2005-08-31 | 中国人民解放军第二军医大学 | Method for preparing dehydrogenated cavidine, dehydrogenated apocavidine and combination |
| CN105287539A (en) * | 2015-11-12 | 2016-02-03 | 江苏康缘药业股份有限公司 | Novel application of corydaline |
Non-Patent Citations (3)
| Title |
|---|
| SHIQIANG ZHOU等: "Three-Step Catalytic Asymmetric Total Syntheses of 13-Methyltetrahydroprotoberberine Alkaloids", 《ORGANIC LETTERS》 * |
| 张梦媛等: "小檗碱对甲型流感病毒 FM1 株感染小鼠肺中 RLH 信号通路的影响", 《中山大学学报（ 医学科学版）》 * |
| 方波等: "小檗碱类抗微生物化合物研究进展", 《国际药学研究杂志》 * |
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