CN107513072A - Application of the polyketone in HIV is suppressed - Google Patents
Application of the polyketone in HIV is suppressed Download PDFInfo
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- CN107513072A CN107513072A CN201610425273.XA CN201610425273A CN107513072A CN 107513072 A CN107513072 A CN 107513072A CN 201610425273 A CN201610425273 A CN 201610425273A CN 107513072 A CN107513072 A CN 107513072A
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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Abstract
The invention discloses application of the polyketone in HIV is suppressed.The application is application of the compound in hiv inhibitor or hiv integrase inhibitor is prepared shown in formula I.Inhibiting rate of the compound when concentration is 100 μM to HIV 1 shown in formula I is 99.91% ± 0.02%, the IC of the compound shown in formula I50For 6.6 μM, the CC of the compound shown in formula I50More than 100 μM.Compound shown in formula I has stronger inhibitory activity to HIV 1, and toxicity is smaller, has preferable drug selectivity.
Description
Technical field
The present invention relates to application of the polyketone in HIV is suppressed.
Background technology
HIV (Human Immunodeficiency Virus) viruses, Chinese full name human immunodeficiency virus, belong to inverse
One kind of Retroviral.After inhibition of HIV intrusion host cell, meeting heavy damage host body immunologic function is serious so as to cause
Opportunistic infections so that causing the secondary immunodeficiency characterized by malignant disease, as acquired immunodeficiency syndrome
(acquired immunodeficiency syndrome, AIDS).Patient With Aids case was found in 1981 in the U.S.,
Then begin to propagate rapidly in the world, at present as one of significant threat of human health.Inhibition of HIV can be divided into
HIV-1 types and HIV-2 types, both genomes have very big difference, and wherein HIV-1 is the master for causing global spread of aids
Want pathogen.
Chemotherapy is still currently the most important ones HIV-1 treatment means, has 26 kinds of chemical entities to enter so far clinical
Using.Classify by its mechanism of action, be mainly RTI and protease inhibitors in these marketed drugs, add up
To 22 kinds.And only 4 kinds of other mechanism of action, including 2 kinds of integrase inhibitors and 2 kinds of viral entry inhibitors.With anti-
The continuous research and development application of HIV medicines, is particularly treated by Cocktail treatment mode to AIDS patient, can be had
Effect reduces virus load, significantly extends the survival of patients time, but the problems such as drug resistance, adverse reaction and the compliance of patient still
Often cause the failure of clinical treatment.Therefore, it is still necessary to which the new anti-HIV-1 medicines of Persisting exploitation are for clinical practice.
Microbe species are huge, have the metabolite of enriched types, are find high-efficiency low-toxicity HIV-resistant activity material one
Individual important sources.The bacterial strain of HIV-resistant activity material is produced by establishing the quick screening of screening model, then from its metabolite
It is guide to find the compound with HIV-resistant activity or the compound with certain activity, carries out structure simplification and modification, discloses
The mechanism of action and structure-activity relationship, it is the effective way for finding new inverase.
Epicocconigrone A are a kind of polyketone, and its structural formula is as shown in formula IEpicocconigrone A are reported in 2014 first, its be from
It is isolated in plant endogenesis epiphyte Epicoccum nigrum metabolite, have and suppress multiple protein kinases and suppression group
Albumen deacetylation enzymatic activity, at the same can suppress RAJI and U-937 human lymphomas growth (EI Amrani M, Lai D,
Debbab A,et al.Protein Kinase and HDAC Inhibitors from the Endophytic Fungus
Epicoccum nigrum.Journal of Natural Products,2014,77:49-56)。
The content of the invention
The technical problems to be solved by the invention are how to suppress HIV.
In order to solve the above technical problems, the invention provides the compound shown in formula I or its pharmaceutically acceptable salt
Application in hiv inhibitor or hiv integrase inhibitor is prepared,
Present invention also offers the compound shown in formula I or its pharmaceutically acceptable salt to prepare treatment or/and prevention
Application in acquired immunodeficiency syndrome medicine.
Present invention also offers medicinal compound, the medicinal compound be formula I shown in compound or its pharmaceutically may be used
The salt of receiving.
In above-mentioned medicinal compound, the medicinal compound or its pharmaceutically acceptable salt move for suppressing HIV
Thing, or the medicinal compound or its pharmaceutically acceptable salt are used to treat or/and prevent acquired immunodeficiency syndrome
Or the medicinal compound or its pharmaceutically acceptable salt are integrated into zooblast for suppressing HIV.
Present invention also offers M1) or method M2)：
M1) suppress HIV animal method, including to receptor apply formula I shown in compound or its pharmaceutically
Acceptable salt is to suppress HIV animal；
M2 the method for) treating or/and preventing acquired immunodeficiency syndrome, including applied to receptor shown in formula I
Compound or its pharmaceutically acceptable salt treated or/and prevented acquired immunodeficiency syndrome.
Above, the compound shown in formula I can be from aspergillus (Aspergillus sp.) CPCC400735 (also known as aspergillus
(Aspergillus sp.) CPCC 400735) culture in isolated, aspergillus (Aspergillus sp.)
CPCC400735 is CGMCC in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center
No.12376。
Above-mentioned aspergillus (Aspergillus sp.) CPCC400735 culture is by above-mentioned aspergillus (Aspergillus
Sp.) CPCC400735 cultivates the material in obtained culture vessel in microbiological culture media.
In above-mentioned aspergillus (Aspergillus sp.) CPCC400735 culture, the microbiological culture media can be song
Mould culture medium (culture medium that can cultivate aspergillus), it can be solid medium, semisolid culturemedium or fluid nutrient medium.It is described micro-
Biological medium can be rice culture medium, potato dextrose agar or other fungies well known to those skilled in the art
Fermentation medium etc..In above-mentioned aspergillus (Aspergillus sp.) CPCC 400735 culture, the rice culture medium can
Be made up of rice and water, can be also made up of rice, water and inorganic salts, can be also made up of rice, water and nitrogen source, also can by rice,
Water, nitrogen source and inorganic salts are made.The potato dextrose agar can be made up of potato, glucose, agar and water,
Also it can be made up of potato, glucose, agar, water and inorganic salts, can be also made up of potato, carbon source, agar, water and nitrogen source,
Also can be made up of potato, carbon source, agar, nitrogen source and inorganic salts.Other described fungi fermentations well known to those skilled in the art
Culture medium can by it is a certain or it is several it is quick-acting, imitate late carbon source, a certain kind or it is several it is quick-acting, imitate nitrogen source, water and/or inorganic salts etc. late
It is made.
Above, the rice can be rice or brown rice.Rice or brown rice are the products of paddy.Paddy refers to not go
Except the fruit of the husk of paddy rice, paddy is made up of husk, pericarp, kind skin, perisperm, aleurone, endosperm and embryo.Brown rice refers to paddy
Husk is sloughed, retains the product of the other each several parts of paddy；Rice refers to only retain endosperm, and paddy remainder is all sloughed
Product.Carbon source is that microorganism grows a kind of nutrients, is carbon compound, including carbohydrate, grease, organic acid and organic acid esters
With small molecular alcohol etc. it is quick-acting, imitate carbon source late.Nitrogen source refers to the material for providing nitrogen needed for microbial nutrition, including peanut cake
Powder, soybean cake powder, dusty yeast, peptone, ammoniacal liquor, ammonium salt and nitrate etc. are quick-acting, imitate nitrogen source late.
In above-mentioned aspergillus (Aspergillus sp.) CPCC400735 culture, the temperature of the culture can be 20-30
DEG C, incubation time can be 10-40 days.
Can be according to including A1)-A5) the step of culture of the method by the compound shown in formula I from aspergillus CPCC400735
It is isolated in thing：
A1) extracted to obtain from above-mentioned aspergillus (Aspergillus sp.) CPCC400735 culture with methanol molten
In the material of methanol；
A2 it is) that the material is water-dispersible, then it is extracted with ethyl acetate, collects ethyl acetate phase, removes ethyl acetate
Ethyl acetate in phase, obtains medicinal extract；The medicinal extract is subjected to silica gel column chromatography separation, elution journey used in silica gel column chromatography
Sequence is first to elute 2 column volumes with chloroform；The following linear gradient elution of 13 column volumes is carried out again：Mobile phase used be by
The mixed liquor of chloroform and methanol composition, the volume ratio of chloroform and methanol is by 20 in linear gradient elution mobile phase:1 is linearly down to 7:
1；Last again with methanol elutes two column volumes；The liquid that the 5.1st to the 6.3rd column volume elutes is collected, is named as
Fr.18-21；
A3 Fr.18-21) is subjected to silica gel column chromatography, in the silica gel column chromatography elution program used be with by chloroform and
The chloroform of methanol composition and the volume ratio of methanol are 7:1 mixing liquid is eluted as mobile phase, collects the 1st to the 1.5th
The liquid that individual column volume elutes is named as Tubes 9-12；
A4 Tubes 9-12) are subjected to preparative high performance liquid chromatography separation, preparative high performance liquid chromatography separation institute
Filler is octadecylsilane chemically bonded silica filler, and the particle diameter of filler is 5 μm, preparative high performance liquid chromatography separation institute
Chromatographic column a diameter of 8mm, a height of 250mm；Mobile phase is that the volume ratio of the first alcohol and water of first alcohol and water composition is 1:1 it is mixed
Close liquid；Flow velocity is 3.0mL/min；All eluting peaks that retention time is 10.4 to 11.3 minutes are collected, are named as
Tubes 9-12-P2；
A5 Tubes 9-12-P2) are subjected to preparative high performance liquid chromatography separation, preparative high performance liquid chromatography separation
Filler used is octadecylsilane chemically bonded silica filler, and the particle diameter of filler is 5 μm, preparative high performance liquid chromatography separation
Chromatographic column used a diameter of 8mm, a height of 250mm；Mobile phase is that the volume ratio of the first alcohol and water of first alcohol and water composition is 9:11
Mixing liquid；Flow velocity is 2.5mL/min, collects the eluting peak that retention time is 20.7min, obtains the compound shown in formula I.
Above-mentioned aspergillus (Aspergillus sp.) CPCC400735 can with conidium, mycelia or containing conidium and/
Or the mycelial form of mycelia is present.
Above, the HIV can be the HIV of HIV-1 types, and the acquired immunodeficiency syndrome can be drawn by HIV-1
Rise.
Above, the animal can be mammal, such as people.
Above, the hiv inhibitor, hiv integrase inhibitor and the treatment or/and prevention acquired immunodeficiency
Syndrome medicine, in addition to containing active component, also contain suitable carrier or excipient.Here carrier material is included but not
It is limited to water soluble carrier material (such as polyethylene glycol, polyvinylpyrrolidone, organic acid), slightly solubility carrier material (such as ethyl
Cellulose, cholesterol ester stearic acid etc.), enteric solubility carrier material (such as CAP and the cellulose of carboxylic first and second).
Wherein it is preferred that water soluble carrier material.A variety of formulations, including but not limited to tablet, glue can be made using these materials
Capsule, dripping pill, aerosol, pill, pulvis, solution, supensoid agent, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository,
Freeze drying powder injection etc..Can be ordinary preparation, sustained release preparation, controlled release preparation and various particulate delivery systems.In order to which unit is given
Tablet is made in pharmaceutically dosage form, can widely use various carriers well known in the art.Example on carrier is, for example, diluent with
Absorbent, as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white bole,
Microcrystalline cellulose, alumina silicate etc.；Wetting agent and adhesive, as water, glycerine, polyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin,
Syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethylcellulose, lac, methylcellulose, potassium phosphate,
Polyvinylpyrrolidone etc.；Disintegrant, such as dry starch, alginate, agar powder, laminaran, sodium acid carbonate and citron
Acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.；Collapse
Solve inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil and fat etc.；Sorbefacient, such as quaternary ammonium salt, dodecane
Base sodium sulphate etc.；Lubricant, such as talcum powder, silica, cornstarch, stearate, boric acid, atoleine, poly- second two
Alcohol etc..Tablet can also be further made to coating tablet, such as sugar coated tablet, thin membrane coated tablet, enteric coated tablets, or double-layer tablets
And multilayer tablet.In order to which unit dosage forms for administration is made into pill, various carriers well known in the art can be widely used.On carrier
Example be such as diluent and absorbent, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidine
Ketone, Gelucire, kaolin, talcum powder etc.；Adhesive such as Arabic gum, bassora gum, gelatin, ethanol, honey, liquid sugar, rice paste
Or batter etc.；Disintegrant, such as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose
Element etc..In order to which unit dosage forms for administration is made into suppository, various carriers well known in the art can be widely used.Example on carrier
Son is, such as the ester of polyethylene glycol, lecithin, cocoa butter, higher alcohol, higher alcohol, gelatin, semi-synthetic glyceride etc..In order to incite somebody to action
Injection preparation is made in unit dosage forms for administration, such as solution, emulsion, freeze drying powder injection and supensoid agent, this area can be used normal
All diluents, for example, water, ethanol, polyethylene glycol, 1,3-PD, the isooctadecanol, polyoxygenated different of ethoxylation
Stearyl alcohol, Polyoxyethylene Sorbitol Fatty Acid Esters etc..In addition, in order to prepare isotonic parenteral solution, can add into injection preparation
Add appropriate sodium chloride, glucose or glycerine, further, it is also possible to add conventional cosolvent, buffer, pH adjusting agent etc..This
Outside, if desired, colouring agent, preservative, spices, flavouring, sweetener or other materials can also be added into pharmaceutical preparation.Make
Can be with administrated by injection with above-mentioned formulation, including be subcutaneously injected, be injected intravenously, intramuscular injection and intracavitary administration etc.；Cavity/canal drug administration,
Such as per rectum and vagina；Respiratory tract administration, such as via intranasal application；Mucosa delivery.Above-mentioned method of administration is preferably drug administration by injection.
It is demonstrated experimentally that the compound shown in formula I be 99.91% to HIV-1 inhibiting rate when concentration is 100 μM ±
0.02%, the IC of the compound shown in formula I50For 6.6 μM, the CC of the compound shown in formula I50More than 100 μM.Shown in formula I
Compound there is stronger inhibitory activity to HIV-1, and toxicity is smaller, and drug selectivity is preferable.
Preservation explanation
Strain name：Aspergillus (Aspergillus sp.)
Strain number：CPCC400735
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On May 5th, 2016
Collection is registered on the books numbering：CGMCC No.12376
Brief description of the drawings
Fig. 1 is the compound shown in formula I1H-NMR is composed.
Fig. 2 is the compound shown in formula I13C-NMR is composed.
Fig. 3 is the integration of the compound suppression HIV-1 shown in formula I.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, it is
Conventional method.Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Potato dextrose agar (PDA) culture medium used in following embodiments：Peeling potatoes, 200g are cut into small
Block, add boiling to boil 30min, 4 layers of filtered through gauze, add 20g glucose, agar 17g, distilled water constant volume to 1000mL, boil mixing,
Sterilized 20 minutes under the conditions of 121 DEG C, obtain PDA culture medium.
PNL4-3Luc (R-E-) and pHIT/G (Zhang et al.Retrovirology (2016) in following embodiments
13:13) public can obtain the biomaterial from NIH or applicant, the biomaterial only attach most importance to duplicate invention related experiment institute
With can not be used as other purposes.
The separation and identification of embodiment 1, aspergillus (Aspergillus sp.) CPCC400735
1.1 strain isolation
Bacterial strain CPCC400735 is isolated from kadsura longepedunculata (Kadsura longipedunculata) stem.Collection south five
Taste plant loads in valve bag, takes back laboratory treatment.1g kadsura longepedunculata stems are weighed, 30s, sterilized water are rinsed with 75% alcohol
Cleaning 3 times, is shredded stem with sterile scissors, is put into sterilized mortar and is added quartz sand to be ground.Lapping liquid is added
In centrifuge tube equipped with 9mL sterilized waters, sample concentration 10-1, after shaking up, 10 are made into successively-2、10-3、10-4、10-5Gradient is dilute
Release liquid.The different μ L of gradient dilution liquid 200 are drawn respectively, are respectively coated on PDA plate, are inverted in after drying in 25 DEG C of incubators
Culture 3-5 days, the fungi being separated to is transferred on PDA inclined-planes and cultivated, and after length is good, is placed in refrigerator and is preserved.The numbering is taken to be
CPCC400735 bacterial strain carries out following identifications.
1.2 bacterial strains are identified
1.2.1 strain morphology is observed
Bacterial strain CPCC400735 is chosen to PDA culture medium flat board center with transfer needle, cultivated in 28 DEG C of incubators.Bacterial strain
CPCC400735 well-growns in PDA culture medium, during culture 5-7 days bacterium colony in light yellow, quality velvet shape to cotton-shaped, compared with
Thickness, have or do not have radial rill；Bacterium colony reverse side yellowish-brown, conidium is more or few, is just fawn, it is brown to be bordering on light powder
Color, deepened after old, be bordering on pink cinnamon, conidial head is just Radiation, afterwards in loose cylindricality, the top capsule of conidial head
Spherical, the fusion overlapping layer that top capsule is obstructed by come into being stigma fused layer and bottle is covered, and bottle stalk produces conidia chain, conidium
Spherical or subsphaeroidal, wall is smooth.In view of above-mentioned morphological feature, Preliminary Identification bacterial strain CPCC400735 is aspergillus fungi.
1.2.2 molecular biology identification
Bacterial strain CPCC400735 genomic DNA, PCR amplification 18S rRNA genes are extracted, and is sequenced.By gained sequence
(sequence 1 in sequence table) is compared with sequence in GenBank databases, the results showed that bacterial strain CPCC400735 and aspergillus
The similitude of (Aspergillus sp.) is 99%, therefore, with reference to morphological feature before, determines that bacterial strain CPCC400735 is
Aspergillus fungi.
Aspergillus (Aspergillus sp.) CPCC400735 has been preserved in Chinese microorganism strain guarantor on May 5th, 2016
Administration committee's common micro-organisms center is hidden, preserving number is CGMCC No.12376.Hereinafter referred aspergillus (Aspergillus
sp.)CPCC400735。
Embodiment 2, utilize aspergillus (Aspergillus sp.) compound shown in CPCC400735 formulas I
The present embodiment is prepared for compound shown in formula I using aspergillus (Aspergillus sp.) CPCC400735
Its specific preparation method is as follows：
1st, aspergillus (Aspergillus sp.) CPCC400735 culture is prepared
Aspergillus (Aspergillus sp.) CPCC400735 connects in 28 DEG C of PDA inclined-planes culture 5 days, then picking mycelia block
Kind in equipped with 100ml seed culture mediums (consisting of：Glucose 2%, sucrose 1%, analysis for soybean powder 0.2%, peptone 1%, phosphoric acid
Hydrogen dipotassium 0.03%, polyethylene glycol 0.25%, sodium nitrate 0.3%, ammonium sulfate 0.3%, surplus are water, pH 6.0.121 DEG C of sterilizings
20 minutes, obtain seed culture medium) 500ml triangular flasks in, 28-30 DEG C of concussion and cultivate is used as seed liquor in 2 days.Then, will plant
Sub- liquid 10ml access equipped with rice medium 500ml triangular flasks (500ml triangular flasks be equipped with 80g rice, addition 100ml go from
Sub- water immersion, 121 DEG C of sterilizings obtain rice medium in 20 minutes) in, 28 DEG C are cultivated 40 days, obtain aspergillus (Aspergillus
Sp.) CPCC400735 solid fermentation cultures.
2nd, the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture mediums is prepared
Take aspergillus (Aspergillus sp.) the CPCC400735 solid fermentation cultures glass bar of 2000g steps 1 will
It is blended, and adds 4L methanol, is extracted 3 times, each 30min at 28 DEG C, merges methanol extract liquid, with Rotary Evaporators (temperature：40
DEG C, revolution：70 turns) rotary evaporation remove methanol extract liquid in methanol, obtained material is aspergillus (Aspergillus sp.)
The methanolic extract of CPCC400735 fermentation culture mediums.
3rd, the compound shown in formula I
3.1st, the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture mediums of step 2 is used
Moisture dissipates, and is then extracted with ethyl acetate, and ethyl acetate phase is collected, with Rotary Evaporators (temperature：40 DEG C, revolution：70 turns) rotation
Turn the ethyl acetate in evaporation removing ethyl acetate phase, obtain medicinal extract.50g medicinal extract is taken to carry out silica gel column chromatography after being dissolved with methanol
Separation.Silica gel used is silica gel H in silica gel column chromatography, and the specification of silicagel column used is 6.5 × 20cm, and column volume is
663mL.Elution program used is first to elute 2 column volumes with chloroform in silica gel column chromatography；Carry out again 13 column volumes as
Lower linear gradient elution：The mixed liquor that mobile phase used is made up of chloroform and methanol, chlorine in linear gradient elution mobile phase
Imitative and methanol volume ratio is by 20:1 is linearly down to 7:1；Last again with methanol elutes two column volumes.Since elution program not
Interruption collects the liquid eluted, and often pipe collects 200ml (200ml/ fractions), continuously collects 56 fractions, is designated as：Fr.1、
Fr.2, Fr.3, Fr.4 ..., Fr.56, TLC detection instruct to merge identical fraction, finally obtain 12 merging components：Fr.1-
(by Fr.18 to Fr.21, this 4 flow points merge to obtain, and are by 3, Fr.4-12, Fr.13-14, Fr.15, Fr.16-17, Fr.18-21
The liquid that 5.1st to the 6.3rd column volume elutes), Fr.22-25, Fr.26-31, Fr.32-34, Fr.35-42, Fr.43-
54, Fr.55-56.
3.2 Fr.18-21 for obtaining step 3.1 carry out pressurized silica gel column chromatography.Silicon used in pressurized silica gel column chromatography
Glue is silica gel H, and the specification of silicagel column used is 4 × 13cm (a diameter of 4cm, a height of 13cm), column volume 163mL.Pressurization
It with the chloroform and the volume ratio of methanol being made up of chloroform and methanol is 7 that elution program used, which is, in silica gel column chromatography:1 mixing
Liquid is eluted as mobile phase, and the liquid eluted is uninterruptedly collected since elution program, and often pipe collects 20ml
(20ml/ fractions), 12 fractions are continuously collected, are designated as：Tube.1、Tube.2、Tube.3、……、Tube.12.By Tube.9
To this 4 flow point mixing of Tube.12, the component for obtaining entitled Tubes 9-12 (is that the 1st to the 1.5th column volume elutes
Liquid).
Tubes9-12 is subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phases preparative separation).The preparative is high
Effect liquid phase chromatogram separation filler used is octadecylsilane chemically bonded silica filler, and the particle diameter of filler is 5 μm, and the preparative is high
Effect liquid phase chromatogram separation chromatographic column a diameter of 8mm, a height of 250mm used.Mobile phase is the first alcohol and water of first alcohol and water composition
Volume ratio be 1:1 mixing liquid；Flow velocity is 3.0mL/min.Collect tR(retention time) is the institute of 10.4 to 11.3 minutes
There is eluting peak, be named as Tubes 9-12-P2.
Tubes 9-12-P2 are subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phases preparative separation).The preparation
Filler used in type high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the particle diameter of filler is 5 μm, the preparation
Chromatographic column a diameter of 8mm, a height of 250mm used in type high performance liquid chromatography separation.Mobile phase is the methanol of first alcohol and water composition
Volume ratio with water is 9:11 mixing liquid；Flow velocity is 2.5mL/min.Collect tR(retention time) is 20.7min elution
Peak, with Rotary Evaporators (temperature：40 DEG C, revolution：70 turns) rotary evaporation removing first alcohol and water, obtain shown in 15.8mg formula I
Compound.
The physicochemical property and spectral data of compound shown in 3.4 formulas I
Compound shown in formula I is faint yellow solid powder, is soluble in methanol, ethanol equal solvent, is insoluble in water.
1H NMR(600MHz,DMSO-d6)δ:10.32(1H,s,H-7),6.77(1H,s,H-9),6.32(1H,s,H-
10),2.28(3H,s,H-8),2.22(3H,s,H-18)；13C NMR(150MHz,DMSO-d6)δ:121.6(C-1),121.6
(C-2),144.2(C-3),138.3(C-4),135.7(C-5),112.9(C-6),191.2(C-7),11.8(C-8),90.0
(C-9),68.7(C-10),197.0(C-11),104.1(C-12),148.6(C-13),132.5(C-14),152.8(C-15),
115.4(C-16),126.6(C-17),10.2(C-18)。
Compound shown in formula I1H-NMR compose and13C-NMR spectrums difference is as depicted in figs. 1 and 2.
4th, to HIV-1 inhibitory activity
(1) cell culture and transfection：293T cell culture is in the DMEM culture mediums containing 10%FBS.6 orifice plates are inoculated with per hole
Cell number is 4 × 105It is individual, transfect, transfection reagent Lipofectamine 2000, turned according to operation instruction after cultivating 24h
Dye；SupT1 cells (SUP-T1 cells, human T lymphoma cell) are incubated in the RPMI1640 culture mediums containing 10%FBS, are placed in
37 DEG C of 5%CO2Quiescent culture in incubator.
(2) preparation of HIV-1 pseudovirus：It is 1.5 × 10 by cell concentration5Individual/ml amount is by 293T cells before transfection
It is inoculated in 6 orifice plates within one day, 24h is cultivated in 2mL culture mediums and is transfected.Plasmid dosage is：PNL4- is transfected in six orifice plates per hole
3Luc (R-E-) 300ng and pHIT/G plasmid 210ng, transfection reagent Lipofectamine2000, by transfection reagent illustrate into
Row transfection.Supernatant is collected after 48 hours, crosses 0.45 μm of filter membrane, virus liquid is collected and obtains the coated HIV-1 cape horn fevers of VSV-G
Poison.
(3) measure of viral infection：The inoculation 5 × 10 per hole of 96 orifice plates5Individual/ml μ the L of SupT1 cells 200.Take 10 μ l
Gained virus liquid infects SupT1 cells.After cultivating 48h, 50 μ l cell suspensions are taken out, add isometric 2 × Luciferase
Cell Culture Lysis cell lysis.Use the fluorescence of kit Luciferiase Assay System measure lysates
Plain enzyme (Luciferase) activity.The amount of infectious virus is drawn according to the size of reporter gene Luciferase activity.
(4) Anti-HIV-1 Active determines：SupT1 cell concentrations are 5 × 105Individual/ml, 96 orifice plates are directly spread, per the μ L of hole 200,
Adding the HIV-1 pseudovirus of above-mentioned preparation simultaneously, (virus is 1 with the volume ratio of cell:20).Adding sample, (sample is dissolved in
In DMSO), each sample sets 3 repetitions, while sets blank control and negative control (DMSO), 37 DEG C of incubation 48h.Take 50 μ L
Cell suspension, the μ L of 2 × Luciferase Cell Culture Lysis lysates 50 are added, after 37 DEG C are incubated half an hour, are taken
Wherein 6 μ L cell pyrolysis liquids add 40 μ L luciferase substrates, and luciferase is determined using the ELIASAs of Centro XS3LB 960
It is worth (RLUs), calculates inhibiting rate of the sample to HIV.HIV inhibiting rate calculation formula are as follows：
Wherein, sample is the compound shown in formula I prepared by step 3.Inhibiting rate by sample to HIV-1, is calculated
Go out the IC of the compound shown in the formula I of step 3 preparation50(refer to and suppress the drug concentration that HIV-1 replicates reduction 50%).
(5) drug toxicity detection utilizes Cell Counting Kit-8 (CCK-8 kits) detection drug toxicities：SupT1
Cell concentration is 5 × 105Individual/ml, 96 orifice plates are directly spread, per the μ L of hole 100.Sample (sample is dissolved in DMSO) is added, each
Sample sets 3 repetitions, while sets blank control and negative control (DMSO), 37 DEG C of incubation 48h.96 orifice plates are taken out, are added per hole
Enter 10 μ L CCK-8,37 DEG C are continued after being incubated 1 hour, and detecting each hole using the multi-function microplate readers of Enspire 2300 exists
Absorbance value (OD) at 450nm wavelength, calculate the cell survival rate of sample well.Cell survival rate calculation formula：
Wherein, sample is the compound shown in formula I prepared by step 3.By the cell survival rate of sample well, it is calculated
The CC of compound shown in formula I prepared by step 350(referring to the drug concentration for causing 50% cell death).
As a result show the compound shown in formula I be 99.91% to HIV-1 inhibiting rate when concentration is 100 μM ±
0.02%, the IC of the compound shown in formula I50For 6.6 μM, the CC of the compound shown in formula I50More than 100 μM.Shown in formula I
Compound there is stronger inhibitory activity to HIV-1, and toxicity is smaller.
5th, the compound shown in formula I is as HIV-1 integrase inhibitors
Experiment (Time of addition) is added using the time to grind the action target spot of the compound shown in formula I
Study carefully.It is specific as follows：
1) experimental method
A. the preparation of pseudovirus:6 orifice plates are inoculated in before 293T cell transfectings, cell concentration is 1.5 × 105/ ml, in 2ml
Cultivate in culture medium, transfected after 24h.Plasmid pNL4-3Luc (R-E-) and pHIT/G cotransfection 293T cells, per hole dosage difference
For 300ng and 210ng.Transfection reagent is Lipofectamine 2000, is transfected according to operation instruction.Collected after 48h
Clear virus liquid, 0.22 μm of membrane filtration, collect virus liquid and obtain HIV-1 pseudovirus.
B. assay of viral infectivity:The inoculation 5 × 10 per hole of 96 orifice plates5SupT1 (200 μ l) cell.Take virus obtained by 10 μ l
Liquid inductance contaminates SupT1 cells.After cultivating 48h, pipettor blows and beats mixing cell repeatedly, takes out 50 μ l cell suspensions, adds isometric 2
× Luciferase Cell Culture Lysis cell lysis.Surveyed using kit Luciferiase Assay System
Determine luciferase (Luciferase) activity of lysate.Infectivity is drawn according to the size of reporter gene Luciferase activity
The amount of virus.
C. influence of the reactive compound to virus replicative cycle is determined:Respectively choose virus infection after 0,0.5,1,2,4,6,
7th, cell is placed 1h by 21h as administration timing of drug point, and after virus infection at 4 DEG C.Cell is collected by centrifugation, by precipitation PBS
Wash 2 times, to remove the virus do not adsorbed on cell, it is ensured that virus enters life cycle in the same time.
2) experimental result
Pressed down during experiment using efabirenz 3TC, non-nucleoside reverse transcriptase inhibitor EFV and integrase
Preparation RAL is as positive drug.As a result show, after virus infection after 1h and 2h during dosing, 3TC and EFV antiviral activity are opened
Begin to decline；It is basically identical with the action target spot of medicine and RAL dosings before virus infects 7h show good activity
(compound shown in Fig. 3, Epicocconigrone A representative formula I).Compound shown in formula I to HIV-1 inhibitory action with
Integrase inhibitor RAL ten divides the integration process that the action target spot of similar compound shown in formula I is probably viral DNA.
Claims (10)
1. the compound or its pharmaceutically acceptable salt shown in formula I are in hiv inhibitor or hiv integrase inhibitor is prepared
Using,
2. application according to claim 1, it is characterised in that：The HIV is HIV-1.
3. the compound or its pharmaceutically acceptable salt shown in formula I are comprehensive in preparation treatment or/and prevention acquired immunodeficiency
Application in simulator sickness medicine,
4. application according to claim 3, it is characterised in that：The acquired immunodeficiency syndrome is caused by HIV-1.
5. according to any described application in claim 1-4, it is characterised in that：Compound shown in formula I is from aspergillus
Isolated in CPCC400735 culture, aspergillus CPCC400735 is common in China Committee for Culture Collection of Microorganisms
The numbering of registering on the books at microorganism center is CGMCC No.12376.
6. medicinal compound, it is characterised in that：The medicinal compound is compound shown in formula I or its is pharmaceutically acceptable
Salt,
7. medicinal compound according to claim 6, it is characterised in that：The medicinal compound or its is pharmaceutically acceptable
Salt be used to suppress HIV animal, or the medicinal compound or its pharmaceutically acceptable salt are used to treat or/and prevent
Acquired immunodeficiency syndrome or the medicinal compound or its pharmaceutically acceptable salt are integrated into animal for suppressing HIV
Cell.
8. medicinal compound according to claim 7, it is characterised in that：The HIV is HIV-1, the acquired immunity
Deficit syndrome is caused by HIV-1.
9.M1) or method M2)：
M1) suppress HIV animal method, including to receptor apply formula I shown in compound or its can pharmaceutically connect
The salt received is to suppress HIV animal；
M2 the method for) treating or/and preventing acquired immunodeficiency syndrome, including apply the change shown in formula I to receptor
Acquired immunodeficiency syndrome is treated or/and prevented to compound or its pharmaceutically acceptable salt；
10. according to any described medicinal compound in claim 6-8 or the method described in claim 9, it is characterised in that：
Compound shown in formula I is isolated from aspergillus CPCC400735 culture, and aspergillus CPCC400735 is in China Microbiological
The numbering of registering on the books of culture presevation administration committee common micro-organisms center is CGMCC No.12376.
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| ATUL A. MORE: "o‑Quinone methides via oxone oxone-mediated benzofuran oxidative dearomatization and their intramolecular cycloaddition with carbonyl groups: an expeditious construction of the central tetracyclic core of integrastatins, epicoccolide A", 《ORGANIC LETTERS》 * |
| MUSTAPHA EL AMRANI: "Protein Kinase and HDAC Inhibitors from the Endophytic Fungus Epicoccum nigrum", 《JOURNAL OF NATURAL PRODUCTS》 * |
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| CN113480557B (en) * | 2021-09-06 | 2021-11-09 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Polyketone compounds, preparation method thereof and application thereof in preparation of antitumor drugs |
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