CN107513072B - Polyketone is inhibiting the application in HIV - Google Patents
Polyketone is inhibiting the application in HIV Download PDFInfo
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- CN107513072B CN107513072B CN201610425273.XA CN201610425273A CN107513072B CN 107513072 B CN107513072 B CN 107513072B CN 201610425273 A CN201610425273 A CN 201610425273A CN 107513072 B CN107513072 B CN 107513072B
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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 polyketone to inhibit the application in HIV.The application is that I compound represented of formula is preparing the application in hiv inhibitor or hiv integrase inhibitor.I compound represented of formula is 99.91% ± 0.02% to the inhibiting rate of HIV-1 when concentration is 100 μM, the IC of I compound represented of formula50It is 6.6 μM, the CC of I compound represented of formula50Greater than 100 μM.I compound represented of formula has stronger inhibitory activity to HIV-1, and toxicity is smaller, has preferable drug selectivity.
Description
Technical field
The present invention relates to polyketone to inhibit the application in HIV.
Background technique
HIV (Human Immunodeficiency Virus) virus, Chinese name human immunodeficiency virus belong to inverse
One kind of Retroviral.After inhibition of HIV invades host cell, host body immune function can be destroyed, seriously so as to cause serious
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.,
It then begins to propagate rapidly in the world, has become one of the significant threat of human health at present.Inhibition of HIV can be divided into
HIV-1 type and HIV-2 type, the two genome 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 clinic so far
Using.Classify by its mechanism of action, is mainly reverse transcriptase inhibitor and protease inhibitors in these marketed drugs, adds 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 drug is especially treated AIDS patient by Cocktail treatment mode, can be had
Effect reduces virus load, significant to extend 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 application.
Microbe species are huge, the metabolite with enriched types, are find high-efficiency low-toxicity HIV-resistant activity substance one
A important sources.It is quickly screened by establishing screening model and produces the bacterial strain of HIV-resistant activity substance, then from its metabolite
Finding compound with HIV-resistant activity or having certain active compound is guide, carries out that structure is simplified and modification, announcement
The mechanism of action and structure-activity relationship are the effective ways for finding new inverase.
Epicocconigrone A is a kind of polyketone, and structural formula is as shown in formula I
Epicocconigrone A is reported in 2014 for the first time, is produced from the metabolism of plant endogenesis epiphyte Epicoccum nigrum
It is isolated in object, have and inhibit multiple protein kinases and inhibition of histone deacetylation enzymatic activity, while being able to suppress RAJI
(EI Amrani M, Lai D, Debbab A, et al.Protein Kinase and is grown with U-937 human lymphoma
HDAC Inhibitors from the Endophytic Fungus Epicoccum nigrum.Journal of
Natural Products,2014,77:49-56)。
Summary of the invention
The technical problem to be solved by the present invention is to how inhibit HIV.
In order to solve the above technical problems, the present invention provides I compound represented of formula or its pharmaceutically acceptable salts
The application in hiv inhibitor or hiv integrase inhibitor is being prepared,
The present invention also provides I compounds represented of formula or its pharmaceutically acceptable salt in preparation treatment or/and prevention
Application in acquired immunodeficiency syndrome drug.
The present invention also provides medicinal compound, the medicinal compound is I compound represented of formula or it pharmaceutically may be used
The salt of receiving.
In above-mentioned medicinal compound, the medicinal compound or its pharmaceutically acceptable salt are for inhibiting HIV infection dynamic
Object or the medicinal compound or its pharmaceutically acceptable salt are for treating or/and preventing acquired immunodeficiency syndrome
Or the medicinal compound or its pharmaceutically acceptable salt are for inhibiting HIV to be integrated into zooblast.
The present invention also provides M1) or method M2):
M1) inhibit HIV infection animal method, including to receptor apply I compound represented of formula or its pharmaceutically
Acceptable salt is to inhibit HIV infection animal；
M2 the method for) treating or/and preventing acquired immunodeficiency syndrome, including to shown in receptor application formula I
Compound or its pharmaceutically acceptable salt treated or/and prevented acquired immunodeficiency syndrome.
Above, I compound represented of formula 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 number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center
No.12376。
The culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 is by above-mentioned aspergillus (Aspergillus
Sp.) the substance in the culture vessel that CPCC400735 is cultivated in microbiological culture media.
In the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735, the microbiological culture media can be song
Mould culture medium (culture medium that can cultivate aspergillus) 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 the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC 400735, the rice culture medium can
Be made, can also be made of rice, water and inorganic salts of rice and water, can also be made of rice, water and nitrogen source, can also by rice,
Water, nitrogen source and inorganic salts are made.The potato dextrose agar can be made of potato, glucose, agar and water,
It can also be made, can also be made of potato, carbon source, agar, water and nitrogen source of potato, glucose, agar, water and inorganic salts,
It can also be made 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, late imitate 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, which refers to, not to be gone
Except the fruit of the husk of paddy rice, paddy is made of husk, pericarp, kind skin, perisperm, aleurone, endosperm and embryo.Brown rice refers to paddy
Husk is sloughed, the product of the other each sections of paddy is retained；Rice, which refers to, only retains endosperm, and paddy rest part 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.The substance of nitrogen needed for nitrogen source refers to offer microbial nutrition, including peanut cake
Powder, soybean cake powder, yeast powder, peptone, ammonium hydroxide, ammonium salt and nitrate etc. are quick-acting, imitate nitrogen source late.
In the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735, the temperature of the culture can be 20-30
DEG C, incubation time can be 10-40 days.
Can be according to including the steps that A1)-A5) culture of the method by I compound represented of formula from aspergillus CPCC400735
It is isolated in object:
A1 it) is extracted from the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 with methanol molten
In the substance of methanol；
A2) that the substance is water-dispersible, it is then extracted with ethyl acetate, collects ethyl acetate phase, remove ethyl acetate
Ethyl acetate in phase, obtains medicinal extract；The medicinal extract is subjected to silica gel column chromatography separation, elutes journey used in silica gel column chromatography
Sequence is first to elute 2 column volumes with chloroform；Carry out the following linear gradient elution of 13 column volumes again: mobile phase used be by
The mixed liquor of chloroform and methanol composition, the volume ratio of chloroform and methanol is linearly down to 7 by 20:1 in linear gradient elution mobile phase:
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, elution program used in the silica gel column chromatography be with by chloroform and
The mixing liquid that the chloroform of methanol composition and the volume ratio of methanol are 7:1 is eluted as mobile phase, collects the 1st to the 1.5th
The liquid that a column volume elutes is named as Tubes 9-12；
A4 Tubes 9-12) is subjected to preparative high performance liquid chromatography separation, which separates institute
Filler is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, which separates institute
Chromatography column diameter is 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 the mixed of 1:1
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) is subjected to preparative high performance liquid chromatography separation, preparative high performance liquid chromatography separation
Filler used is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, preparative high performance liquid chromatography separation
Chromatography column diameter used is 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 I compound represented of formula.
Above-mentioned aspergillus (Aspergillus sp.) CPCC400735 can with conidium, mycelia or containing conidium and/
Or the mycelial form of mycelia exists.
Above, the HIV can be the HIV of HIV-1 type, and the acquired immunodeficiency syndrome can be drawn by HIV-1
It rises.
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 drug, in addition to containing active constituent, also containing suitable carrier or excipient.Here carrier material includes 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 the first and second cellulose of cellulose acetate phthalate and carboxylic).
Wherein it is preferred that water soluble carrier material.A variety of dosage forms, including but not limited to tablet, glue can be made using these materials
Capsule, dripping pill, aerosol, pill, pulvis, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository,
Freeze drying powder injection etc..It 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, and various carriers well known in the art can be widely used.Example about 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, glycerol, polyethylene glycol, ethyl alcohol, propyl alcohol, starch slurry, dextrin,
Syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethylcellulose, lac, methylcellulose, potassium phosphate,
Polyvinylpyrrolidone etc.；Disintegrating agent, such as dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citron
Acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.；It collapses
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 pill is made in unit dosage forms for administration, various carriers well known in the art can be widely used.About 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, ethyl alcohol, honey, liquid sugar, rice paste
Or batter etc.；Disintegrating agent, such as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose
Element etc..In order to which suppository is made in unit dosage forms for administration, various carriers well known in the art can be widely used.Example about carrier
Son is, such as ester, gelatin, semi-synthetic glyceride of polyethylene glycol, lecithin, cocoa butter, higher alcohol, higher alcohol etc..In order to incite somebody to action
Injection preparation is made in unit dosage forms for administration, and such as solution, emulsion, freeze drying powder injection and suspension, it is normal that this field can be used
All diluents, for example, water, ethyl alcohol, polyethylene glycol, 1,3-PD, ethoxylation isooctadecanol, polyoxygenated different
Stearyl alcohol, Polyoxyethylene Sorbitol Fatty Acid Esters etc..In addition, can add into injection preparation to prepare isotonic injection
Add suitable sodium chloride, glucose or glycerol, further, it is also possible to add conventional cosolvent, buffer, pH adjusting agent etc..This
Outside, if desired, colorant, preservative, fragrance, corrigent, sweetener or other materials can also be added into pharmaceutical preparation.Make
It can be with administrated by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary administration etc. with above-mentioned dosage form；Cavity/canal drug administration,
Such as per rectum and vagina；Respiratory tract administration, such as via intranasal application；Mucosa delivery.Above-mentioned administration route is preferably drug administration by injection.
It is demonstrated experimentally that I compound represented of formula when concentration is 100 μM to the inhibiting rate of HIV-1 be 99.91% ±
0.02%, the IC of I compound represented of formula50It is 6.6 μM, the CC of I compound represented of formula50Greater 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 abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on May 5th, 2016
Collection is registered on the books number: CGMCC No.12376
Detailed description of the invention
Fig. 1 is I compound represented of formula1H-NMR spectrum.
Fig. 2 is I compound represented of formula13C-NMR spectrum.
Fig. 3 is the integration that I compound represented of formula inhibits HIV-1.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Potato dextrose agar (PDA) culture medium as used in the following examples: peeling potatoes, 200g are cut into small
Block adds boiling to boil 30min, and 4 layers of filtered through gauze add 20g glucose, agar 17g, and distilled water constant volume to 1000mL boils mixing,
It sterilizes 20 minutes under the conditions of 121 DEG C, obtains 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 not can be used as other purposes and use.
The separation and identification of embodiment 1, aspergillus (Aspergillus sp.) CPCC400735
1.1 strain isolation
Bacterial strain CPCC400735 is isolated from the stem of kadsura longepedunculata (Kadsura longipedunculata).Acquisition south five
Taste plant is packed into valve bag, takes back laboratory treatment.1g kadsura longepedunculata stem is weighed, rinses 30s, sterile water with 75% alcohol
Cleaning 3 times, is shredded stem with sterile scissors, puts into sterilized mortar and quartz sand is added to be ground.Lapping liquid is added
In centrifuge tube equipped with 9mL sterile water, sample concentration 10-1, after shaking up, successively it is made into 10-2、10-3、10-4、10-5Gradient is dilute
Release liquid.Different 200 μ L of gradient dilution liquid are drawn respectively, are respectively coated on PDA plate, are inverted in 25 DEG C of incubators after drying
Culture 3-5 days, the fungi being separated to is transferred on the inclined-plane PDA and is cultivated, and after length is good, is placed in refrigerator and is saved.The number is taken to be
The bacterial strain of CPCC400735 carries out following identifications.
The identification of 1.2 bacterial strains
1.2.1 strain morphology is observed
Bacterial strain CPCC400735 is chosen with transfer needle to PDA culture medium plate center, is cultivated in 28 DEG C of incubators.Bacterial strain
CPCC400735 well-grown in PDA culture medium, bacterium colony is in light yellow at culture 5-7 days, quality velvet shape to cotton-shaped, compared with
Thickness has or does 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 deepens after old, is bordering on pink cinnamon, and it is in loose cylindricality, the top capsule of conidial head that conidial head is just radiation shape afterwards
Spherical, top capsule is covered by the fusion overlapping layer that stigma fused layer and bottle obstruct of coming into being, and bottle stalk generates 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
The genomic DNA of bacterial strain CPCC400735, PCR amplification 18S rRNA gene are extracted, and is sequenced.By gained sequence
(sequence 1 in sequence table) is compared with sequence in GenBank database, the results showed that bacterial strain CPCC400735 and aspergillus
The similitude of (Aspergillus sp.) is 99%, therefore, in conjunction with 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, deposit number is CGMCC No.12376.Hereinafter referred aspergillus (Aspergillus
sp.)CPCC400735。
Embodiment 2 utilizes compound shown in aspergillus (Aspergillus sp.) CPCC400735 preparation formula I
The present embodiment is prepared for compound shown in formula I using aspergillus (Aspergillus sp.) CPCC400735
Its it is specific the preparation method is as follows:
1, the culture of aspergillus (Aspergillus sp.) CPCC400735 is prepared
Aspergillus (Aspergillus sp.) CPCC400735 is cultivated 5 days for 28 DEG C on the inclined-plane PDA, and then picking mycelia block connects
Kind in equipped with 100ml seed culture medium (consisting of: glucose 2%, sucrose 1%, 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 flask in, 28-30 DEG C shake culture 2 days be used as seed liquor.Then, it will plant
Sub- liquid 10ml access equipped with rice medium 500ml triangular flask (500ml triangular flask is equipped with 80g rice, be added 100ml go from
Sub- water impregnates, and 121 DEG C of sterilizings obtain rice medium in 20 minutes) in, 28 DEG C are cultivated 40 days, and aspergillus (Aspergillus is obtained
Sp.) CPCC400735 solid fermentation culture.
2, the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium is prepared
Take aspergillus (Aspergillus sp.) the CPCC400735 solid fermentation culture glass bar of 2000g step 1 will
It is blended, and 4L methanol is added, and 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 substance is aspergillus (Aspergillus sp.)
The methanolic extract of CPCC400735 fermentation culture medium.
3, I compound represented of preparation formula
3.1, the methanolic extract of the aspergillus of step 2 (Aspergillus sp.) CPCC400735 fermentation culture medium is used
Then water dispersion is extracted with ethyl acetate, collect ethyl acetate phase, with Rotary Evaporators (temperature: 40 DEG C, revolution: 70 turns) rotation
Turn the ethyl acetate in evaporation removing ethyl acetate phase, obtains medicinal extract.Silica gel column chromatography is carried out after taking 50g medicinal extract to be dissolved with methanol
Separation.Silica gel used in silica gel column chromatography is silica gel H, and the specification of silicagel column used is 6.5 × 20cm, and column volume is
663mL.Elution program used in silica gel column chromatography is first to elute 2 column volumes with chloroform；Carry out again 13 column volumes as
Lower linear gradient elution: the mixed liquor that mobile phase used is made of chloroform and methanol, chlorine in linear gradient elution mobile phase
Imitative and methanol volume ratio is linearly down to 7:1 by 20:1；Last again with methanol elutes two column volumes.Since elution program not
Interruption collects the liquid eluted, and every pipe collects 200ml (200ml/ fraction), continuously collects 56 fractions, is denoted as: Fr.1,
Fr.2, Fr.3, Fr.4 ..., Fr.56, TLC detection guidance merges identical fraction, finally obtains 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 (diameter 4cm, a height of 13cm), column volume 163mL.Pressurization
Elution program used in silica gel column chromatography is the mixing for being 7:1 with the volume ratio of the chloroform and methanol that are made of chloroform and methanol
Liquid is eluted as mobile phase, and the liquid eluted is uninterruptedly collected since elution program, and every pipe collects 20ml
(20ml/ fraction), continuously collect 12 fractions, be denoted 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 phase preparative separation).The preparative is high
Effect liquid phase chromatogram separation filler used is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, and the preparative is high
Effect liquid phase chromatogram separation chromatography column diameter used is 8mm, a height of 250mm.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 10.4 to 11.3 minutes institutes
There is eluting peak, is named as Tubes 9-12-P2.
Tubes 9-12-P2 is subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation).The preparation
Filler used in type high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, the preparation
Chromatography column diameter used in type high performance liquid chromatography separation is 8mm, a height of 250mm.Mobile phase is the methanol of first alcohol and water composition
The mixing liquid that volume ratio with water is 9:11；Flow velocity is 2.5mL/min.Collect tR(retention time) is the elution of 20.7min
Peak removes first alcohol and water with Rotary Evaporators (temperature: 40 DEG C, revolution: 70 turns) rotary evaporation, obtains shown in the formula I of 15.8mg
Compound.
The physicochemical property and spectral data of 3.4 formula, I compound represented
I compound represented of formula is faint yellow solid powder, is soluble in methanol, in ethyl alcohol 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)。
I compound represented of formula1H-NMR spectrum and13C-NMR spectrum is as depicted in figs. 1 and 2 respectively.
4, to the inhibitory activity of HIV-1
(1) cell culture and transfection: 293T cell culture is in the DMEM culture medium containing 10%FBS.The every hole inoculation of 6 orifice plates
Cell number is 4 × 105A, culture transfects afterwards for 24 hours, and transfection reagent Lipofectamine 2000 is turned according to operation instruction
Dye；SupT1 cell (SUP-T1 cell, human T lymphoma cell) is incubated in the RPMI1640 culture medium containing 10%FBS, is placed in
37 DEG C of 5%CO2Stationary culture in incubator.
(2) preparation of HIV-1 pseudovirus: being 1.5 × 10 by cell concentration5The amount of a/ml is by 293T cell before transfection
It is inoculated in 6 orifice plates within one day, cultivates in 2mL culture medium and transfected for 24 hours.Plasmid dosage are as follows: every hole transfects pNL4- in six orifice plates
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, is collected virus liquid and is obtained the coated HIV-1 cape horn fever of VSV-G
Poison.
(3) measurement of viral infection: the every hole inoculation 5 × 10 of 96 orifice plates5The 200 μ L of SupT1 cell of a/ml.Take 10 μ l
Gained virus liquid infects SupT1 cell.After cultivating 48h, 50 μ l cell suspensions are taken out, isometric 2 × Luciferase is added
Cell Culture Lysis lytic cell.Use the fluorescence of kit Luciferiase Assay System measurement lysate
Plain enzyme (Luciferase) activity.The amount of infectious virus is obtained according to the active size of reporter gene Luciferase.
(4) Anti-HIV-1 Active measures: SupT1 cell concentration is 5 × 105A/ml, directly 96 orifice plates of paving, every 200 μ L of hole,
The HIV-1 pseudovirus of above-mentioned preparation is added simultaneously (the viral volume ratio with cell is 1:20).Sample is added, and (sample is dissolved in
In DMSO), each sample sets 3 repetitions, while blank control and negative control (DMSO) is arranged, 37 DEG C of incubation 48h.Take 50 μ L
Cell suspension is added 2 × Luciferase Cell Culture Lysis lysate, 50 μ L and takes after 37 DEG C of incubation half an hours
Wherein 40 μ L luciferase substrates are added in 6 μ L cell pyrolysis liquids, measure luciferase using 960 microplate reader of Centro XS3LB
It is worth (RLUs), calculates sample to the inhibiting rate of HIV.HIV inhibiting rate calculation formula is as follows:
Wherein, sample is I compound represented of formula prepared by step 3.By sample to the inhibiting rate of HIV-1, calculate
The IC for I compound represented of formula that out prepared by step 350(referring to the drug concentration for inhibiting HIV-1 duplication to reduce 50%).
(5) drug toxicity detection detects drug toxicity: SupT1 using Cell Counting Kit-8 (CCK-8 kit)
Cell concentration is 5 × 105A/ml, directly 96 orifice plates of paving, every 100 μ L of hole.It is added sample (sample is dissolved in DMSO), each
Sample sets 3 repetitions, while blank control and negative control (DMSO) is arranged, 37 DEG C of incubation 48h.96 orifice plates are taken out, every hole adds
Enter 10 μ L CCK-8,37 DEG C are continued after being incubated for 1 hour, are detected each hole using 2300 multi-function microplate reader of Enspire and are existed
Absorbance value (OD) at 450nm wavelength, calculates the cell survival rate of sample well.Cell survival rate calculation formula:
Wherein, sample is I compound represented of formula prepared by step 3.By the cell survival rate of sample well, it is calculated
The CC of I compound represented of formula prepared by step 350(referring to the drug concentration for causing 50% cell death).
The result shows that I compound represented of formula when concentration is 100 μM to the inhibiting rate of HIV-1 be 99.91% ±
0.02%, the IC of I compound represented of formula50It is 6.6 μM, the CC of I compound represented of formula50Greater than 100 μM.Shown in formula I
Compound there is stronger inhibitory activity to HIV-1, and toxicity is smaller.
5, I compound represented of formula is as HIV-1 integrase inhibitor
Experiment (Time of addition) is added using the time to grind the action target spot of I compound represented of formula
Study carefully.It is specific as follows:
1) experimental method
A. the preparation of pseudovirus: being inoculated in 6 orifice plates before 293T cell transfecting, cell concentration is 1.5 × 105/ ml, in 2ml
It cultivates in culture medium, transfects afterwards for 24 hours.Plasmid pNL4-3Luc (R-E-) and pHIT/G cotransfection 293T cell, every hole dosage difference
For 300ng and 210ng.Transfection reagent is Lipofectamine 2000, is transfected according to operation instruction.It is 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 every hole inoculation 5 × 10 of 96 orifice plates5SupT1 (200 μ l) cell.Take virus obtained by 10 μ l
Liquid inductance contaminates SupT1 cell.After cultivating 48h, pipettor is blown and beaten repeatedly mixes cell, takes out 50 μ l cell suspensions, is added isometric 2
× Luciferase Cell Culture Lysis lytic cell.It is surveyed using kit Luciferiase Assay System
Determine luciferase (Luciferase) activity of lysate.Infectivity is obtained according to the active size of reporter gene Luciferase
The amount of virus.
C. influence of the measurement reactive compound to virus replicative cycle: choose 0 after virus infection respectively, 0.5,1,2,4,6,
7,21h is as administration timing of drug point, and after virus infection, by cell in 4 DEG C of placement 1h.Cell is collected by centrifugation, by precipitating PBS
It washes 2 times, to remove the virus not being adsorbed on cell, it is ensured that virus enters life cycle in the same time.
2) experimental result
Pressed down when experiment using efabirenz 3TC, non-nucleoside reverse transcriptase inhibitor EFV and integrase
Preparation RAL is as positive drug.The results show that the antiviral activity of 3TC and EFV are opened after virus infection after 1h and 2h when dosing
Begin to decline；And RAL dosing until virus infection 7h shows good activity, it is almost the same with the action target spot of drug
(I compound represented of Fig. 3, Epicocconigrone A representative formula).I compound represented of formula to the inhibiting effect of HIV-1 with
Integrase inhibitor RAL ten divides similar, and the action target spot of I compound represented of formula may be the integration process of viral DNA.
Claims (5)
1. I compound represented of formula or its pharmaceutically acceptable salt are in preparing hiv inhibitor or hiv integrase inhibitor
Using,
The HIV is HIV-1.
2. application according to claim 1, it is characterised in that: training of I compound represented of formula from aspergillus CPCC400735
Isolated in feeding object, aspergillus CPCC400735 is stepped on China Committee for Culture Collection of Microorganisms's common micro-organisms center
Charging to volume number is CGMCC No.12376.
3. I compound represented of formula or its pharmaceutically acceptable salt are comprehensive in preparation treatment or/and prevention acquired immunodeficiency
Application in simulator sickness drug,
4. application according to claim 3, it is characterised in that: the acquired immunodeficiency syndrome is caused by HIV-1.
5. application according to claim 3 or 4, it is characterised in that: I compound represented of formula is from aspergillus CPCC400735's
Isolated in culture, aspergillus CPCC400735 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Number of registering on the books is CGMCC No.12376.
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| Protein Kinase and HDAC Inhibitors from the Endophytic Fungus Epicoccum nigrum;Mustapha El Amrani;《Journal of natural products》;20131216;第77卷(第1期);第49-56页 * |
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