CN101434627B - 2'-fluoro-4'-substituted nucleoside analogue, synthetic method and use thereof - Google Patents
2'-fluoro-4'-substituted nucleoside analogue, synthetic method and use thereof Download PDFInfo
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Abstract
The invention discloses a nucleoside analogue and a preparation method and application thereof, more particularly relates to 2'-fluorin-4'-substituted nucleoside analogue or salts thereof or 5'-phosphate ester thereof and a preparation method and application thereof. The nucleoside analogue has a general formula shown below, wherein R is CH3, CN, N3, ethynyl; X is F, OH, NH2; Y is H, CH3, F, OH, NH2. and can be applied to treat virus infections, particularly the infections of HBV, HCV or HIV, with good activity.
Description
This case is the dividing an application of patent of invention " 2 '-fluoro-4 '-replacement-nucleoside analog, its preparation method and application " (2007101375480) of on August 7th, 2007 application.
Technical field
The present invention relates to nucleoside analog, its preparation method and application thereof, relate in particular to 2 '-fluoro-4 '-substituted nucleosides analogue, its preparation method and application.
Background technology
The U.S. found the first routine AIDS patient in 1981, was that isolation identification has gone out hiv virus (HIV) to the nineteen eighty-three scientist, found that HIV belongs to the lentiviridae of Retroviridae, only is made up of 9749 Nucleotide.At present HIV replication cycle process is got clear substantially, discover that its reproductive process roughly is divided into following steps: absorption, intrusion and shelling, reverse transcription, integration, viral RNA and proteinic synthetic, assembling, discharge and ripe, wherein each link all can be used as the target spot of screening HIV medicine, and wherein proteinic synthetic duplicating with virus gene genome nucleic acid is the step of most critical.The focus of screening inverase concentrates on the inhibitor of seeking these specific enzymess at present, comprises that reversed transcriptive enzyme (RT) inhibitor, protein synthesis enzyme inhibitors and reversed transcriptive enzyme start factor inhibitors etc.The reversed transcriptive enzyme of HIV is a kind of multifunctional enzyme, has the archaeal dna polymerase of dependenc RNA, archaeal dna polymerase and the Rhase H activity of dependence DNA simultaneously.HIV is a template with virus genomic RNA at first, is primer with the running RNA of host cell, carries out the synthetic of (-) chain DNA, synthetic in the same way then (+) chain DNA.After reverse transcription finished, the entrained whole genetic information of HIV were converted into double-stranded mDNA from single stranded RNA.Reverse transcriptase inhibitors can stop the prolongation of mDNA, disturbs the reverse transcription process of HIV, thereby becomes the medicine of chemotherapy acquired immune deficiency syndrome (AIDS).Mechanism of action difference according to inhibitor, can be divided into two classes to them: 1) efabirenz, it acts in the HIV DNA reverse transcription process, by being embedded in the viral DNA, impel it to become defective dna and can't duplicate after the DNA of the host cell of HIV is integrated, thereby reach the effect of anti-HIV, existing medicine is zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), Abacavir (Abacavir); 2) non-nucleoside reverse transcriptase inhibitor, its mechanism of action are to stop HIV RNA to be directly connected to RT, do not allow it be encoded into DNA.Existing medicine nevirapine (Nevirapine), Delavirdine and Efavirenz.
The inverase zidovudine of first listing in 1987 is efabirenz, though this drug toxicity is very big, and fails to cure a routine patient, and it can mitigation symptoms, prolongs patient's life.Several nucleoside analog inverase listings are arranged again subsequently, and therefore, nucleoside analog is the important compound that may have HIV (human immunodeficiency virus)-resistant activity of a class.All there is certain defective in these medicines at present, are that drug effect is limited on the one hand, are to take serious toxic side effect for a long time on the other hand, and can produce resistance.Therefore, the synthetic of the similar thing of novel nucleoside still is an important research direction.In order to find more effective ucleosides antiviral, people have carried out various modifications to nucleosides, and the nucleoside analog that wherein contains fluorine is a class wherein.(Clark，J.PCT?Patent?Appl.，WO?2005003174；Ismaili，H.M.A.PCTPatent?Appl.，WO?0160315A22001)。Studies show that this compounds all has antiviral activity in various degree, is the novel compound with antiviral activity of a class.But in existing literature, that does not see the present invention so far as yet contains the synthetic of 2 '-fluoro-4 '-replacement-D-and L-nucleoside analog and the relevant report of using it for the anti-HIV of preparation and anti-HBV and HCV virus drugs.
Summary of the invention
Main purpose of the present invention is to provide a kind of 2 '-fluoro-4 '-substituted nucleosides analogue that contains; Another purpose is to provide the synthetic method of this compounds; Another purpose is to provide the application of this compounds aspect medicine.
For realizing the object of the invention, the following realization of technical scheme:
This present invention 2 '-fluoro-4 '-substituted nucleosides analogue has general formula (I) structure:
Wherein
R＝CH
3，CN，N
3，C≡CH；
R′＝H，F；
X＝F，OH，NH
2；
Y＝H，CH
3，F，OH，NH
2。
It can pass through active compound (I) or its pro-drug or its 5 '-phosphoric acid ester and organic acid or inorganic acid reaction and form salt, exists with the form of salt.
It can be following compound once be not limited only to these compounds:
The present invention contains the application of 2 '-fluoro-4 '-replacement-nucleoside analog in the preparation medicine, is applied to prepare in anti-HBV or anti-HCV or the anti HIV-1 virus medicine, and described resisting HBV virus medicine is an anti-hbv drug; Described anti-HCV virus drugs is the anti-third liver medicine; Described anti HIV-1 virus medicine is an anti-AIDS drug.The present invention contains the preparation feedback of 2 '-fluoro-4 '-substituted nucleosides analogue, works as R=N
3The time path as follows:
Compound i i's is synthetic: compound i (D-ribose or L-ribose) is dissolved among the HCl/MeOH, and the airtight stirring of insulation adds the pyridine termination reaction under water-bath, and decompressing and extracting gets buff syrup shape materialization compound ii;
Compound i ii's is synthetic: compound i i is dissolved in the exsiccant pyridine, and cryosel is bathed and is slowly dripped Benzoyl chloride down, and stirring reaction under the room temperature is poured reaction solution in the frozen water into chloroform extraction after reaction finishes.Organic layer washs successively to water layer with sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, and be washed till water layer with frozen water again and be neutral, anhydrous sodium sulfate drying, decompressing and extracting gets buff syrup shape materialization compound iii.
Compound i v's is synthetic: ii is dissolved in the chloroform with the benzoylate compound i, adds red phosphorus, and ice-water bath stirs slow dripping bromine down; stir about 30min, slowly drip frozen water, stirring at room; add trash ice then and stir and melt, again reaction solution is poured in the frozen water into chloroform extraction.The organic layer saturated sodium bicarbonate is washed till water layer and is weakly alkaline, and frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, decompressing and extracting gets the deep yellow syrup, with silicagel column separate white dry syrup compound i v.
Compound v's is synthetic: compound i v is dissolved in the exsiccant pyridine, and ice-water bath slowly drips aceticanhydride down, stirs about 30min, removes ice-water bath, and the about 7h of stirring at room is warmed up to 40 ℃ and keeps about 1h.The adding trash ice is poured reaction solution in the frozen water into chloroform extraction after stirring and melting.Organic layer is washed till water layer successively with sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, and frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, and decompressing and extracting gets buff syrup shape compound v.
Compound vi's is synthetic: compound v is dissolved in the exsiccant methylene dichloride, ice-water bath slowly feeds exsiccant HCl gas down, add the frozen water washing, tell organic layer, be washed till water layer with saturated sodium bicarbonate and be weakly alkaline, frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, and decompressing and extracting gets buff syrup.Recrystallization gets white solid compound vi.
Compound vii's is synthetic: be dissolved in compound vi in the exsiccant methylene dichloride and exsiccant DMF in, stir about-15 ℃ and slowly add SO down
2Cl
2,-15 ℃ are stirred about 30min, rise to room temperature reaction naturally, at 0 ℃ of following minute 3 times adding imidazoles, and the mixed solution stirring at room, reaction finishes the back to reaction solution adding CH
2Cl
2Dilution is with frozen water washing, water layer CH
2Cl
2Extract, organic layer merges the back and uses anhydrous sodium sulfate drying, and the decompressing and extracting solvent gets buff syrup.Get white solid compound vii with the silicagel column separation and purification.
Compound viii's is synthetic: compound vii is dissolved in the ethyl acetate, stirs adding Et down
3N3HF is warming up to 60 ℃ of left and right sides stir about 3h, 70 ℃ of stir about 1.5h.Add the icy salt solution termination reaction, use dichloromethane extraction then, wash with salt solution, water, saturated sodium bicarbonate successively after organic layer merges, anhydrous sodium sulfate drying, the decompressing and extracting solvent gets the deep yellow syrup.Purifying gets buff syrup, and the crude product crystallization gets white crystal compound viii.
Compound i x's is synthetic: compound viii is dissolved in the anhydrous methylene chloride, adds the mixing solutions of HBr-AcOH, the stirring at room reaction.With the mixture evaporate to dryness, dissolve residuum with methylene dichloride, and wash dichloromethane solution after reaction finishes, methylene dichloride is boiled off, obtain syrup with sodium bicarbonate.Simultaneously, with the cytosine(Cyt) and the (NH that protected
4)
2SO
4Back flow reaction under the nitrogen protection in HMDS, reaction finishes, and the pressure reducing and steaming solvent obtains silylated cytosine(Cyt).The syrup that obtains in the above-mentioned reaction is dissolved in the ethylene dichloride, joins in the silylated cytosine(Cyt), mixture is at N
2Protection is back flow reaction down.With the ice termination reaction, dichloromethane extraction, dichloromethane layer are used saturated sodium bicarbonate and salt water washing, anhydrous sodium sulfate drying successively.Dry back is steamed and is desolventized, and obtains white solid, and column chromatography separating purification obtains compound i x.
Compound x's is synthetic: compound i x is dissolved in saturated NH
3-CH
3OH, the stirring at room reaction.With the solvent evaporate to dryness, the gained residuum obtained compound x by the column chromatography purifying after reaction finished.
Compound xi's is synthetic: with compound x, imidazoles, triphenylphosphine are dissolved in the tetrahydrofuran (THF), to wherein slowly adding the tetrahydrofuran solution that is dissolved with iodine.The stirring at room reaction.Steaming desolventized after reaction finished, and added ethyl acetate in residuum, filtered.Steam and remove ethyl acetate, the separation of residuum column chromatography obtains compound xi.
Compound xii's is synthetic: compound xi is dissolved in the tetrahydrofuran (THF), to wherein adding DBU.At 60 ℃ of left and right sides stirring reactions.Solvent evaporated after reaction finishes, the separation of residuum column chromatography obtains compound xii.
Compound xiii's is synthetic: about 0 ℃, the DMF solution that is dissolved with ICl joined be dissolved with NaN
3DMF solution in, freezing point stir about 10 minutes slowly joins above-mentioned solution with the DMF that is dissolved with compound xii, continues reaction.After finishing, reaction in mixing solutions, adds S-WAT, up to the color completely dissolve of iodine.Remove solvent under reduced pressure, the residuum column chromatography separating purification obtains compound xii
Compound xiv's is synthetic: compound xiii is dissolved among the DMF, stirs adding Silver monoacetate down, room temperature reaction reacts the after-filtration that finishes.Under reduced pressure remove solvent, residuum column chromatography purifying obtains compound xiv.
Compound xv's is synthetic: compound xiv is dissolved in the triethylamine methanol solution, and the stirring at room reaction, reaction end back is steamed and is desolventized, and residuum column chromatography purifying obtains compound xv.
The present invention contains the preparation feedback of 2 '-fluoro-4 '-substituted nucleosides analogue, works as R=CH
3, CN, the path is as follows during C ≡ CH:
Synthesizing of compound b: get compound a, add acetone, slowly add the vitriol oil under the agitation condition, stirring reaction under the room temperature is after the TLC detection reaction finishes, regulate the pH value with strong aqua, filter, the most of acetone of pressure reducing and steaming adds 0.4% dilute hydrochloric acid under agitation condition, react under the acetone reflux conditions, compound a all is hydrolyzed into compound b.Use solid NaHCO
3Neutralization reaction liquid filters, and filtrate under reduced pressure boils off solvent, and residuum dissolves with methylene dichloride, uses anhydrous Na
2SO
4Dried overnight is filtered, boil off solvent after, obtain yellow thickness oily matter b.
Synthesizing of compound c: compound b and triethylamine are dissolved in methylene dichloride, and cryosel is bathed down and is slowly dripped parachlorobenzoyl chloride, keeps 0 ℃ of following mechanical stirring reaction.After finishing, reaction adds saturated NaHCO
3The aqueous solution, dichloromethane layer water, saturated common salt water washing, anhydrous MgSO
4Drying is filtered, and pressure reducing and steaming solvent, and recrystallization residuum get the white crystal compound c.
Synthesizing of compound d: compound c is dissolved in the anhydrous methylene chloride, under nitrogen protection, adds imidazoles and t butyldimethylsilyl chloride (TBDMSC1).Room temperature reaction after reaction finishes, use the hydrochloric acid neutralization reaction, tells two-layerly, and organic layer is used anhydrous Na after distinguishing water and saturated common salt water washing
2SO
4Drying, steaming desolventizes, and residuum carries out separation and purification by column chromatography, obtains compound d.
Synthesizing of Verbindung: compound d joins in sodium methylate-methanol mixture liquid, room temperature reaction.React and finish the back with the dilute acetic acid neutralization, filter, use methanol wash, remove solvent under reduced pressure, the residuum column chromatography separating purification gets Verbindung.
Compound f's is synthetic: dropwise dripping DMSO under-60 ℃ of conditions in the dichloromethane solution of oxalyl chloride approximately, under identical temperature, stir about dripped the dichloromethane solution of Verbindung after 15 minutes.Making an appointment with-65 ℃ of stirring reactions about 30 minutes, and adding triethylamine, the stirring at room reaction.Reaction finishes the back and add entry in reaction solution, tells organic layer, the organic layer anhydrous magnesium sulfate drying.Steaming desolventizes, and residuum gets compound f with column chromatography separating purification.
Compound g's is synthetic: put into sodium hydroxide and water in reaction flask, after stirring, add formalin, 95% ethanol, and then add compound f.About 35 ℃ of following stirring reactions.Reaction is cooled off reaction flask with frozen water after finishing while stirring, separates out fully until product.Suction filtration, solid are washed with water to neutral and oven dry, and the gained solid is dissolved in the anhydrous methanol, adds sodium borohydride again, back flow reaction, and reaction finishes the back with dilute hydrochloric acid neutralization reaction liquid, uses the dichloromethane extraction reaction solution, anhydrous Na
2SO
4Dry back is steamed and is desolventized, and gets compound g.
Compound h's is synthetic: compound g is dissolved in the methyl alcohol, adds Dowex H
+(using methanol wash in advance), room temperature reaction, reaction is removed resin filter after finishing, and with methyl alcohol repetitive scrubbing resin, the solution evaporate to dryness gets solid.The gained solid is dissolved in the acetone, slowly adds the vitriol oil (catalytic amount) under the agitation condition, the stirring at room reaction is regulated the pH value with strong aqua, filters, and pressure reducing and steaming acetone obtains compound h.
Synthesizing of compound i: under about-60 ℃, in the dichloromethane solution of oxalyl chloride, dropwise drip DMSO, drip off back stir about 15 minutes, drip the dichloromethane solution of compound h, drip off about 30 minutes of about-60 ℃ of reactions in back, add triethylamine, room temperature reaction adds the entry termination reaction after about 30 minutes, tells organic layer, the organic layer anhydrous Na
2SO
4Dry.Steaming desolventizes, and residuum gets solid with column chromatography separating purification.With ClCH
2P (C
6H
5)
3Cl joins in the anhydrous tetrahydro furan, is cooled to-78 ℃ approximately, adds the about 1h of hexane solution stirring reaction of n-BuLi, adds gained solid dry tetrahydrofuran solution then.Temperature is elevated to about 0 ℃ gradually, continues reaction.The saturated NH of careful dropping after reaction finishes
4Cl solution is used ethyl acetate extraction, and organic layer salt solution washed twice is used anhydrous MgSO at last
4Drying removes solvent under reduced pressure, and the residuum that obtains is dissolved in the anhydrous tetrahydro furan, is cooled to slowly drip the hexane solution of n-BuLi about-78 ℃.About stirring reaction 2h, use saturated NH carefully
4Cl solution termination reaction.Organic layer saturated common salt water washing, anhydrous MgSO
4Drying removes solvent under reduced pressure, and residuum gets compound i with quick silicagel column separation and purification.
Compound j's is synthetic: compound i is joined in the dilute hydrochloric acid solution, and the stirring at room reaction is used solid NaHCO after reaction finishes
3Neutralization is filtered, and filtrate boils off solvent under reduced pressure, and residuum dissolves with methylene dichloride, and uses anhydrous Na
2SO
4Dry after-filtration boils off solvent, gets compound j.
Compound k's is synthetic: compound j is dissolved in the pyridine, drips BzCl, and room temperature reaction, solvent evaporated after reaction finishes gets compound k behind the residuum purifying.
Compound l's is synthetic: with compound k and 2,2,6,6-tetramethyl piperidine-N-oxygen is dissolved in the methylene dichloride, is chilled to about 0 ℃, with blended chlorine bleach liquor, NaHCO
3Join reaction solution with water mixed solution, reaction is about 30 minutes about 0 ℃, adds Virahol, stirring at room.The dichloromethane layer of telling washes with water 2 times, anhydrous MgSO
4Drying is filtered, and boils off the residuum that solvent obtains and carries out recrystallization, obtains compound 1.
Compound m's is synthetic: compound k is joined in the mixed solution of dehydrated alcohol and ethyl acetate, be chilled to about 0 ℃, add sodium borohydride in batches, stirring reaction, reaction finish the back with the dilute acetic acid neutralization, filter, the filtrate evaporate to dryness, residuum with the methylene dichloride dissolving, is washed the anhydrous MgSO of dichloromethane layer more then
4Drying is filtered, the pressure reducing and steaming methylene dichloride, and residuum obtains solids with the mixed solvent recrystallization of methylene dichloride and sherwood oil.The gained solids is dissolved in the pyridine, about 0 ℃, drips BzCl, after dropwising, room temperature reaction, steaming desolventized residuum ethyl acetate extraction, saturated NaHCO after reaction finished
3Solution washing, anhydrous Na
2SO
4Drying is filtered, and boils off solvent, and the residuum column chromatography separating purification gets compound m.
Compound n's is synthetic: compound m is dissolved in the methyl alcohol, adds the mixing solutions of concentrated hydrochloric acid/dioxane, room temperature reaction is used NaHCO after reaction finishes
3Neutralization reaction liquid filters, and the filtrate decompression distillation removes desolvates, and residuum is dissolved in an amount of methylene dichloride, uses anhydrous Na
2SO
4Dried overnight, the filtrate evaporate to dryness gets compound n with column chromatography separating purification.
Compound O's is synthetic: compound n is dissolved in the methylene dichloride, and room temperature condition adds the DAST stirring reaction down, pours mixture into saturated NaHCO after reaction finishes
3In the solution, tell organic layer, and use saturated NaHCO
3Solution washing, anhydrous Na
2SO
4Drying, column chromatography separating purification obtain compound o.
Compound p's is synthetic: with compound.Join in the formic acid solution, at room temperature stirring reaction reacts the back pressure reducing and steaming solvent that finishes, residuum and toluene steam altogether solids, the gained solids is dissolved in dry pyridine and acetic anhydride solution, room temperature reaction, after reaction finishes reaction solution is concentrated, steam altogether with toluene then, obtain compound p.
Compound q's is synthetic: compound p is dissolved in the anhydrous methylene chloride, and it is chilled to about 0 ℃, and slowly logical then HCl gas is to saturated, and vacuum boils off solvent under the room temperature, and residuum vacuum-drying obtains compound q.
Compound r's is synthetic: 2,6-diaminopurine and hexa methyl silazane (HMDS) are at (NH
4)
2SO
4Reflux is to obtaining clear solution under existing; boil off solvent under the vacuum and obtain white solid; this solid is dissolved in 1, in the 2-ethylene dichloride, adds 1 of compound q again; 2-dichloroethane solution and molecular sieve; after the stirring at room reaction under nitrogen protection, TLC detection reaction fully, in reaction solution, add methylene dichloride; and use diatomite filtration, filtrate is used saturated NaHCO respectively
3With salt solution washing, organic layer anhydrous Na
2SO
4Dried overnight boils off residuum that solvent obtains and separates with the decompression silicagel column and obtain compound r.
Compound s's is synthetic: r is dissolved in NH with compound
3-CH
3In the saturated solution of OH, stirring at room reaction, the reaction back that finishes is steamed and is desolventized, and residuum obtains compound s by column chromatography separating purification.
Beneficial effect of the present invention is, by the modification to glycosyl and base, makes special constructions such as this D-and L-nucleoside analog fluoro-containing group, alkynyl, nitrine, cyano group remedy present D-and big, the active less deficiency of L-nucleoside analog toxic side effect; And this simple synthetic method is feasible, and yield is higher, is applied to prepare anti-HBV or anti-HCV or anti HIV-1 virus medicine, has using value preferably.
Embodiment
The synthetic route of general formula compound I also further specifies invention in conjunction with the embodiments according to the present invention, but is not to limit the scope of the invention.
Compound 15 was synthetic when embodiment 1 was cytosine(Cyt) when base
Compound i i's is synthetic: compound i (D-ribose or L-ribose) (8.66mmol) is dissolved among the 31.2mlHCl/MeOH (0.2mmol/L), the airtight stirring 3h of insulation under 27 ℃ of water-baths, add 7.8ml pyridine termination reaction, decompressing and extracting gets buff syrup shape materialization compound ii (96%);
Compound i ii's is synthetic: compound i i (7.92mmol) is dissolved in the 21ml exsiccant pyridine, and cryosel is bathed slow Dropwise 5 .2ml Benzoyl chloride (0.0447mol) down, reaction solution is poured in the 39ml frozen water into chloroform extraction behind the stirring 17h under the room temperature.Organic layer washs successively to water layer with 3mol/L sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, is washed till water layer with frozen water again and is neutral, and more than the anhydrous sodium sulfate drying 4h, decompressing and extracting gets buff syrup shape materialization compound iii (80.0%).
1H?NMR(CDCl
3)δ?ppm：7.28～8.10(m，15H，OBz)，5.87(m，1H，H-3)，5.68(d，1H，H-2)，5.16(s，1H，H-1)，4.72(m，2H，H-5)，4.52(q，1H，H-4)，3.42(s，3H，OCH
3)。
Compound i v's is synthetic: benzoylate compound i ii (0.0027mmol) is dissolved in the 13mol chloroform; add red phosphorus (390mg; 0.0126mol) the following slow dripping bromine (1.3ml of ice-water bath stirring; 0.025mmol), behind the stirring 30min, slowly drip frozen water 1.3ml; stirring at room 4h; add the 7g trash ice then and stir and melt, again reaction solution is poured in the 14ml frozen water into chloroform extraction.The organic layer saturated sodium bicarbonate is washed till water layer and is weakly alkaline, frozen water is washed till water layer and is neutral, more than the anhydrous sodium sulfate drying 4h, decompressing and extracting gets the deep yellow syrup, separates (sherwood oil: the acetone gradient elution) get white dry syrup compound i v (64.2%) with silicagel column.
1H?NMR(CDCl
3)δ?ppm：7.32～8.10(m，15H，OBz)，5.90(m，1H，H-3)，5.70(d，1H，H-2)，5.63(s，1H，H-1)，4.55～4.79(m，3H，H-5，H-4)。
Compound v's is synthetic: compound i v (2.73mmol) is dissolved in the 5ml exsiccant pyridine, and ice-water bath slowly drips aceticanhydride (0.0276mol) down, behind the stirring 30min, removes ice-water bath, and stirring at room 7h is warmed up to 40 ℃ and keeps 1h.Adding 6.5g trash ice is poured reaction solution in the 13ml frozen water into chloroform extraction after stirring and melting.Organic layer is washed till water layer successively with 3mol/L sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, and frozen water is washed till water layer and is neutral, and more than the anhydrous sodium sulfate drying 4h, decompressing and extracting gets buff syrup shape compound v (92.1%).
1H?NMR(CDCl
3)δ?ppm：7.30～8.10(m，15H，OBz)，6.43(s，1H，H-1)，5.90(m，1H，H-3)，5.80(d，1H，H-2)，4.50～4.80(m，2H，H-5)，2.00(s，3H，?CH
3COO-)。
Compound vi's is synthetic: compound v (2.57mmol) is dissolved in the 26ml exsiccant methylene dichloride, ice-water bath slowly feeds exsiccant HCl gas 2.5h down, add frozen water 19.5ml washing, tell organic layer, be washed till water layer with saturated sodium bicarbonate and be weakly alkaline, frozen water is washed till water layer and is neutral, and more than the anhydrous sodium sulfate drying 4h, decompressing and extracting gets buff syrup.Get white solid compound vi (65.1%) with normal hexane, methylene dichloride mixed solvent recrystallization.
1H?NMR(CDCl
3)δ?ppm：7.36～8.14(m，15H，OBz)，6.68(d，J＝4.4Hz，1H，H-1)，5.59(dd，1H，H-3)，4.64～4.80(m，4H，H-2，H-4?and?H-5)。M.p.139～140℃。
Compound vii's is synthetic: be dissolved in compound vi (2.81mmol) in the 13ml exsiccant methylene dichloride and 3.5ml exsiccant DMF in ,-15 ℃ of stirrings slowly add SO down
2Cl
2(0.0079mmol), behind-15 ℃ of stirring 30min, rise to room temperature naturally, reaction 3h, at 0 ℃ of following minute 3 times adding imidazoles (0.0407mmol), mixed solution stirring at room 15h, reaction solution adds CH
2Cl
2(26ml) dilution is with frozen water (35ml) washing, water layer CH
2Cl
2Extract, organic layer merges the back with more than the anhydrous sodium sulfate drying 4h, and the decompressing and extracting solvent gets buff syrup.With silicagel column separation and purification (sherwood oil: the acetone gradient elution) get white solid compound vii (76.0%).
1H?NMR(CDCl
3)δ?ppm：7.00～8.10(m，15H，OBz)，6.71(d，J＝4.4Hz，1H，H-1)，5.59(dd，1H，H-3)，5.25(dd，1H，H-2)，4.56～4.81(m，3H，H-4，H-5)。M.p.128～129℃。
Compound viii's is synthetic: compound vii (2.2mmol) is dissolved in the ethyl acetate (54ml), and (2.08ml 0.013mmol), is warming up to 60 ℃ and stirs 3h, and 70 ℃ are stirred 1.5h to add Et3N3HF under stirring.Add icy salt solution (10ml) termination reaction, use dichloromethane extraction then, successively with salt solution, water, saturated sodium bicarbonate washing, more than the anhydrous sodium sulfate drying 4h, the decompressing and extracting solvent got the deep yellow syrup after organic layer merged.(5cm * 5cm) purifying (methylene dichloride wash-out) gets buff syrup (86.8%), and crude product crystallization in 95% ethanolic soln gets white crystal compound viii (66.4%) with the silica gel funnel.
1H?NMR(CDCl
3)δppm：7.31～8.10(m，15H，OBz)，6.71(d，J＝9.0Hz，1H，H-1)，5.68(dd，J＝19.44Hz，1H，H-3)，5.32(d，J＝48.2Hz，1H，H-2)，4.65～4.77(m，3H，H-4，H-5)。M.p.80～82℃。
Compound i x's is synthetic: compound viii (6.0mmol) is dissolved in the anhydrous methylene chloride (20ml), adding HBr-AcOH (45%, V/V, 4.6ml, mixing solutions 25mmol), stirring at room reaction 20h.With the mixture evaporate to dryness, dissolve residuum with methylene dichloride (50ml), and (3 * 30ml) washing dichloromethane solutions boil off methylene dichloride, obtain syrup with sodium bicarbonate.Simultaneously, with the cytosine(Cyt) (15mmol) and (NH that protected
4)
2SO
4(0.1g) back flow reaction 17h under the nitrogen protection in HMDS (30ml), the pressure reducing and steaming solvent obtains silylated cytosine(Cyt).The syrup that obtains in the above-mentioned reaction is dissolved in the ethylene dichloride (25ml), joins in the silylated cytosine(Cyt), mixture is back flow reaction 15h under the N2 protection.With the ice termination reaction, (3 * 45ml) extractions, dichloromethane layer is used saturated sodium bicarbonate and salt water washing, anhydrous sodium sulfate drying successively with methylene dichloride.Dry back is steamed and is desolventized, and obtains white solid, and column chromatography is separated (1%MeOH-CHCl
3) purifying, obtain compound i x (71%).
1H?NMR(CDCl
3)δppm：8.07(d，J＝9.86Hz，1H)，7.45(d，J＝9.82Hz，1H)，7.26～8.10(m，10H，OBz)，6.03(d，J＝9.0Hz，1H，H-1)，5.64(dd，J＝19.44Hz，1H，H-3)，5.26(d，J＝48.2Hz，1H，H-2)，4.65～4.77(m，3H，H-4，H-5)。
Compound x's is synthetic: compound i x (3.60mmol) is dissolved in saturated NH
3-CH
3OH (30ml) stirring at room reaction 15h.With the solvent evaporate to dryness, the gained residuum is by column chromatography (15:1CHCl
3-MeOH) purifying obtains compound x (80%).
1H?NMR(CDCl
3)δ?ppm：8.10(d，J＝9.86Hz，1H)，7.40(d，J＝9.82Hz，1H)，6.03(d，J＝9.0Hz，1H，H-1)，5.64(dd，J＝19.44Hz，1H，H-3)，5.26(d，J＝48.2Hz，1H，H-2)，4.50(m，1H，H-4)，3.70～3.77(m，2H，H-5)。
Compound xi's is synthetic: with compound x (9.46mmol), imidazoles (18.93mmol), triphenylphosphine (14.19mmol) are dissolved in the tetrahydrofuran (THF) (50ml), to wherein slowly adding the tetrahydrofuran solution 15ml that is dissolved with iodine (14.18mmol).Stirring at room reaction 3h.Steaming desolventizes, and adds ethyl acetate (100ml) in residuum, filters.Steam and remove ethyl acetate, the separation of residuum column chromatography obtains compound xi (83.9%).
1HNMR(CDCl
3)δ?ppm：8.06(d，J＝9.86Hz，1H)，7.43(d，J＝9.82Hz，1H)，6.01(d，J＝9.0Hz，1H，H-1)，5.66(dd，J＝19.44Hz，1H，H-3)，5.22(d，J＝48.2Hz，1H，H-2)，4.57(m，1H，H-4)，3.58～3.69(m，2H，H-5)。
Compound xii's is synthetic: compound xi (5.88mmol) is dissolved in the tetrahydrofuran (THF) (50ml), to wherein adding DBU (6.44mmol).Stir 3h at 60 ℃.Solvent evaporated, the separation of residuum column chromatography obtains compound xii (75.1%).Be directly used in next step reaction.
Compound xiii's is synthetic: at 0 ℃, the 15ml DMF solution that will be dissolved with ICl (13.9mmol) joins in DMF (15ml) solution that is dissolved with NaN3 (9.75mmol), 0 ℃ was stirred 10 minutes, and the DMF (20ml) that will be dissolved with compound xii (6.8mmol) slowly joins above-mentioned solution.0 ℃ was reacted 1 hour.In mixing solutions, add S-WAT, up to the color completely dissolve of iodine.Remove solvent under reduced pressure, the residuum column chromatography separating purification obtains compound xii (77.6%).Be directly used in next step reaction.
Compound xiv's is synthetic: compound xiii (5mmol) is dissolved among the DMF (15ml), stirs adding Silver monoacetate (6mmol) down, room temperature reaction 8h filters.Under reduced pressure remove solvent (below 50 ℃), residuum column chromatography purifying obtains compound xiv (71.3%).
1H?NMR(DMSO-d
6，300MHz)δ：8.12(d，1H)，7.40(d，1H)，7.26(br，2H)，6.12(dd，1H)，5.87(d，1H)，5.09(t，1H)，4.90(dt，1H)，4.18(dt，1H)，3.74-3.87(m，2H)，2.12(s，3H，CH
3)。
Compound xv's is synthetic: compound xiv (5mmol) is dissolved in 5% the triethylamine methanol solution (100ml), and stirring at room 12h, steaming desolventizes, and residuum column chromatography purifying obtains compound xv (89.0%).ESI-MS:287[M+H]。
1H?NMR(DMSO-d
6，300MHz)δ：8.14(d，1H)，7.32(d，1H)，7.21(br，2H)，6.08(dd，1H)，5.82(d，1H)，5.11(t，1H)，4.92(dt，1H)，4.16(dt，1H)，3.62-3.69(m，2H)。
It is alkynyl that embodiment 2 works as 4 '-bit substituent, and base is 1, and compound 25 is synthetic during the 6-diaminopurine
Synthesizing of compound b: get compound a (60g), add acetone (2L), slowly add the vitriol oil (40ml) under the agitation condition, stirring reaction is 40 minutes under the room temperature, the TLC detection reaction finishes, regulate pH=7～8 with strong aqua, filter, the most of acetone of pressure reducing and steaming, residue acetone and the about 150ml of water, add 0.4% dilute hydrochloric acid (150ml) under agitation condition, reaction is 20 minutes under the acetone reflux conditions, and compound a all is hydrolyzed into compound b.Use solid NaHCO
3Neutralization reaction liquid filters to pH=7～8, and filtrate under reduced pressure boils off solvent, and residuum is used anhydrous Na with methylene dichloride dissolving (200ml)
2SO
4Dried overnight is filtered, boil off solvent after, obtain yellow thickness oily matter b (73g, 95%), ESI-MS:191[M+H].
Compound c synthetic: with compound b (154g, 0.81mol) and triethylamine (339ml 2.43mol) is dissolved in the methylene dichloride (1.50L), cryosel is bathed and to be chilled to 0 ℃, slowly drip parachlorobenzoyl chloride (113ml, 0.891mol), keep 0 ℃ of following mechanical stirring reaction 4h, add saturated NaHCO
3The aqueous solution (500ml), dichloromethane layer water, saturated common salt water washing, anhydrous MgSO
4Drying is filtered, and pressure reducing and steaming solvent, and recrystallization residuum get white crystal compound c (196g, 74.6%), ESI-MS:330[M+H].
Compound d synthetic: compound c (0.114mol) is dissolved in the anhydrous methylene chloride (600ml), under nitrogen protection, add imidazoles (15.5g, 0.228mol) and t butyldimethylsilyl chloride (TBDMSCl) (18.9g, 0.125mol).Behind the room temperature reaction 3h, with 1N hydrochloric acid neutralization reaction, tell two-layerly, organic layer after water and the saturated common salt water washing, is used anhydrous Na respectively
2SO
4Drying, steaming desolventizes, residuum carries out separation and purification by column chromatography, obtains compound d, productive rate 69.2%, ESI-MS:444[M+H].
Synthesizing of Verbindung: compound d (0.64mmol) joins in the mixture liquid of 0.2N sodium methylate-methyl alcohol (15ml), room temperature reaction 4h, neutralize with dilute acetic acid, filter, use methanol wash, remove solvent under reduced pressure, the residuum column chromatography separating purification, get Verbindung, productive rate 75%, ESI-MS:305[M+H].
Compound f's is synthetic: under-60 ℃ to oxalyl chloride (3.4ml, 38.7mmol) methylene dichloride (80.0ml) solution in dropwise drip DMSO (5.5ml, 77.5mmol), under identical temperature, stir after 15 minutes, drip methylene dichloride (100ml) solution of Verbindung (25.7mmol).-65 ℃ of stirring reactions 30 minutes, add triethylamine (10.9ml, 78.2mmol), stirring at room reaction 30 minutes.Add entry in the reaction mixture, tell organic layer, the organic layer anhydrous magnesium sulfate drying.Steaming desolventizes, residuum with column chromatography separate refine compound f, productive rate 94.6%, ESI-MS:303[M+H].
Compound g's is synthetic: put into sodium hydroxide (1.3g) and water (11.5ml) in reaction flask, the back that stirs adds 30% formalin, 7.2ml95% ethanol, adds compound f (25mmol) then.Control reaction temperature stirs 2h down at 30～35 ℃.Reaction is cooled off reaction flask with frozen water after finishing while stirring, separates out fully until product.Suction filtration, solid are washed with water to neutral and oven dry, and the gained solid is dissolved in the anhydrous methanol, add again sodium borohydride (0.925g, 25mmol), back flow reaction 1h, with dilute hydrochloric acid neutralization reaction liquid, dichloromethane extraction (3 * 50ml), add anhydrous Na
2SO
4Drying, steaming desolventizes, and gets compound g, productive rate 90.5%, ESI-MS:335[M+H].
Compound h's is synthetic: compound g (9.7mmol) is dissolved in the methyl alcohol (260ml), adds DowexH
+(40ml uses methanol wash in advance), room temperature reaction 4h removes resin filter, and with methyl alcohol repetitive scrubbing resin, the solution evaporate to dryness gets solid.The gained solid is dissolved in the acetone (200ml), slowly add the vitriol oil (catalytic amount under the agitation condition, 2ml), the about 0.5h of stirring at room reaction regulates pH=7～8 with strong aqua, filters, the most of acetone of pressure reducing and steaming, add 0.4% dilute hydrochloric acid (40ml) again, the acetone reflux conditions reacted 20 minutes down, used NaHCO
3Neutralization reaction liquid filters to pH=7～8, and decompression is steamed down and desolventized, and residuum dissolves with an amount of methylene dichloride, uses anhydrous Na
2SO
4After the drying, boil off solvent, obtain compound h, productive rate 80.2%, ESI-MS:261[M+H].
Synthesizing of compound i :-60 ℃ to oxalyl chloride (3.4ml, 38.7mmol) methylene dichloride (80ml) solution in dropwise drip DMSO (5.5ml, 77.5mmol), drip off and stir after 15 minutes, drip methylene dichloride (100ml) solution of compound h (25.7mmol), drip off-60 ℃ of reactions in back 30 minutes, the adding triethylamine (10.9ml, 78.2mmol), room temperature reaction adds the entry termination reaction after 30 minutes, tell organic layer, the organic layer anhydrous Na
2SO
4Dry.Steaming desolventizes, and residuum gets solid with column chromatography separating purification.With ClCH
2P (C
6H
5)
3Cl (2.15g, 6.19mmol) join in the anhydrous tetrahydro furan (50ml), be cooled to-78 ℃, add the hexane solution (1.6M of n-BuLi, 4.0ml) stirring reaction 1h, add the dry tetrahydrofuran solution (50ml) of gained solid (1.51mmol) then.Cooling temperature is elevated to 0 ℃ gradually, continues reaction 3h.The saturated NH of careful dropping
4Cl solution (10ml), the usefulness ethyl acetate extraction (2 * 100ml), and organic layer salt solution washed twice (2 * 75ml), use anhydrous MgSO at last
4Drying removes solvent under reduced pressure, and the residuum that obtains is dissolved in the anhydrous tetrahydro furan (40ml), is cooled to-78 ℃, slowly drip n-BuLi hexane solution (1.6M, 20ml).Stirring reaction 2h uses saturated NH carefully
4Cl solution (20ml) termination reaction.Organic layer saturated common salt water washing, anhydrous MgSO
4Drying removes solvent under reduced pressure, and residuum gets compound i, productive rate 48.5%, ESI-MS:255[M+H with quick silicagel column separation and purification].
Compound j's is synthetic: compound i (10mmol) is joined in 0.2% the hydrochloric acid soln (50ml), stirring at room reaction 6h uses solid NaHCO
3Neutralization reaction is filtered to pH=7～8, and filtrate boils off solvent under reduced pressure, and residuum dissolves with methylene dichloride, and uses anhydrous Na
2SO
4Dry after-filtration boils off solvent, gets compound j, productive rate 99.0%, ESI-MS:215[M+H].
Compound k's is synthetic: compound j (5mmol) is dissolved in the pyridine (20ml), drips BzCl (5mmol), and room temperature reaction 6h, evaporate to dryness gets compound k, productive rate 85.3%, ESI-MS:319[M+H behind the residuum purifying].
Synthesizing of compound 1: with compound k (59.3mmol) and 2,2,6,6-tetramethyl piperidine-N-oxygen (0.059mmol) is dissolved in the methylene dichloride (99ml), is chilled to 0 ℃, with chlorine bleach liquor's (33.6ml, 8.5～13.5% available chlorine), NaHCO
3(11.2g) and water (190ml) mixing solutions join reaction solution, 0 ℃ of reaction 30 minutes adds Virahol (1.95ml), room temperature reaction 10 minutes, the dichloromethane layer of telling wash with water 2 times, anhydrous MgSO
4Drying is filtered, and boils off the residuum that solvent obtains and carries out recrystallization, obtains compound l, productive rate 87.8%, ESI-MS:317[M+H].
Compound m's is synthetic: compound k (0.05mol) is joined in the mixed solution of dehydrated alcohol (60ml) and ethyl acetate (30ml), be chilled to 0 ℃, add in batches sodium borohydride (23.3g, 612mmol), then at 0 ℃ of stirring reaction 1h, be neutralized to pH=7～8 with dilute acetic acid, filter, the filtrate evaporate to dryness, residuum dissolves with methylene dichloride again, washing then, the anhydrous MgSO of dichloromethane layer
4Drying is filtered, the pressure reducing and steaming methylene dichloride, and residuum obtains solids with the mixed solvent recrystallization of methylene dichloride and sherwood oil.The gained solids is dissolved in the pyridine, under 0 ℃ of condition, drips BzCl (50mmol), after dropwising, room temperature reaction 3h, steaming desolventizes, residuum ethyl acetate extraction, saturated NaHCO
3Solution washing, anhydrous Na
2SO
4Drying is filtered, and boils off solvent, and the residuum column chromatography separating purification gets compound m, productive rate 89.7%, ESI-MS:423[M+H].
Compound n's is synthetic: compound m (4mmol) is dissolved in the methyl alcohol (22ml), adds hydrochloric acid/dioxane mixing solutions (20ml) of 4.0M, room temperature reaction 10h uses NaHCO
3Neutralization reaction liquid filters to pH=7～8, and the filtrate decompression distillation removes desolvates, and residuum is dissolved in an amount of methylene dichloride, uses anhydrous Na
2SO
4Dried overnight, the filtrate evaporate to dryness gets compound n with column chromatography separating purification, productive rate 89.5%, ESI-MS:397[M+H].
Compound.Synthetic: compound n (1.76mmol) is dissolved in the methylene dichloride, and room temperature condition adds DAST down, and (0.4ml 3.03mmol) stirs 12h, pours mixture into 10ml saturated NaHCO
3In the solution, tell organic layer, and use saturated NaHCO
3Solution washing, anhydrous Na
2SO
4Drying, column chromatography separating purification obtain compound o, productive rate 11.5%, ESI-MS:399[M+H].
Compound p's is synthetic: it is in 80% the formic acid solution that compound o (10mmol) is joined 20ml concentration, stirring reaction 12h at room temperature, the pressure reducing and steaming solvent, residuum and toluene steam altogether solids, the gained solids is dissolved in dry pyridine and acetic anhydride solution, room temperature reaction 4h, reaction solution concentrates the back and steams altogether with toluene, obtain compound p, productive rate 90.3%, ESI-MS:427[M+H].
1H?NMR(CDCl
3，300MHz)δ:2.07(s，3H)，2.85(s，1H)，4.62-4.77(m，2H)，4.89-5.07(m，3H)，7.32-8.17(m，10H)。
Compound q's is synthetic: compound p (4.76mmol) is dissolved in the anhydrous methylene chloride (40ml), it is chilled to 0 ℃, slowly logical then HCl gas is to saturated (about 3h), vacuum boils off solvent under the room temperature, residuum vacuum-drying obtained compound q in 2 hours, productive rate 87.1%, compound q is directly used in next step reaction.
Compound r's is synthetic: (HMDS is 9ml) at (NH with hexa methyl silazane for 2,6-diaminopurine (4.78mmol)
4)
2SO
4(50mg) exist following reflux to obtaining clear solution (about 5h); boil off solvent under the vacuum and obtain white solid, this solid is dissolved in 1, in the 2-ethylene dichloride (35ml); add 1 of compound q again; the 2-dichloroethane solution (4.56mmol, 30ml) and 0.4nm molecular sieve (2.6g), stirring at room is 6 days under nitrogen protection; after the TLC detection reaction is complete; add methylene dichloride (80ml) in reaction solution, and use diatomite filtration, filtrate is used saturated NaHCO respectively
3With salt solution washing, organic layer anhydrous Na
2SO
4Dried overnight, boil off residuum that solvent obtains separate with the decompression silicagel column obtain compound r, productive rate 78.5%, ESI-MS:517[M+H].
1H?NMR(CDCl
3，300MHz)δ：2.85(s，1H)，4.67-4.79(m，2H)，4.92-5.13(m，3H)，5.79(br，1H)，7.32-8.17(m，9H)，8.35(s，1H)。
Synthesizing of compound 25: compound r (3.60mmol) is dissolved in the saturated NH3-methanol solution (30ml), stirring at room reaction 15h, steaming desolventizes, and residuum obtains compound 25 by column chromatography separating purification, productive rate 78.9%, ESI-MS:309[M+H].
1H?NMR(CDCl
3，300MHz)δ：2.85(s，1H)，3.69-3.79(m，2H)，4.79-5.07(m，3H)，5.60(br，1H)，5.79(br，1H)，7.32-8.17(m，4H)，8.35(s，1H)。
The external HIV (human immunodeficiency virus)-resistant activity experiment of the present invention:
The anti-HIV-1 activity of compound 1,15,19,36,39,46,50,52 in cell cultures
1. material and method
1.1 material
1.1.1 medicine:
Treat the reagent thing: compound 1,9,15,19,36,39,46,50,52, no lot number, water is insoluble, and the DMSO solubilized is made into behind the proper concn with the substratum dilution with DMSO dissolving during experiment, adds cell cultures immediately.
Positive control drug:
(1) zidovudine (Zidovudine AZT) for known nucleoside HIV-1 reverse transcriptase inhibitors of clinical application, is white powder, available from Shanghai Disainuo Chemical Pharmaceutical Co., Ltd, lot number: 040201b,
(2) (nevirapine is NVP) available from pool, Nanjing many doctorization Information Research Centers (lot number: 0301001), be the known non-nucleoside reverse transcriptase inhibitor of clinical application for nevirapine.
1.1.2 virus
HIV-1IIIB tests virus strain, for U.S. Mount Sinai Medical Center doctor Jiang Jiandong grants, increases in the H9 cell, and-196 ℃ frozen.
1.1.3 cell
The human T lymphocyte MT-4 that goes down to posterity, for medical research center professor Zhang Xingquan of Univ Colorado-Boulder USA gives, go down to posterity in this chamber ,-196 ℃ of preservations.Cell culture medium: RPMI Medium1640 substratum adds 10% foetal calf serum, 100IU/ml penicillin, 100 μ g/ml Streptomycin sulphates and kantlex and contains L-glutaminate.37 ℃, 5%CO
2Cultivate in the incubator, went down to posterity once in per three days.
1.1.4 main agents
RPMI Medium1640 substratum is a U.S. GIBCO company product;
Foetal calf serum (Fetal Bovine Serum) is river, a Tianjin page or leaf biochemical product company limited product;
Penicillin and Streptomycin sulphate are North China Pharmaceutical Factory's product; Kantlex is Shanghai Xudong Hipu Medicine Co., Ltd's product;
Tetrazole indigo plant (Thiazolyl blue, MTT), citric acid is a U.S. Sigma company product;
Triton X-100 is a Sweden KEBO AB STOCKHOLM company product;
N, N dimethyl formamide (N, N-Dimethyl Formamide) is Beijing Chemical Plant's product;
HIV-1P24 antigen detecting agent box: with Dutch BioMerieux company product.
1.1.5 key instrument
Emax
TMMicroplate reader is a U.S. MolICular Devices company product;
Experiment is carried out in 3 grades of biocontainment laboratories.
1.2 experimental technique
96 well culture plates, if medicine and positive control drug suppress viral experimental group, different time was got the different dilution groups of blood after every hole added the different concns DMSO solution 100ul of medicine to be measured or positive control drug AZT and NVP or rat oral pharmaceutical various dose group, every hole 100ul establishes cell contrast and virus control group positive drug AZT and NVP control group simultaneously.MT-4 cell 100TCID
50HIV-1IIIB infected after 1.5 hours, after substratum washing 1 time, was made into 2 * 10
5Cell/ml ' is inoculated in medicine to be suppressed in 96 well culture plates of viral experimental group and positive control medicine group and virus control group.The cell control group adds the equivalent nutrient solution, cultivates after 4 days.Use the microscope observing cell pathology, measure toxicity with MTT dyeing simultaneously, supernatant liquor by specification method is measured P24 antigen.Calculate inhibiting rate, the poisonous concentration (CC of half respectively
50) and medium effective concentration (IC
50) and SI.
2 results
2.1 compound 1,9,15,19,36,39,46,50,52 in the MT-4 cell cultures to the restraining effect of HIV-1
Measure compound 1,9,15,19,36,39,46,50 with mtt assay, the toxicity of 52 pair cells and anti-HIV-1 activity, and comparative drug is measured cytotoxicity to CC with HIV-1 cells infected and non-infected cells
50Influence with SI.The results are shown in Table 1.
Table 1 compound 1,9,15,19,36,39,46,50,52 and positive drug AZT and NVP anti-HIV-1 activity in the MT-4 cell cultures
*: cytotoxicity: mtt assay
Compound 1,15 and 19 usefulness wild-type HIV-1 duplicate pharmacological model and carry out the pharmacological screening result
Table 2 compound 1,15 and 19 usefulness wild-type HIV-1 duplicate pharmacological model and carry out pharmacological screening table as a result
By table 1 and table 2 as can be known, novel 2 '-fluoro-4 '-replacement-nucleosides all has good HIV (human immunodeficiency virus)-resistant activity, and especially compound 1, compound 15 and compound 19 have good application and development prospect.The exploitation of this compounds will bring glad tidings for the AIDS patient.
The external anti-HCV activity experiment of the present invention
1. material and method
1.1 cell cultures
HCV replicating cell (Avva.5) is cultivated in the Eagle medium of the improved G418 that comprises 10% fetal bovine serum and 1mg/mL of Dulbecco ' s.The 293-Sip-L cell is cultivated in the Eagle medium of the hygromycin B of improved G418 that contains 10% fetal bovine serum and 250 μ g/mL of Dulbecco ' s and 150 μ g/mL.12HCV infects measuring method (RT-PCR method)
Cultivated 12 hours in comprising the HCV male substratum of 100 μ L with the cell in the Petri culture dish of 60mm diameter.Cell is cultivated in the fresh medium that does not comprise HCV and is changed every day once then.When cell detected HCV-RNA in infection after 7 days, cell needed trypsinized and cleans twice with the improved Eagle medium of centrifuging Dulbecco ' s.The top section (in contrast) of the washing second time and the cell of washing collect to carry out the RNA extraction in pairs and RT-PCR detects.Beta-actin mRNA measures simultaneously to do contrast.
1.3HCV-RNA detection by quantitative
Adopt full-automatic PCR euzymelinked immunosorbent assay (ELISA) come quantitative assay HCV-RNA (2.0 editions, RocheDiagnostics, Branchburg, NJ).
2. result
Compound 17 has inhibition effect (seeing the following form) when 6,0.6 and 0.06 μ g/mL.The HCV-RNA level is measured with full-automatic PCR euzymelinked immunosorbent assay (ELISA).
In view of this, 17 couples of HCV of compound have stronger restraining effect.
Claims (6)
1. it has the 2 '-fluoro-4 '-replacement-nucleoside analog of general formula (I) structure, or itself and organic acid or the inorganic acid reaction salt or its 5 '-phosphoric acid ester that form, it is characterized in that general formula (I) structure is as follows:
Wherein
R＝CH
3，CN，N
3，C≡CH；
X＝F，OH，NH
2；
Y＝H，CH
3，F，OH，NH
2。
2. 2 '-fluoro-4 ' as claimed in claim 1-substituted nucleosides analogue is characterized in that, described compound is one of following compound:
3. the R=N that works as claimed in claim 1
3The preparation method of nucleoside analog is characterized in that, comprises the following steps:
Compound i i's is synthetic: compound i D-ribose or L-ribose are dissolved among the HCl/MeOH, and the airtight stirring of insulation adds the pyridine termination reaction under water-bath, and decompressing and extracting gets buff syrup shape materialization compound ii;
Compound i ii's is synthetic: compound i i is dissolved in the exsiccant pyridine, and cryosel is bathed and is slowly dripped Benzoyl chloride down, and stirring reaction under the room temperature is poured reaction solution in the frozen water into chloroform extraction after reaction finishes; Organic layer washs successively to water layer with sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, and be washed till water layer with frozen water again and be neutral, anhydrous sodium sulfate drying, decompressing and extracting gets buff syrup shape materialization compound iii;
Compound i v's is synthetic: ii is dissolved in the chloroform with the benzoylate compound i, adds red phosphorus, and ice-water bath stirs slow dripping bromine down, stir 30min, slowly drip frozen water, stirring at room, add trash ice then and stir and melt, again reaction solution is poured in the frozen water into chloroform extraction; The organic layer saturated sodium bicarbonate is washed till water layer and is weakly alkaline, and frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, decompressing and extracting gets the deep yellow syrup, with silicagel column separate white dry syrup compound i v;
Compound v's is synthetic: compound i v is dissolved in the exsiccant pyridine, and ice-water bath slowly drips aceticanhydride down, stirs 30min, removes ice-water bath, and stirring at room 7h is warmed up to 40 ℃ and keeps 1h; The adding trash ice is poured reaction solution in the frozen water into chloroform extraction after stirring and melting; Organic layer is washed till water layer successively with sulfuric acid, the saturated sodium bicarbonate of frozen water, precooling and is weakly alkaline, and frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, and decompressing and extracting gets buff syrup shape compound v;
Compound vi's is synthetic: compound v is dissolved in the exsiccant methylene dichloride, ice-water bath slowly feeds exsiccant HCl gas down, add the frozen water washing, tell organic layer, be washed till water layer with saturated sodium bicarbonate and be weakly alkaline, frozen water is washed till water layer and is neutral, anhydrous sodium sulfate drying, and decompressing and extracting gets buff syrup; Recrystallization gets white solid compound vi;
Compound vii's is synthetic: be dissolved in compound vi in the exsiccant methylene dichloride and exsiccant DMF in ,-15 ℃ of stirrings slowly add SO down
2Cl
2,-15 ℃ are stirred 30min, rise to room temperature reaction naturally, at 0 ℃ of following minute 3 times adding imidazoles, and the mixed solution stirring at room, reaction finishes the back to reaction solution adding CH
2Cl
2Dilution is with frozen water washing, water layer CH
2Cl
2Extract, organic layer merges the back and uses anhydrous sodium sulfate drying, and the decompressing and extracting solvent gets buff syrup, gets white solid compound vii with the silicagel column separation and purification;
Compound viii's is synthetic: compound vii is dissolved in the ethyl acetate, stirs adding Et down
3N3HF is warming up to 60 ℃ and stirs 3h, and 70 ℃ are stirred 1.5h; Add the icy salt solution termination reaction, use dichloromethane extraction then, wash with salt solution, water, saturated sodium bicarbonate successively after organic layer merges, anhydrous sodium sulfate drying, the decompressing and extracting solvent gets the deep yellow syrup; Purifying gets buff syrup, and the crude product crystallization gets white crystal compound viii;
Compound i x's is synthetic: compound viii is dissolved in the anhydrous methylene chloride, adds the mixing solutions of HBr-AcOH, the stirring at room reaction; With the mixture evaporate to dryness, dissolve residuum with methylene dichloride, and wash dichloromethane solution after reaction finishes, methylene dichloride is boiled off, obtain syrup with sodium bicarbonate; Simultaneously, with the adenine and the (NH that protected
4)
2SO
4Back flow reaction under the nitrogen protection in HMDS, reaction finishes, and the pressure reducing and steaming solvent obtains silylated adenine; The syrup that obtains in the above-mentioned reaction is dissolved in the ethylene dichloride, joins in the silylated adenine, mixture is at N
2Protection is back flow reaction down; With the ice termination reaction, dichloromethane extraction, dichloromethane layer are used saturated sodium bicarbonate and salt water washing successively, anhydrous sodium sulfate drying, and dry back is steamed and is desolventized, and obtains white solid, and column chromatography separating purification obtains compound i x;
Compound x's is synthetic: compound i x is dissolved in saturated NH
3-CH
3OH, the stirring at room reaction, with the solvent evaporate to dryness, the gained residuum obtained compound x by the column chromatography purifying after reaction finished;
Compound xi's is synthetic: with compound x, imidazoles, triphenylphosphine are dissolved in the tetrahydrofuran (THF), to wherein slowly adding the tetrahydrofuran solution that is dissolved with iodine; The stirring at room reaction; Steaming desolventized after reaction finished, and added ethyl acetate in residuum, filtered, and steamed and removed ethyl acetate, and the separation of residuum column chromatography obtains compound xi;
Compound xii's is synthetic: compound xi is dissolved in the tetrahydrofuran (THF), to wherein adding DBU; At 60 ℃ of stirring reactions, solvent evaporated after reaction finishes, the separation of residuum column chromatography obtains compound xii;
Compound xiii's is synthetic: at 0 ℃, the DMF solution that is dissolved with ICl joined be dissolved with NaN
3DMF solution in, freezing point stirred 10 minutes, and the DMF that is dissolved with compound xii is slowly joined above-mentioned solution, continued reaction; After finishing, reaction in mixing solutions, adds S-WAT, up to the color completely dissolve of iodine; Remove solvent under reduced pressure, the residuum column chromatography separating purification obtains compound xiii;
Compound xiv's is synthetic: compound xiii is dissolved among the DMF, stirs adding Silver monoacetate down, room temperature reaction reacts the after-filtration that finishes; Under reduced pressure remove solvent, residuum column chromatography purifying obtains compound xiv;
Compound xv's is synthetic; Compound xiv is dissolved in the triethylamine methanol solution, and stirring at room reaction, reaction finish the back steaming and desolventize, and residuum column chromatography purifying obtains compound xv.
4. as claimed in claim 1 when R is C ≡ CH the preparation method of nucleoside analog, it is characterized in that, comprise the following steps:
Synthesizing of compound b: get compound a, add acetone, slowly add the vitriol oil under the agitation condition, stirring reaction under the room temperature is after the TLC detection reaction finishes, regulate the pH value with strong aqua, filter, the most of acetone of pressure reducing and steaming adds 0.4% dilute hydrochloric acid under agitation condition, react under the acetone reflux conditions, compound a all is hydrolyzed into compound b; Use solid NaHCO
3Neutralization reaction liquid filters, and filtrate under reduced pressure boils off solvent, and residuum dissolves with methylene dichloride, uses anhydrous Na
2SO
4Dried overnight is filtered, boil off solvent after, obtain yellow thickness oily matter b;
Synthesizing of compound c: compound b and triethylamine are dissolved in methylene dichloride, and cryosel is bathed down and is slowly dripped parachlorobenzoyl chloride, keeps 0 ℃ of following mechanical stirring reaction; After finishing, reaction adds saturated NaHCO
3The aqueous solution, dichloromethane layer water, saturated common salt water washing, anhydrous MgSO
4Drying is filtered, and pressure reducing and steaming solvent, and recrystallization residuum get the white crystal compound c;
Synthesizing of compound d: compound c is dissolved in the anhydrous methylene chloride, under nitrogen protection, adds imidazoles and t butyldimethylsilyl chloride; Room temperature reaction after reaction finishes, use the hydrochloric acid neutralization reaction, tells two-layerly, and organic layer is used anhydrous Na after distinguishing water and saturated common salt water washing
2SO
4Drying, steaming desolventizes, and residuum carries out separation and purification by column chromatography, obtains compound d;
Synthesizing of Verbindung: compound d joins in sodium methylate-methanol mixture liquid, room temperature reaction; React and finish the back with the dilute acetic acid neutralization, filter, use methanol wash, remove solvent under reduced pressure, the residuum column chromatography separating purification gets Verbindung;
Compound f's is synthetic: dropwise drip DMSO under-60 ℃ of conditions, under identical temperature, stir after 15 minutes in the dichloromethane solution of oxalyl chloride, drip the dichloromethane solution of Verbindung;-65 ℃ of stirring reactions 30 minutes, add triethylamine, the stirring at room reaction; Reaction finishes the back and add entry in reaction solution, tells organic layer, the organic layer anhydrous magnesium sulfate drying, and steaming desolventizes, and residuum gets compound f with column chromatography separating purification;
Compound g's is synthetic: put into sodium hydroxide and water in reaction flask, after stirring, add formalin, 95% ethanol, and then add compound f, 35 ℃ of following stirring reactions; Reaction is cooled off reaction flask with frozen water after finishing while stirring, separates out fully until product; Suction filtration, solid are washed with water to neutral and oven dry, and the gained solid is dissolved in the anhydrous methanol, adds sodium borohydride again, back flow reaction, and reaction finishes the back with dilute hydrochloric acid neutralization reaction liquid, uses the dichloromethane extraction reaction solution, anhydrous Na
2SO
4Dry back is steamed and is desolventized, and gets compound g;
Compound h's is synthetic: compound g is dissolved in the methyl alcohol, adds the Dowex H that uses methanol wash in advance
+, room temperature reaction, reaction is removed resin filter after finishing, and with methyl alcohol repetitive scrubbing resin, the solution evaporate to dryness gets solid; The gained solid is dissolved in the acetone, slowly adds the vitriol oil of catalytic amount under the agitation condition, the stirring at room reaction is regulated the pH value with strong aqua, filters, and pressure reducing and steaming acetone obtains compound h;
Synthesizing of compound i: in the dichloromethane solution of oxalyl chloride, dropwise dripping DMSO under-60 ℃, dripping off the back stirred 15 minutes, drip the dichloromethane solution of compound h, drip off-60 ℃ of reactions in back 30 minutes, add triethylamine, room temperature reaction adds the entry termination reaction after 30 minutes, tell organic layer, the organic layer anhydrous Na
2SO
4Drying, steaming desolventizes, and residuum gets solid with column chromatography separating purification; With ClCH
2P (C
6H
5)
3Cl joins in the anhydrous tetrahydro furan, is cooled to-78 ℃, adds the hexane solution stirring reaction 1h of n-BuLi, adds gained solid dry tetrahydrofuran solution then, and temperature is elevated to 0 ℃ gradually, continues reaction; The saturated NH of careful dropping after reaction finishes
4Cl solution is used ethyl acetate extraction, and organic layer salt solution washed twice is used anhydrous MgSO at last
4Drying removes solvent under reduced pressure, and the residuum that obtains is dissolved in the anhydrous tetrahydro furan, is cooled to-78 ℃, slowly drips the hexane solution of n-BuLi; Stirring reaction 2h uses saturated NH carefully
4Cl solution termination reaction; Organic layer saturated common salt water washing, anhydrous MgSO
4Drying removes solvent under reduced pressure, and residuum gets compound i with quick silicagel column separation and purification;
Synthesizing of compound i: compound i is joined in the dilute hydrochloric acid solution, and the stirring at room reaction is used solid NaHCO after reaction finishes
3Neutralization is filtered, and filtrate boils off solvent under reduced pressure, and residuum dissolves with methylene dichloride, and uses anhydrous Na
2SO
4Dry after-filtration boils off solvent, gets compound j;
Compound k's is synthetic: compound j is dissolved in the pyridine, drips BzCl, and room temperature reaction, solvent evaporated after reaction finishes gets compound k behind the residuum purifying;
Synthesizing of compound 1: with compound k and 2,2,6,6-tetramethyl piperidine-N-oxygen is dissolved in the methylene dichloride, is chilled to 0 ℃, with blended chlorine bleach liquor, NaHCO
3Join reaction solution with water mixed solution, 0 ℃ was reacted 30 minutes, added Virahol, stirring at room, and the dichloromethane layer of telling washes with water 2 times, anhydrous MgSO
4Drying is filtered, and boils off the residuum that solvent obtains and carries out recrystallization, obtains compound l;
Compound m's is synthetic: compound k is joined in the mixed solution of dehydrated alcohol and ethyl acetate, be chilled to 0 ℃, add sodium borohydride in batches, stirring reaction, reaction finish the back with the dilute acetic acid neutralization, filter, the filtrate evaporate to dryness, residuum with the methylene dichloride dissolving, is washed the anhydrous MgSO of dichloromethane layer more then
4Drying is filtered, the pressure reducing and steaming methylene dichloride, and residuum obtains solids with the mixed solvent recrystallization of methylene dichloride and sherwood oil; The gained solids is dissolved in the pyridine, drips BzCl at 0 ℃, after dropwising, room temperature reaction, steaming desolventized residuum ethyl acetate extraction, saturated NaHCO after reaction finished
3Solution washing, anhydrous Na
2SO
4Drying is filtered, and boils off solvent, and the residuum column chromatography separating purification gets compound m;
Compound n's is synthetic: compound m is dissolved in the methyl alcohol, adds the mixing solutions of concentrated hydrochloric acid/dioxane, room temperature reaction is used NaHCO after reaction finishes
3Neutralization reaction liquid filters, and the filtrate decompression distillation removes desolvates, and residuum is dissolved in an amount of methylene dichloride, uses anhydrous Na
2SO
4Dried overnight, the filtrate evaporate to dryness gets compound n with column chromatography separating purification;
Compound o's is synthetic: compound n is dissolved in the methylene dichloride, and room temperature condition adds the DAST stirring reaction down, pours mixture into saturated NaHCO after reaction finishes
3In the solution, tell organic layer, and use saturated NaHCO
3Solution washing, anhydrous Na
2SO
4Drying, column chromatography separating purification obtain compound o;
Compound p's is synthetic: compound o is joined in the formic acid solution, stirring reaction at room temperature, pressure reducing and steaming solvent after reaction finishes, residuum and toluene steam altogether solids, the gained solids is dissolved in dry pyridine and acetic anhydride solution, and room temperature reaction after reaction finishes concentrates reaction solution, steam altogether with toluene then, obtain compound p;
Compound q's is synthetic: compound p is dissolved in the anhydrous methylene chloride, and it is chilled to 0 ℃, and slowly logical then HCl gas is to saturated, and vacuum boils off solvent under the room temperature, and residuum vacuum-drying obtains compound q;
Compound r's is synthetic: 2,6-diaminopurine and hexa methyl silazane are at (NH
4)
2SO
4Reflux is to obtaining clear solution under existing; boil off solvent under the vacuum and obtain white solid; this solid is dissolved in 1, in the 2-ethylene dichloride, adds 1 of compound q again; 2-dichloroethane solution and molecular sieve; after the stirring at room reaction under nitrogen protection, TLC detection reaction fully, in reaction solution, add methylene dichloride; and use diatomite filtration, filtrate is used saturated NaHCO respectively
3With salt solution washing, organic layer anhydrous Na
2SO
4Dried overnight boils off residuum that solvent obtains and separates with the decompression silicagel column and obtain compound r;
Compound s's is synthetic: r is dissolved in NH with compound
3-CH
3In the saturated solution of OH, stirring at room reaction, the reaction back that finishes is steamed and is desolventized, and residuum obtains compound s by column chromatography separating purification.
5. the application of the 2 '-fluoro-4 ' described in claim 1 or 2-substituted nucleosides analogue in the preparation medicine is characterized in that, is applied to prepare resisting HBV virus medicine or anti-HCV virus drugs or anti HIV-1 virus medicine.
6. the application of the 2 '-fluoro-4 ' described in claim 5-replacement-nucleoside analog in the preparation medicine is characterized in that the HBV virus drugs is an anti-hbv drug, and the HCV virus drugs is the anti-third liver medicine, and the HIV virus drugs is an anti-AIDS drug.
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| WO2020007070A1 (en) * | 2018-07-02 | 2020-01-09 | 河南真实生物科技有限公司 | Crystal form, preparation method, and application of 4'-substituted nucleoside |
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| Title |
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| Nucleic Acids》.2001,第20卷(第8期),1583-1598页. * |
| Shortnacy-Fowler, Anita T.等.Synthesis and biological activity of 4"-C-hydroxymethyl-2"-fluoro-D-arabinofuranosylpurine nucleosides.《 Nucleosides, Nucleotides & Nucleic Acids》.2001,第20卷(第8期),1583-1598页. |
| Shortnacy-Fowler, Anita T.等.Synthesis and biological activity of 4"-C-hydroxymethyl-2"-fluoro-D-arabinofuranosylpurine nucleosides.《 Nucleosides, Nucleotides & * |
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