CN102603836A - Schisandrin C simplifier, schisandrin analogue, preparation method and applications thereof - Google Patents
Schisandrin C simplifier, schisandrin analogue, preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a schisandrin C simplifier, a schisandrin C analogue, a preparation method and applications of the schisandrin C simplifier and the schisandrin C analogue, and relates to pyrimidine nucleoside derivatives formed by seven-position substitution of biphenyl compounds, a preparation method of the pyrimidine nucleoside derivatives and applications of the pyrimidine nucleoside derivatives in antiviral aspect, belonging to the field of pharmaceutical chemistry. The schisandrin C simplifier and the schisandrin analogue have the following general structure formula shown as the pictures in the abstract, wherein BR1=CH3, C2H5, i-C3H7; when R2=CH3, R3+R4=-CH2-; and when R4=CH3, R2+R3=-CH2-. The compounds have the activities of antiviral, resistance to infection and proliferation and the like, is feasible in the preparation method, is applied to medicaments for treating virus and has good application prospect.
Description
Technical field
The present invention relates to Wuweizisu C and simplify thing, analogue, relate in particular to 7 substituted miazines nucleoside derivates of biphenyl compound, its preparation method and, belong to the pharmaceutical chemistry field in the application of anti-virus aspect.
Background technology
Various viruses are like hepatitis B, third liver and AIDS etc., very serious to the harm that the mankind bring.Bird flu is arrived period, does not almost have medicine and can treat.In 6,000,000,000 populations of the whole world, 1/2 people lives in the high popular district of hepatitis B virus (HBV), and about 2,000,000,000 people have been proved to be HBV and have infected, and 3～400,000,000 people are the HBV chronic infection, wherein 15%～25% finally will die from liver cirrhosis and liver cancer.In preceding 10 the disease causes of the death in the whole world, hepatitis B accounts for the 7th, and be about 100 ten thousand examples because of hepatitis B death person every year.Hepatitis B virus is the major disease that threatens human health.In treatment HBV medicine, be faced with many difficult problems, like resistance, drug target is unclear, produces resistance etc.Still need research and develop anti-HBV new drug efficient, low toxicity.
It is global popular that hepatitis C is, and is the main reason of America and Europe and Japan and other countries hepatopathy in whole latter stage.Suffer from third liver according to about 1.8 hundred million people in the statistics whole world in 2009 of the World Health Organization, and about 3.5 ten thousand examples of annual New Development hepatitis C case.The whole world was doubled by the death toll that the relevant hepatopathy of third liver causes in per 10 years approximately, and the present third liver mortality ratio comes the tenth in all diseases of the whole world.Also there is not hepatitis C vaccine at present.The ribavirin (ribavirin) and the glycol interferon alpha-2b combination therapy chronic hepatitis C of U.S. Valeant invention; Be considered to conventional treatment; HCV rate of virological response (SVR) is merely 47%～54%, and also has the part patient to have to discontinue medication because of its severe side effect.Also have numerous patient's myrrhs to use.Therefore, research and development are efficient, the anti-third liver new drug of low toxicity is extremely urgent.
Virus of AIDS (HIV) is the major disease of serious threat human health.Though the misery that existing medicine is used to slow down aids patient, mostly curative effect is not high, and toxic side effect is big, and is very easy to produce resistance.Therefore, the research new drug remains the key areas that the mankind defeat serious disease.
Research both at home and abroad shows: Wuweizisu C and simplification thing have antivirus action preferably, the natural product class medicine that natural product picodna DNA and nucleic acid RNA monomer nucleosides obtain through modification, and toxic side effect is lower.Up to the present, there be more than 70 nucleoside medicine to be widely used in treating diseases such as cancer, virus.Yet, still can not satisfy the needs of safeguarding human health, still need research and develop out better new drug, optimal medicine should be to treat virus, can play even more important effect to human health.
Summary of the invention
The object of the invention is to provide one type of new Wuweizisu C with antiviral activity to simplify thing and analogue; Another purpose is to provide its preparation method; Purpose is to provide its application aspect the preparation antiviral again.
Be to realize the object of the invention, the present invention has carried out structural modification to biphenyl compound, has synthesized 7 substituted miazines nucleoside derivates of biphenyl compound (A) and (B), and has comprised its spendable various salt and verivate in pharmacy.It has following general structure A, B and synthetic through following reaction scheme, and this compounds comprises operable salt in its pharmacy, ester, prodrug or its metabolite.
R
1=CH
3, C
2H
5, i-C
3H
7Work as R
2=CH
3The time R
3+ R
4=-CH
2-; Work as R
4=CH
3The time R
2+ R
3=-CH
2-.
(1) with compound I, 1-ethyl-3-(3-dimethylamine propyl) carbodiimide) and the 4-Dimethylamino pyridine be dissolved in the anhydrous methylene chloride stirring reaction at room temperature; In this system, add compound I I then; Continue the stirring at room reaction, the TLC monitoring is after question response is accomplished; Through extraction, drying, separation and purification gets compound III; (2) compound III is dissolved in the anhydrous tetrahydro furan, adds tetrabutyl ammonium fluoride and Glacial acetic acid min. 99.5, stirring reaction at room temperature, the TLC monitoring, after question response is accomplished, solvent evaporated, the separation and purification of gained crude product obtains compd A; (3) with compound III, triazole and pyridine are dissolved in the anhydrous methylene chloride, in this system, add POCl3 under the ice bath; At room temperature stirring reaction steams the solvent decompression then, in remaining solid, adds ammoniacal liquor and 1; The 4-dioxane at room temperature continues stirring reaction, after reaction finishes; Through extraction, drying, separation and purification gets compound IV; (4) compound IV is dissolved in the anhydrous tetrahydro furan, adds tetrabutyl ammonium fluoride and Glacial acetic acid min. 99.5, stirring reaction at room temperature, the TLC monitoring, after question response is accomplished, solvent evaporated, the separation and purification of gained crude product obtains compd B.
It can pass through active compound (A) and (B) or its prodrug or its 5 '-SULPHOSUCCINIC ACID ESTER and organic acid or inorganic acid reaction form salt, exist with the form of salt.
Compound of being contained in the general formula according to the invention and salt thereof and prodrug are used to prepare treatment, antiviral or pharmaceutical prepn.Compound among the general formula 1-3 according to the invention also can be used for treating or prevents or alleviate the disease that virus causes.The preferred hepatitis B of indication virus, hepatitis C, virus of AIDS, yellow fever virus (YFV), respiratory syncytial virus (RSV), hsv (HSV), bovine viral diarrhea virus (BVDV), hepatitis G virus (HGV), GB virus-B (GBV-B), dengue virus (Dengue), ERC group virus (HRV), poliovirus (Poliovirus), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) etc.
The compound salt form can be by negative ion formation suitable in group that has positive charge in the molecule (like amido) and the pharmacy in the general formula according to the invention.Suitable negative ion includes, but are not limited to fluorochemical, muriate, bromide, iodide, vitriol, nitrate salt, phosphoric acid salt, tartrate, metilsulfate, trifluoroacetate, PHENRAMINE MALEATE, acetate etc.Spendable salt can also be formed by electronegative group (carboxyl) in the compound and positive ion in the pharmacy.Can be used for the positively charged ion of this purpose, like sodium salt, sylvite, magnesium salts, calcium salt and organic amine salt ion such as tetramethyl-ammonium salt ion, TBuA salt ion and other organic ion.
Innovative point of the present invention and advantage are: biphenyl compound is modified, and 7 substituted miazines nucleoside derivates of serial biphenyl compound have been synthesized in invention, and these compounds have antiviral infection and anti-hyperplasia diffusion isoreactivity; The preparation method is feasible; Be applied to treat virus drugs, have good application prospect.
Embodiment
Through following specific embodiment the present invention is better explained.
The synthetic route of general formula compound and combine embodiment that invention is further specified according to the present invention, but and unrestricted scope of the present invention.
Embodiment 1: compound 9,11 synthetic
(1), compound 2 is synthetic
(2), compound 3 is synthetic
With compound 2 (100mg 0.35mmol) is dissolved in the 20mL pyridine, under 0 ℃, drip respectively imidazoles (71mg, 1.0mmol) and TERT-BUTYL DIMETHYL CHLORO SILANE (79mg; 0.53mmol), under this temperature, continue to stir the TLC monitoring; Reaction needs 13 hours approximately, and after question response was accomplished, decompression steamed solvent; Crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:1), obtain white solid compound 3 (115mg, 82%);
1H NMR (300 MHz, CDCl
3) δ: 9.78 (1H, s, NH), 7.41 (d, 1H, J=8.1Hz), 6.39 (dd, 1H; J=12.6,4.8Hz), 5.62 (d, 1H, J=8.1Hz), 5.08 (dt, 1H, J=52.8; 4.5Hz), 4.32 (dd, 2H, J=20.7,3.9Hz), 3.80 (s, 2H); 3.39 (bs, 1H ,-OH), 0.79 (s, 9H), 0.00 (s, 6H).
(3), compound 5 is synthetic
With the two methyl esters 3 of biphenyl (5g, 12mmol), Pottasium Hydroxide (2.7g; 48mmol) be dissolved in the 50mL absolute ethyl alcohol, reflux was chilled to room temperature after 8 hours; Hydrochloric acid with 10% is transferred pH=1, and the solid of generation is used the B suction filtration, collects filter cake; Drying obtains white solid compound 5 (4.7g, 99%).
1H?NMR?(300?MHz,?DMSO):?12.35?(2H,?s,?2×OH),?7.27?(2H,?s,?2×Ar-H),?5.98?(4H,?d,?
J?=?11.1?Hz,?2×OCH
2O),?3.88?(6H,?s,?2×OCH
3).
(4), compound 6 is synthetic
Get compound 5 (4.5g, 11.5mmol) and diacetyl oxide (60mL 635mmol) adds in the flask; Cost reflux, nitrogen protection was reacted 10 hours down at 140 ℃; Decompression steams the unnecessary diacetyl oxide and the acetate of generation, obtains brown white solid, in residuum, adds dry toluene 20mL; Steam except that behind the toluene and obtain white solid compound 6 (3.7g, 87%), step reaction under directly being used for.
(5), compound 7 is synthetic
Get compound 6 (3.7g, 10mmol), absolute ethyl alcohol 40mL, 4-Dimethylamino pyridine (10mg; 0.07mmol) add in the flask, cost reflux, reflux is 12 hours under nitrogen protection, the TLC monitoring (methylene dichloride: methyl alcohol=10:1); After question response is accomplished, cooling, solution is with ETHYLE ACETATE (3 * 20mL) extractions (3 * 20mL); Merge organic phase and use anhydrous sodium sulfate drying, obtain white solid compound 7 (3.7g, 95%) behind the evaporate to dryness;
1H NMR (300 MHz, DMSO): 9.22 (bs, 1H), 7.43 (s, 1H), 7.34 (s, 1H), 5.97-5.92 (m,
4H), 4.10-4.06 (m, 2H), 3.95 (s, 6H), 1.05 (t, 3H, J=7.2Hz).
(6), compound 8 is synthetic
With compound 7 (230mg, 0.55mmol), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (141mg; 0.74mmol) and the 4-Dimethylamino pyridine (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours; (147mg 0.37mmol), continued stirring at room 6 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 8 (144mg, 49%);
1H NMR (300 MHz, CDCl
3) δ: 9.83 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H, J=4.5Hz); 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz), 5.99-6.02 (m, 4H); 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3,2.1Hz), 4.97 and 4.75 (dt, 1H; J=39,2.7Hz), 4.13-4.09 (m, 2H), 3.99 (s, 6H), 3.77-3.73 (m, 2H); 1.11-1.09 (m, 3H), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz)
.
(7), compound 9 is synthetic
(144mg 0.18mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.25mL with compound 8; 0.85mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 13 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:1); Obtain white solid compound 9 (105mg, 85%).
?1H?NMR?(300?MHz,?CDCl
3)?δ:?9.53?(bs,?1H,?NH),?7.59-7.55?(m,?1H)?,?7.54-7.40?(m,?2H),?6.39?(td,?1H,?J?=22.2,?11.1,?5.1Hz),?6.04-5.98?(m,?4H),?5.76?(d,?1H,?J?=8.4Hz),?5.49(dq,?1H,?J?=22.5,?4.5Hz),?5.14-4.79?(m,?1H),?4.13-4.09?(m,?2H),?3.99?(s,?6H),?3.98-3.87(m,?2H),?1.11-1.09(m,?3H);?ESI-MS:?688.1533[M+1]
+,?710.1345[M+23]
+.
(8), compound 10 is synthetic
With compound 8 (145mg, 0.18mmol), triazole (100mg, 1.45mmol); Pyridine (0.8mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 24 hours; Then the solvent decompression is steamed, in remaining solid, add ammoniacal liquor and 1,4-dioxane; Continuation was at room temperature stirred 21 hours, and adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase; Anhydrous sodium sulfate drying obtains white solid compound 10 (78mg, 54%) with thin layer silica gel separation and purification (ETHYLE ACETATE) behind the evaporate to dryness.
1H?NMR?(300?MHz,?CDCl
3)?δ:?7.60?(d,?1H,?J?=7.5Hz),?7.47?(s,?1H),?7.43(d,?1H,?J?=1.8Hz),?6.44?and?6.34?(dd,?1H,?J?=15.6,?4.2Hz),?5.98-6.20?(m,?4H),?5.72(d,?1H,?J?=7.5Hz),?5.49?and?5.42?(dd,?1H,?J?=10.5,?2.7Hz),?5.05?and?4.84?(dt,?1H,?J?=?6.9,?2.7Hz),?4.13-4.09?(m,?2H),?3.99?(s,?6H),?3.98-3.87(m,?2H),?2.05?(bs,?2H,?-NH
2),?1.11-1.09(m,?3H),?0.89?(d,?9H,?J=2.4Hz),?0.05-0.09?(m,?6H).
(9), compound 11 is synthetic
(78mg 0.10mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.20mL with compound 10; 0.68mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 12 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 11 (27mg, 40%).
1H NMR (300 MHz, CDCl
3) δ: 7.60 (d, 1H, J=7.5Hz), 7.43-7.42 (m, 2H), 6.51-6.36 (m, 1H), 6.02-5.99 (m; 4H), 5.81 (d, 1H, J=7.5Hz), 5.49 (dd, 1H, J=21; 8.1Hz), 4.88 (d, 1H, J=52.2Hz), 4.13-4.09 (m, 2H), 3.98 (d; 6H, J=2.1Hz), 3.78-3.73 (m, 2H), 2.05 (bs, 2H ,-NH
2), 1.11-1.09 (m, 3H); ESI-MS:687.1697 [M+1]
+.
Embodiment 2: compound 14,16 synthetic
(1), compound 13 is synthetic
Is raw material with compound 4 with methyl alcohol, with the method synthetic compound 12 of synthetic compound 7, with compound 12 (100mg, 0.25mmol); 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (57mg, 0.30mmol) (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours with the 4-Dimethylamino pyridine; (149mg 0.37mmol), continued stirring at room 12 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 13 (96mg, 49%);
1H NMR (300 MHz, CDCl
3) δ: 9.83 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H, J=4.5Hz); 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz), 5.99-6.02 (m, 4H); 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3,2.1Hz), 4.97 and, 4.75 (dt; 1H, J=39,2.7Hz), 3.99 (s, 6H), 3.84-3.94 (m, 2H), 3.68 (d; 3H, J=2.7Hz), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz); ESI-MS:810.2074 [M+23]
+, 826.1875 [M+39]
+ .
(2), compound 14 is synthetic
(100mg 0.13mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.15mL with compound 13; 0.51mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 16 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:2); Obtain white solid compound 14 (83mg, 95%).
?1H?NMR?(300?MHz,?CDCl
3)?δ:?9.99?(bs,?1H,?NH),?7.54-7.59?(m,?1H)?,?7.37-7.43?(m,?2H),?6.33-6.42?(m,?1H),?5.95-5.99?(m,?4H),?5.73?(d,?1H,?J?=8.1Hz),?5.51(dq,?1H,?J?=22.5Hz),?4.76-5.13?(m,?1H),?3.94-3.95?(m,?6H),?3.80-3.90?(m,?2H),?3.66?(d,?3H,?J?=3.6Hz);?ESI-MS:?674.1393[M+1]
+,?696.1178[M+23]
+,?712.0928[M+39]
+.
(3), compound 15 is synthetic
With compound 13 (100mg, 0.13mmol), triazole (70mg, 1.01mmol); Pyridine (0.6mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 23 hours, then the solvent decompression is steamed; In remaining solid, add ammoniacal liquor and 1, the 4-dioxane at room temperature continues to stir 5.5 hours; Adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase, anhydrous sodium sulfate drying; With thin layer silica gel separation and purification (ETHYLE ACETATE), obtain white solid compound 15 (59mg, 59.1%) behind the evaporate to dryness.
1H?NMR?(300?MHz,?CDCl
3)?δ:?7.60?(d,?1H,?J?=7.5Hz),?7.47?(s,?1H),?7.43(d,?1H,?J?=1.8Hz),?6.44?and?6.34?(dd,?1H,?J?=15.6,?4.2Hz),?5.98-6.20?(m,?4H),?5.72(d,?1H,?J?=7.5Hz),?5.49?and?5.42?(dd,?1H,?J?=10.5,?2.7Hz),?5.05?and?4.84?(dt,?1H,?J?=?6.9,?2.7Hz),?3.98-3.99?(m,?6H),?3.87-3.91?(m,?2H),?3.68?(d,?3H,?J?=2.4Hz),?2.05?(bs,?2H,?-NH2),?0.89?(d,?9H,?J=2.4Hz),?0.05-0.09?(m,?6H).
(4), compound 16 is synthetic
(59mg 0.08mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.08mL with compound 15; 0.27mmol) and 5 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 16 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 16 (23mg, 46%).
1H NMR (300 MHz, CDCl
3) δ: 7.27-7.36 (m, 3H), 6.27-6.36 (m, 1H), 5.95-6.05 (m, 4H); 5.79 (d, 1H, J=7.5Hz), 5.52-5.59 (m, 1H), 5.07 and, 4.86 (dt; 1H, J=35.7,3.9Hz), 4.34 (bs, 1H ,-OH); 3.89-3.91 (m, 6H), 3.73 (s, 2H), 3.58 (s, 3H). ESI-MS:673.1555 [M+1]
+, 695.1385 [M+23]
+.ESI-MS:673.1555 [M+1]
+, 695.1385 [M+23]
+.
Embodiment 3: compound 19,21 synthetic
(1), compound 18 is synthetic
Is raw material with compound 4 with Virahol, with the method synthetic compound 17 of synthetic compound 7, with compound 17 (238mg, 0.55mmol); 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (141mg, 0.74mmol) (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours with the 4-Dimethylamino pyridine; (147mg 0.37mmol), continued stirring at room 6 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 18 (157mg, 52%);
1H NMR (300 MHz, CDCl
3) δ: 9.53 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H; J=4.5Hz), 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz); 5.99-6.02 (m, 4H), 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3; 2.1Hz), 5.25-5.14 (m, 1H, J=6Hz), 4.97 and 4.75 (dt, 1H, J=39,2.7Hz); 3.99 (s, 6H), 3.77-3.73 (m, 2H), 1.24 (d, 3H, J=6Hz), 1.22 (d; 3H, J=6Hz), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz)
.
(3), compound 19 is synthetic
(147mg 0.18mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.25mL with compound 18; 0.85mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 13 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:1); Obtain white solid compound 19 (112mg, 89%).
?1H?NMR?(300?MHz,?CDCl
3)?δ:?9.53?(bs,?1H,?NH),?7.59-7.55?(m,?1H)?,?7.54-7.40?(m,?2H),?6.39?(td,?1H,?J?=22.2,?11.1,?5.1Hz),?6.04-5.98?(m,?4H),?5.76?(d,?1H,?J?=8.4Hz),?5.49(dq,?1H,?J?=22.5,?4.5Hz),?5.21-5.10?(m,?1H,?J?=6Hz),?5.14-4.79?(m,?1H),?3.99?(s,?6H),?3.98-3.87(m,?2H),?1.09?(d,?3H,?J?=6Hz),?1.02?(d,?3H,?J?=6Hz).
(4), compound 20 is synthetic
With compound 18 (147mg, 0.18mmol), triazole (100mg, 1.45mmol); Pyridine (0.8mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 24 hours; Then the solvent decompression is steamed, in remaining solid, add ammoniacal liquor and 1,4-dioxane; Continuation was at room temperature stirred 20 hours, and adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase; Anhydrous sodium sulfate drying obtains white solid compound 20 (88mg, 60%) with thin layer silica gel separation and purification (ETHYLE ACETATE) behind the evaporate to dryness.
1H?NMR?(300?MHz,?CDCl
3)?δ:?7.60?(d,?1H,?J?=7.5Hz),?7.47?(s,?1H),?7.43(d,?1H,?J?=1.8Hz),?6.44?and?6.34?(dd,?1H,?J?=15.6,?4.2Hz),?5.98-6.20?(m,?4H),?5.72(d,?1H,?J?=7.5Hz),?5.49?and?5.42?(dd,?1H,?J?=10.5,?2.7Hz),?5.25-5.14?(m,?1H,?J?=6Hz),?5.05?and?4.84?(dt,?1H,?J?=?6.9,?2.7Hz),?3.99?(s,?6H),?3.98-3.87(m,?2H),?2.05?(bs,?2H,?-NH
2),?1.09?(d,?3H,?J?=6Hz),?1.02?(d,?3H,?J?=6Hz),?0.89?(d,?9H,?J=2.4Hz),?0.05-0.09?(m,?6H).
(5), compound 21 is synthetic
(82mg 0.10mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.20mL with 20; 0.68mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 12 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 21 (32mg, 45%).
1H NMR (300 MHz, CDCl
3) δ: 7.60 (d, 1H, J=7.5Hz), 7.43-7.42 (m, 2H), 6.51-6.36 (m, 1H), 6.02-5.99 (m; 4H), 5.81 (d, 1H, J=7.5Hz), 5.49 (dd, 1H, J=21,8.1Hz); 5.22-5.10 (m, 1H, J=6Hz), 4.88 (d, 1H, J=52.2Hz), 3.98 (d; 6H, J=2.1Hz), 3.78-3.73 (m, 2H), 2.05 (bs, 2H ,-NH
2), 1.09 (d, 3H, J=6Hz), 1.02 (d, 3H, J=6Hz).
Embodiment 4: compound 24,26 synthetic
(1), compound 23 is synthetic
With compound 22 (100mg, 0.25mmol), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (57mg; 0.30mmol) and the 4-Dimethylamino pyridine (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours; (149mg 0.37mmol), continued stirring at room 14 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 23 (108mg, 55%);
1H NMR (300 MHz, CDCl
3) δ: 9.80 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H, J=4.5Hz); 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz), 5.99-6.02 (m, 4H); 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3,2.1Hz), 4.97 and, 4.75 (dt; 1H, J=39,2.7Hz), 3.99 (s, 6H), 3.84-3.94 (m, 2H), 3.68 (d; 3H, J=2.7Hz), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz).
(2), compound 24 is synthetic
(100mg 0.13mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.15mL with compound 23; 0.51mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 20 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:2); Obtain white solid compound 24 (19mg, 90%).
?1H?NMR?(300?MHz,?CDCl
3)?δ:?9.99?(bs,?1H,?NH),?7.54-7.59?(m,?1H)?,?7.37-7.43?(m,?2H),?6.33-6.42?(m,?1H),?5.95-5.99?(m,?4H),?5.73?(d,?1H,?J?=8.1Hz),?5.51(dq,?1H,?J?=22.5Hz),?4.76-5.13?(m,?1H),?3.94-3.95?(m,?6H),?3.80-3.90?(m,?2H),?3.66?(d,?3H,?J?=3.6Hz)?;?ESI-MS:?674.1389[M+1]
+,?696.1181[M+23]
+,?712.0930[M+39]
+.
(3), compound 25 is synthetic
With compound 23 (100mg, 0.13mmol), triazole (70mg, 1.01mmol); Pyridine (0.6mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 19 hours, then the solvent decompression is steamed; In remaining solid, add ammoniacal liquor and 1, the 4-dioxane at room temperature continues to stir 5.5 hours; Adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase, anhydrous sodium sulfate drying; With thin layer silica gel separation and purification (ETHYLE ACETATE), obtain white solid compound 25 (62mg, 62%) behind the evaporate to dryness.
1H?NMR?(300?MHz,?CDCl
3)?δ:?7.60?(d,?1H,?J?=7.5Hz),?7.47?(s,?1H),?7.43(d,?1H,?J?=1.8Hz),?6.44?and?6.34?(dd,?1H,?J?=15.6,?4.2Hz),?5.98-6.20?(m,?4H),?5.72(d,?1H,?J?=7.5Hz),?5.49?and?5.42?(dd,?1H,?J?=10.5,?2.7Hz),?5.05?and?4.84?(dt,?1H,?J?=?6.9,?2.7Hz),?3.98-3.99?(m,?6H),?3.87-3.91?(m,?2H),?3.68?(d,?3H,?J?=2.4Hz),?2.05?(bs,?2H,?-NH2),?0.89?(d,?9H,?J=2.4Hz),?0.05-0.09?(m,?6H).
(4), compound 26 is synthetic
(59mg 0.08mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.08mL with compound 25; 0.27mmol) and 5 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 14 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 26 (20mg, 40%).
1H NMR (300 MHz, CDCl
3) δ: 7.27-7.36 (m, 3H), 6.27-6.36 (m, 1H), 5.95-6.05 (m, 4H), 5.79 (d; 1H, J=7.5Hz), 5.52-5.59 (m, 1H), 5.07 and 4.86 (dt, 1H; J=35.7,3.9Hz), 4.34 (bs, 1H ,-OH), 3.89-3.91 (m; 6H), 3.73 (s, 2H), 3.58 (s, 3H), ESI-MS:673.1550 [M+1]
+, 695.1388 [M+23]
+.
Embodiment 5: compound 29,31 synthetic
(1), compound 28 is synthetic
With compound 27 (230mg, 0.55mmol), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (141mg; 0.74mmol) and the 4-Dimethylamino pyridine (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours; (147mg 0.37mmol), continued stirring at room 19 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 28 (150mg, 51%);
1H NMR (300 MHz, CDCl
3) δ: 9.53 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H, J=4.5Hz); 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz), 5.99-6.02 (m, 4H); 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3,2.1Hz), 4.97 and 4.75 (dt, 1H; J=39,2.7Hz), 4.13-4.09 (m, 2H), 3.99 (s, 6H), 3.77-3.73 (m, 2H); 1.11-1.09 (m, 3H), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz)
.
(2), compound 29 is synthetic
(144mg 0.18mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.25mL with compound 28; 0.85mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 15 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:1); Obtain white solid compound 29 (98mg, 79%).
1H NMR (300 MHz, CDCl
3) δ: 9.53 (bs, 1H, NH), 7.59-7.55 (m, 1H), 7.54-7.40 (m, 2H), 6.39 (td; 1H, J=22.2,11.1,5.1Hz), 6.04-5.98 (m, 4H), 5.76 (d, 1H; J=8.4Hz), 5.49 (dq, 1H, J=22.5,4.5Hz), 5.14-4.79 (m, 1H), 4.13-4.09 (m; 2H), 3.99 (s, 6H), 3.98-3.87 (m, 2H), 1.11-1.09 (m, 3H); ESI-MS:688.1529 [M+1]
+, 710.1340 [M+23]
+.
(3), compound 30 is synthetic
With compound 28 (145mg, 0.18mmol), triazole (100mg, 1.45mmol); Pyridine (0.8mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 20 hours; Then the solvent decompression is steamed, in remaining solid, add ammoniacal liquor and 1,4-dioxane; Continuation was at room temperature stirred 25 hours, and adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase; Anhydrous sodium sulfate drying obtains white solid compound 30 (78mg, 54%) with thin layer silica gel separation and purification (ETHYLE ACETATE) behind the evaporate to dryness.
1H?NMR?(300?MHz,?CDCl
3)?δ:?7.60?(d,?1H,?J?=7.5Hz),?7.47?(s,?1H),?7.43(d,?1H,?J?=1.8Hz),?6.44?and?6.34?(dd,?1H,?J?=15.6,?4.2Hz),?5.98-6.20?(m,?4H),?5.72(d,?1H,?J?=7.5Hz),?5.49?and?5.42?(dd,?1H,?J?=10.5,?2.7Hz),?5.05?and?4.84?(dt,?1H,?J?=?6.9,?2.7Hz),?4.13-4.09?(m,?2H),?3.99?(s,?6H),?3.98-3.87(m,?2H),?2.05?(bs,?2H,?-NH
2),?1.11-1.09(m,?3H),?0.89?(d,?9H,?J=2.4Hz),?0.05-0.09?(m,?6H).
(4), compound 31 is synthetic
(78mg 0.10mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.20mL with compound 30; 0.68mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 15 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 31 (34mg, 50%).
1H NMR (300 MHz, CDCl
3) δ: 7.60 (d, 1H, J=7.5Hz), 7.43-7.42 (m, 2H), 6.51-6.36 (m, 1H), 6.02-5.99 (m; 4H), 5.81 (d, 1H, J=7.5Hz), 5.49 (dd, 1H, J=21; 8.1Hz), 4.88 (d, 1H, J=52.2Hz), 4.13-4.09 (m, 2H), 3.98 (d; 6H, J=2.1Hz), 3.78-3.73 (m, 2H), 2.05 (bs, 2H ,-NH
2), 1.11-1.09 (m, 3H); ESI-MS:687.1699 [M+1]
+, 709.1523 [M+23]
+.
Embodiment 6: compound 34,36 synthetic
(1), compound 33 is synthetic
With compound 32 (238mg, 0.55mmol), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (141mg; 0.74mmol) and the 4-Dimethylamino pyridine (40mg 0.33mmol) is dissolved in the 20mL anhydrous methylene chloride, at room temperature stirs 1.5 hours; (147mg 0.37mmol), continued stirring at room 10 hours in this system, to add compound 3 then; The TLC monitoring, after question response was accomplished, this mixture was with dichloromethane extraction (3 * 10mL); Organic phase is used saturated sodium bicarbonate successively, saturated common salt water washing, anhydrous sodium sulfate drying; Behind the evaporate to dryness with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=2:1), obtain white solid compound 33 (169mg, 56%);
1H NMR (300 MHz, CDCl
3) δ: 9.53 (s, 1H, NH), 7.59 (d, 1H, J=6.3Hz), 7.45 (d, 1H; J=4.5Hz), 7.42 (d, 1H, J=5.1Hz), 6.36 and 6.26 (dd, 1H, J=10.8,3.3Hz); 5.99-6.02 (m, 4H), 5.73 (d, 1H, J=6.3Hz), 5.53 and 5.48 (dd, 1H, J=9.3; 2.1Hz), 5.25-5.14 (m, 1H, J=6Hz), 4.97 and 4.75 (dt, 1H, J=39,2.7Hz); 3.99 (s, 6H), 3.77-3.73 (m, 2H), 1.24 (d, 3H, J=6Hz), 1.22 (d; 3H, J=6Hz), 0.90 (d, 9H, J=0.9Hz), 0.10 (d, 6H, J=2.1Hz)
.
(2), compound 34 is synthetic
(147mg 0.18mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.25mL with compound 33; 0.85mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 11 hours; After question response is accomplished, solvent evaporated, the gained crude product is with silica gel column chromatography separating purification (sherwood oil: ETHYLE ACETATE=1:1); Obtain white solid compound 34 (107mg, 85%).
1H NMR (300 MHz, CDCl
3) δ: 9.53 (bs, 1H, NH), 7.59-7.55 (m, 1H), 7.54-7.40 (m, 2H), 6.39 (td, 1H; J=22.2,11.1,5.1Hz), 6.04-5.98 (m, 4H), 5.76 (d, 1H, J=8.4Hz), 5.49 (dq; 1H, J=22.5,4.5Hz), 5.21-5.10 (m, 1H, J=6Hz), 5.14-4.79 (m, 1H), 3.99 (s; 6H), 3.98-3.87 (m, 2H), 1.09 (d, 3H, J=6Hz), 1.02 (d, 3H, J=6Hz).
(3), compound 35 is synthetic
With compound 33 (147mg, 0.18mmol), triazole (100mg, 1.45mmol); Pyridine (0.8mL) is dissolved in the 20mL anhydrous methylene chloride, in this system, slowly adds POCl3 (0.05mL) under the ice bath, at room temperature stirs 19 hours; Then the solvent decompression is steamed, in remaining solid, add ammoniacal liquor and 1,4-dioxane; Continuation was at room temperature stirred 20 hours, and adding ethyl acetate extraction after reaction finishes (3 * 10mL), merge organic phase; Anhydrous sodium sulfate drying obtains white solid compound 35 (87mg, 52%) with thin layer silica gel separation and purification (ETHYLE ACETATE) behind the evaporate to dryness.
1H NMR (300 MHz, CDCl
3) δ: 7.60 (d, 1H, J=7.5Hz), 7.47 (s, 1H), 7.43 (d, 1H; J=1.8Hz), 6.44 and 6.34 (dd, 1H, J=15.6,4.2Hz), 5.98-6.20 (m, 4H); 5.72 (d, 1H, J=7.5Hz), 5.49 and 5.42 (dd, 1H, J=10.5,2.7Hz); 5.25-5.14 (m, 1H, J=6Hz), 5.05 and 4.84 (dt, 1H, J=6.9,2.7Hz); 3.99 (s, 6H), 3.98-3.87 (m, 2H), 2.05 (bs, 2H ,-NH
2), 1.09 (d, 3H, J=6Hz), 1.02 (d, 3H, J=6Hz), 0.89 (d, 9H, J=2.4Hz), 0.05-0.09 (m, 6H).
(4), compound 36 is synthetic
(82mg 0.10mmol) is dissolved in the 15mL anhydrous tetrahydro furan, in this system, adds tetrabutyl ammonium fluoride (0.20mL with compound 35; 0.68mmol) and 10 Glacial acetic acid min. 99.5, at room temperature stirred the TLC monitoring 11 hours; After question response is accomplished, solvent evaporated, the gained crude product is analysed separation and purification (ETHYLE ACETATE) with thin layer silica gel; Obtain white solid compound 36 (36mg, 51%).
1H NMR (300 MHz, CDCl
3) δ: 7.60 (d, 1H, J=7.5Hz), 7.43-7.42 (m, 2H), 6.51-6.36 (m, 1H), 6.02-5.99 (m; 4H), 5.81 (d, 1H, J=7.5Hz), 5.49 (dd, 1H, J=21,8.1Hz); 5.22-5.10 (m, 1H, J=6Hz), 4.88 (d, 1H, J=52.2Hz), 3.98 (d; 6H, J=2.1Hz), 3.78-3.73 (m, 2H), 2.05 (bs, 2H ,-NH
2), 1.09 (d, 3H, J=6Hz), 1.02 (d, 3H, J=6Hz).
The above; Only being part embodiment of the present invention, is not that the present invention is done any pro forma restriction, any simple modification that every foundation technical spirit of the present invention is done the foregoing description; Equivalent variations and modification all belong in the technical scheme scope of the present invention.
Synthetic compound of the present invention can be used for the activeconstituents of antiviral, can use separately, also can with other antiviral drug combinations.In the drug combination therapeutic process of indication of the present invention, comprise at least a The compounds of this invention of utilization with and reactive derivative use to increase general curative effect with other one or more antiviral.Dose during drug combination and administration time should be decided according to the rational therapy effect that is obtained under the condition of different.
The medicament compatibility of being contained comprises the effective dose of the compound in the general formula.This medicament compatibility can contain a kind of effective antiviral Chemical Composition and medicinal formula.Here " effective dose " refers to the consumption that can produce required this compound of result of treatment for institute's treatment target.This effective dose or dosage can be by experience person being arranged according to the suggestion of different situations and difference.Such as, the viral species of being treated is different, and the usage of medicine is different; Whether shared etc. with other treat-ment such as other antiviral, dosage all can change.Can process any spendable preparation formulation.If some has alkalescence or acidic cpd and can form avirulent acid or salt, can use the form of the salt of this compound.Spendable organic acid salt comprises spendable anion salt on the physiology in the pharmacy, like p-methyl benzenesulfonic acid salt, metilsulfate, acetate, benzoate, Citrate trianion, tartrate, PHENRAMINE MALEATE, SUMATRIPTAN SUCCINATE, ascorbate salt and glycerophosphate etc.Spendable inorganic salt comprise muriate, bromide, fluorochemical, iodide, vitriol, nitrate salt, supercarbonate, carbonate, phosphoric acid salt etc.The form that can process described salt if any the compound and the suitable acid of alkalescence as amine.The compound of carboxylic-acid can form spendable salt with basic metal or earth alkali metal.
The compound of containing in the formula of of the present invention generally is prone to be dissolved in the mixed solvent of organic solvent, water-soluble solvent and organic solvent and water-soluble solvent and water.Water-soluble solvent alcohols, poly terepthaloyl moietie, N-methyl-2-pyrrolinone, DMAC N,N, N, dinethylformamide, methyl-sulphoxide, second cyanogen with and share.Described pure particular methanol, ethanol, Virahol, USP Kosher or terepthaloyl moietie.The compounds of this invention can mix with preparations carrier commonly used and process preparation.Compound dissolution is in water miscible organic solvent, non-protonic solvent, water-soluble lipid, Schardinger dextrins, lipid acid, phosphatide or in the mixed solvent of these solvents and make medicament solution; Add saline water again and obtain the glucide of 1-20%, like the aqueous solution of glucose.The preparation stabilization that makes therefrom also is used for animal and clinical.
With compound in the above-mentioned general formula is the product medicine that active ingredient is prepared into; Can administered through oral or parenteral route administration; Also can be through transplant medicine pump in the body and additive method administration, the parenteral route administration of indication here is meant subcutaneous intracutaneous, intramuscular, intravenously, intra-arterial, atrium in, in the synovial membrane, in the breastbone, in the sheath, interior, the intracranial injection of wound site or drip infusion technique etc.Use conventional method proportioning by the technician, mixing finally becomes needed pharmaceutical dosage form.Can be the outstanding solution of tablet, capsule, emulsion, pulvis, the little pin of intravenous administration, infusion solutions, lyophilized powder, dripping pill, emulsion suspension liquid, water, the aqueous solution, colloid, colloidal solution, sustained release preparation, nanometer formulation or be used for animal or clinical with other forms of formulation.
Compound in the general formula of the present invention is used to treat or prevents or alleviate the preparation that virus causes disease medicament.Indication virus includes but are not limited to hepatitis B (HBV); Hepatitis C (HVC); AIDS (HIV) poison; Yellow fever virus (YFV); Respiratory syncytial virus (RSV); Hsv (HSV); Bovine viral diarrhea virus (BVDV); Hepatitis G virus (HGV); GB virus-B (GBV-B); Dengue virus (Dengue); ERC group virus (HRV); Poliovirus (Poliovirus); Varicella zoster virus (VZV); Epstein-Barr virus (EBV); Cytomegalovirus (CMV) etc.
Antiviral activity
The compound of being contained among the present invention has antiviral activity, comprises that anti-AIDS HIV-1, HBV and HCV are active, just lists act part instance below and does an explanation.
One, the anti-HIV-1 replication activity is measured:
The compound of being contained among mensuration the present invention is to the active influence of HIV-1 virus replication.Through embodiment of the invention gained compound and 293T (are originated: after ATCC) cell is hatched 15 minutes, add VSVG/HIV-luc (NL4-3) virus strain, cultivated 48 hours for 37 ℃, with the gradient of infection of luciferase as reporter-gene assays HIV-1.(Zidovudine, AZT) (Xiamen steps and restrains medicine ltd) is as positive control with zidovudine.
Substratum: DMEM substratum (U.S. Gibco company), RPMI-1640, FBS; Compound method: RPMI-1640/DMEM+10%FBS.Medium is used in experiment: DMSO 99.8MIN. (DMSO, U.S. Sigma).
Experimental technique:
1, trial-product, reference substance preparation: given the test agent is weighed and is dissolved among the DMSO, and storage liquid concentration is 10mmol/L; Reference substance: the zidovudine and be dissolved in DMSO of weighing, storage liquid concentration is 10 mmol/L.
2, experimental procedure
2.1 the preparation of wild-type HIV-1 reorganization pseudovirus: transfection previous day, by 2.2 * 10
6The density inoculation 293ET cell of individual cell is in 100 mm petridish, with the calcium phosphate precipitation method cotransfection 3 of improvement
VSV-G plasmid and 8
Wild-type HIV-1 core gene after the transfection 16 hours, with the PBS cells washed and renew bright substratum and continue to cultivate 32 hours, is collected supernatant and through 0.45
Membrane filtration, generate wild-type HIV-1 recombinant virus particle VSVG/HIV-WT.
2.2 the p24 antigen measuring of HIV-1 reorganization pseudovirus: respectively get after the doubling dilution virus stock solution used wild-type 450
; With 50
lysate carry out cracking; According to p24 antigen ELISA test kit specification sheets (ZeptoMetrix; Cat:0801111), measure and calculate the p24 antigen concentration of wild-type recombinant virus stoste.
2.3 medicine detects the HIV-1 inhibition: infect previous day, the 293T cell is pressed every hole 6 * 10
4Density be inoculated on 24 orifice plates, dissolve testing compound with DMSO, added in the cell culture fluid in preceding 15 minutes in infecting, the DMSO solvent is made blank, adds 0.5 ml virus liquid again and (according to p24 concentration virus stock solution used is diluted to 0.1 – 0.5
).Infected back 48 hours; Remove supernatant; Add in every hole 50
cell pyrolysis liquid (Promega) lysing cell; Again with 20
product of cell lysis is added in the 30 μ l luciferase substrates (Promega); Relative reactivity with the plain enzyme of FB15 fluorimetric detector (Sirius) Instrument measuring cell fluorescence; Compare the half-inhibition concentration that the computerized compound duplicates wild-type HIV-1 with DMSO.
2.4 use the influence of MTS method detection compound pair cell survival: the 293T cell of logarithmic phase is seeded in 96 orifice plates every hole 100ul, 37 ℃, 5%CO by the cell density in 8000 ~ 10000/hole
2After cultivating 24 h in the incubator, add testing compound and be blank (final concentration is 0.1%) with DMSO, 37 ℃, 5%CO
2Continue in the incubator to cultivate 44 hours.In every hole, add 20
The mixed solution that MTS/PMS joins at present, 37 ℃, 5%CO
2Develop the color after continuing in the incubator to cultivate 4h.Join on the detector at enzyme, wavelength 490 nm and 650 nm (background) locate to detect the absorbance value (OD) in each hole, and calculate the survival rate of cell.
Record the anti-HIV-1 virus activity (EC of embodiment of the invention synthetic compound
50) and cytotoxicity (CC
50) result sees the following form:
Two, anti-B-mode anti-inflammatory HBV external biological activity test:
2.1, the recovery and the cultivation of cell
From liquid nitrogen container, take out a frozen HepG2.2.15 cell, place 42 ℃ of water-baths to shake rapidly to thawing.In DMEM perfect medium (foetal calf serum 10%; G418 380
; Penicillium mould 100
, Streptomycin sulphate 100
).Culture condition is 5% CO
2, 37 ℃ of cultivations.Went down to posterity once in 2～3 days behind the cell normal growth.
2.2, the mtt assay detection compound is to the toxicity of HepG2.2.15 cell.
The MTT colourimetry detection of drugs of setting up according to Mosmann is to the toxicity of HepG2.2.15 cell.Cultured continuously 72 hours, every hole adds MTT10ul, after 4 hours, abandoning supernatant, every hole adds DMSO200ul, and ELIASA (detecting wavelength 490nm, reference wavelength 630nm) reads each hole OD value, the record result.The computerized compound is to HepG2.2.15 cell inhibiting percentage:
Suppress percentage=
Calculate the half toxic concentration (TC of medicine pair cell according to the inhibition percentage of medicine pair cell
50).
2.3, compound is to the HBV body outer suppressioning experiment
Selection as screening concentration, detects HBV surface antigen s and the antigenic influence of e in its pair cell nutrient solution less than compound half toxic concentration.Select the HepG2.2.15 cell of logarithmic phase for use, trysinization becomes single cell suspension, according to 2 * 10
4The concentration of/ml is inoculated in 24 orifice plates, every hole 1ml, 3 holes of each concentration.Add and to contain the substratum of different pharmaceutical concentration, and to establish the cell that adds complete substratum be normal control.Collect the 3rd day, the 6th day, the 9th day cell conditioned medium liquid and cell, in-20 ℃ of preservations.Measure the 3rd day, the 6th day, the 9th day the inside and outside e antigen of cell and the expression of surface antigen with the ELISA method.
2.4, the ELISA detection compound is to the influence of HBeAg and HBsAg in the HepG2.2.15 cell conditioned medium liquid
Carry out according to the test kit working method;
Measure: use the ELIASA reading, select dual wavelength 450/630nm, read each hole OD value.
The result judges: at first calculate the Cutoff value according to the average OD value of COV=negative control * 2.1, if sample OD value>=COV is positive, sample OD value≤COV is negative.
Three, anti-hepatitis C HCV is active:
The compound that the present invention is contained has the effect of anti-hepatitis C HCV.External anti-HCV activity experiment is following:
3.1, cell cultures
Cultivate in the Eagle medium of HCV replicating cell (Avva.5) improved G418 that comprises 10% fetal bovine serum and 1mg/mL at
.293-Sip-L cells
Improved containing 10% fetal bovine serum and 250
The G418 and 150
hygromycin B cultured in Eagle's medium.
3.2, HCV 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 petridish 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 needs trypsinized and improved Eagle medium cleans twice with
with centrifuging.Top section of washing (as contrast) and the cell of washing collect to carry out RNA extraction and RT-PCR detection in pairs for the second time.Beta-actin mRNA measures to do contrast simultaneously.
3.4, the detection by quantitative of HCV-RNA
Adopt full-automatic PCR ELISA come quantitatively determined HCV-RNA (2.0 editions, Roche Diagnostics, Branchburg, NJ).
The present invention synthesized compounds in 0.3
and 0.03
when inhibitory effect (see table below).The HCV-RNA level is measured with full-automatic PCR ELISA.
The above-claimed cpd sequence number is consistent with embodiment of the invention institute synthetic compound sequence number.
Claims (7)
1. Wuweizisu C is simplified thing, analogue or its salt, it is characterized in that, this Wuweizisu C is simplified thing, analogue has following general structure:
R
1=CH
3, C
2H
5, i-C
3H
7Work as R
2=CH
3The time R
3+ R
4=-CH
2-; Work as R
4=CH
3The time R
2+ R
3=-CH
2-.
2. Wuweizisu C as claimed in claim 1 is simplified thing, analogue or its salt, it is characterized in that it is one of following compound:
。
3. Wuweizisu C as claimed in claim 1 is simplified the compound method of thing, analogue, it is characterized in that, and is synthetic through following steps:
R
1=CH
3, C
2H
5, i-C
3H
7Work as R
2=CH
3The time R
3+ R
4=-CH
2-; Work as R
4=CH
3The time R
2+ R
3=-CH
2-;
(1) with compound I, 1-ethyl-3-(3-dimethylamine propyl) carbodiimide) and the 4-Dimethylamino pyridine be dissolved in the anhydrous methylene chloride stirring reaction at room temperature; In this system, add compound I I then; Continue the stirring at room reaction, the TLC monitoring is after question response is accomplished; Through extraction, drying, separation and purification gets compound III; (2) compound III is dissolved in the anhydrous tetrahydro furan, adds tetrabutyl ammonium fluoride and Glacial acetic acid min. 99.5, stirring reaction at room temperature, the TLC monitoring, after question response is accomplished, solvent evaporated, the separation and purification of gained crude product obtains compd A; (3) with compound III, triazole and pyridine are dissolved in the anhydrous methylene chloride, in this system, add POCl3 under the ice bath; At room temperature stirring reaction steams the solvent decompression then, in remaining solid, adds ammoniacal liquor and 1; The 4-dioxane at room temperature continues stirring reaction, after reaction finishes; Through extraction, drying, separation and purification gets compound IV; (4) compound IV is dissolved in the anhydrous tetrahydro furan, adds tetrabutyl ammonium fluoride and Glacial acetic acid min. 99.5, stirring reaction at room temperature, the TLC monitoring, after question response is accomplished, solvent evaporated, the separation and purification of gained crude product obtains compd B.
4. according to claim 1 or claim 2 Wuweizisu C is simplified the application in the preparation antiviral of thing, analogue or its salt, it is characterized in that, it is applied to preparation treatment, prevention or alleviates antiviral and pharmaceutical prepn as activeconstituents.
5. Wuweizisu C as claimed in claim 4 is simplified the application in the preparation antiviral of thing, analogue or its salt; It is characterized in that; With it is that active ingredient is prepared into oral or the parenteral route form of medication, or passes through the form of medication of transplant medicine pump in the body.
6. simplify the application in the preparation antiviral of thing, analogue or its salt like claim 4 or 5 described Wuweizisu Cs, it is characterized in that active ingredient is one or more The compounds of this invention.
7. simplify the application in the preparation antiviral of thing, analogue or its salt like claim 4 or 5 described Wuweizisu Cs; It is characterized in that, be applied to prepare hepatitis B (HBV), hepatitis C (HVC), AIDS (HIV) poison, yellow fever virus (YFV), respiratory syncytial virus (RSV), hsv (HSV), bovine viral diarrhea virus (BVDV), hepatitis G virus (HGV), GB virus-B (GBV-B), dengue virus (Dengue), ERC group virus (HRV), poliovirus (Poliovirus), varicella zoster virus (VZV) or Epstein-Barr virus (EBV), cytomegalovirus (CMV) medicine or preparation of pharmaceutical formulations.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| US11697666B2 (en) | 2021-04-16 | 2023-07-11 | Gilead Sciences, Inc. | Methods of preparing carbanucleosides using amides |
| US11767337B2 (en) | 2020-02-18 | 2023-09-26 | Gilead Sciences, Inc. | Antiviral compounds |
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| US11767337B2 (en) | 2020-02-18 | 2023-09-26 | Gilead Sciences, Inc. | Antiviral compounds |
| US11697666B2 (en) | 2021-04-16 | 2023-07-11 | Gilead Sciences, Inc. | Methods of preparing carbanucleosides using amides |
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