CN1208407A - [S-(R*,R*)]-3-methyl-2-(3-oxo-3h-benzo[d] isothiazol-2-yl] pentanoic acid and [S-(R*,R*)], L-2-(2-(2-(1-carboxy-2-methylbutyl carbamoyl) phenyldisulfonyl) benzoyl-amino)-3-methylpentanic acid - Google Patents

[S-(R*,R*)]-3-methyl-2-(3-oxo-3h-benzo[d] isothiazol-2-yl] pentanoic acid and [S-(R*,R*)], L-2-(2-(2-(1-carboxy-2-methylbutyl carbamoyl) phenyldisulfonyl) benzoyl-amino)-3-methylpentanic acid Download PDF

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CN1208407A
CN1208407A CN96199009A CN96199009A CN1208407A CN 1208407 A CN1208407 A CN 1208407A CN 96199009 A CN96199009 A CN 96199009A CN 96199009 A CN96199009 A CN 96199009A CN 1208407 A CN1208407 A CN 1208407A
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dithio
methyl
benzoyl
acid
thf
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P·菲奥里
T·P·普斯
J·瓦克尔
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Warner Lambert Co LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D275/00—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings
    • C07D275/04—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/22—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides
    • C07C319/24—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides by reactions involving the formation of sulfur-to-sulfur bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton

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  • Thiazole And Isothizaole Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)

Abstract

The present application discloses a method of synthesizing [S-(R*,R*)]-3-methyl-2-(3-oxo-3H-benzo[d]-isothiazol-2-yl)pentanoic acid and [S-(R*,R*)], L-2-{2-[2-(1-carboxy-2-methylbutylcarbamoyl)phenyldisulfonyl]-benzoylamino}-3-methylpentanoic acid.

Description

Preparation [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid and [S-(R *, R *)], the method for L-2-{ 2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid
Invention field
The invention provides synthetic [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid and [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid.
Background of invention
Compound [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid can be used as antiviral drug and can be used for treating the patient who carries HIV.Compound [S-R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl-phenyl connection alkylsulfonyl] benzamido }-3 methylvaleric acid is synthetic [S-(R *, R *)]-intermediate of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid and be used for the treatment of bacterium and virus infection, comprise the patient who carries HIV.For example see United States Patent (USP) 5463122, the document is incorporated this paper into for referencial use at this.Therefore, the synthetic [S-(R of simple, high productivity *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid and [S-R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl] benzamido }-method of 3 methylvaleric acid is useful.
Summary of the invention
The invention provides preparation [S-(R *, R *)]-method of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid, it comprises makes 2,2 '-dithio-Whitfield's ointment and halogenating agent prepared in reaction 2,2 '-the two benzoyl halogen of dithio-; 2,2 '-the two benzoyl halogen of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with L-Isoleucine prepared in reaction [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid; [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid and halogen oxidant prepared in reaction [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.
In an embodiment preferred, halogenating agent is a thionyl chloride.
In another embodiment preferred, 2,2 '-the two benzoyl halogen of dithio-are 2,2 '-the two Benzoyl chlorides of dithio-.
Alkali is sulfonic acid hydrogen sodium in another embodiment preferred.
Halogen oxidant is chlorine, bromine or iodine in an embodiment preferred.
In a more preferred embodiment, halogen oxidant is a bromine.
The present invention also provides preparation [S-(R *, R *)]-method of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid, it comprises 2,2 '-dithio-Whitfield's ointment and thionyl chloride prepared in reaction 2,2 '-the two Benzoyl chlorides of dithio-; 2,2 '-the two Benzoyl chlorides of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and sodium bicarbonate with at least two equivalent L-Isoleucine prepared in reaction [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid; [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid and bromine and acetic acidreaction preparation [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.
The present invention also provides preparation [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid, it comprises 2,2 '-dithio-Whitfield's ointment and halogenating agent prepared in reaction 2,2 '-dithio-pair benzoyl halogen; 2,2 '-the two benzoyl halogen of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with L-Isoleucine prepared in reaction [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid.
In another preferred embodiment of this method, halide reagent is a thionyl chloride
In another preferred embodiment of this method, 2,2 '-the two benzoyl halogen of dithio-are 2,2 '-the two Benzoyl chlorides of dithio-.
In another preferred embodiment, alkali is sodium bicarbonate.
The present invention also provides preparation [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid, it comprises 2,2 '-dithio-Whitfield's ointment and thionyl chloride prepared in reaction 2,2 '-dithio-pair Benzoyl chlorides; 2,2 '-the two Benzoyl chlorides of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with L-Isoleucine prepared in reaction [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid.
Detailed Description Of The Invention
The invention provides according to following reaction process I preparation [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid and [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid.The reaction process I
Figure A9619900900062
In the synthetic first step, by halogenating agent and 2,2 '-the dithio-salicylism reaction, can be from Aldrich, Milwaukee, Wiswnsin buy 2,2 '-the dithio-Whitfield's ointment, be transformed into its corresponding carboxylic acid halides-2,2 '-dithio-pair benzoyl halogen.Those skilled in the art are familiar with the conversion of acid-carboxylic acid halides.Suitably the example of halogenating agent includes but not limited to: thionyl chloride, phosgene, phosphorus trichloride, phosphorus pentachloride, and phosphorus tribromide.
In preferred embodiments, halogenating agent is a thionyl chloride, and carboxylic acid halides is an acyl chlorides.Term " halogen " comprises chlorine, bromine, fluorine and iodine.
In general, under inert atmosphere, in aprotic solvent, react.The example of suitable aprotic solvent includes but not limited to toluene, heptane, hexane, and acetonitrile.In an embodiment preferred, solvent is a toluene.2,2 '-the two benzoyl halogen of dithio-can be by gained, and promptly the crude product form is used for the step down, or available those skilled in the art's well-known process purifying.
At synthetic second in the step, 2,2 '-the two benzoyl halogen of dithio-generate [S-(R with the reaction of L-Isoleucine in the mixture of tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid.With the solution of tetrahydrofuran (THF) (THF) or THF and alkali than other solvent that is used for generating acid amides from chlorine and a-amino acid more high yield, high purity and hypotoxicity, low carinogenicity.And, be not to be conspicuous with THF as solvent, because the L-Isoleucine only has limited solubleness in THF, and THF is decomposed by the hydrogenchloride that produces in the reaction usually.In addition, this is synthetic and do not require a-amino acid is carried out any protection/deprotection steps and is difficult for optically-activeization, makes product optical activity height.[S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid can be used for the following step by gained maybe can be by the repurity of those skilled in the art's well-known process.Suitably the example of alkali includes but not limited to sodium bicarbonate, yellow soda ash, salt of wormwood, and saleratus.Preferred bases is a sodium bicarbonate.
In synthetic the 3rd goes on foot, [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid and halogen oxidant prepared in reaction [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.In this step, [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-disulfide linkage of 3 methylvaleric acid is oxidized, and the cyclisation of intermediate sulfenyl bromine forms [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.Suitable halogen oxidant includes but not limited to bromine, chlorine and iodine.In an embodiment preferred, halogen oxidant is a bromine.
Generally should stir [S-(R along with adding halogen oxidant in the step at synthetic *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-slurries of 3 methylvaleric acid in acetate.After the stirring, filter the precipitation that is become.Product directly precipitates from reaction mixture with high pure state.
Afterwards, by using ether, preferable methyl tertbutyl ether and water abstraction purification precipitate, and it is [S-(R *, R *)]-crude product of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.Merge and concentrated ether extraction liquid, obtain oil.This oil is dissolved in the ether, adds heptane then.Filter, clean and dry gained precipitation, obtain [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.
Following embodiment is used to illustrate specific embodiments of the present invention, and they do not limit the present invention in any way, and comprises claim.
Embodiment 1
2,2 '-the two Benzoyl chlorides of dithio-
In 2 liter of three neck round-bottomed flask that mechanical stirrer, thermometer and band nitrogen inlet reflux exchanger are housed, add 2,2 '-dithio-Whitfield's ointment (120g), toluene (600ml), dimethyl formamide (1ml), and thionyl chloride (128g).Along with being stirred under the nitrogen, obtain clear yellow solution in 75 ℃ of heated mixt 21 hours.Heated solution to 80 ℃, on B by the diatomite filtration mixture.Clean filter cake with toluene (100ml), filtrate the transferring to of merging is equipped with in 2 liter of 3 neck round-bottomed flask of mechanical stirrer, thermometer and still head.Under the vacuum (5mmHg) hot solution is concentrated into the cumulative volume of 300ml, and the temperature of final material is 65 ℃.Add the new toluene (500ml) that steams in the mixture of reheat to 80 ℃, vacuum (5mmHg) hot solution down is concentrated into the cumulative volume of 300ml, adds new steaming toluene (500ml) again.Mixture reheat to 80 ℃, under the vacuum (5mmHg) with the last volume of solution concentration to 400ml.And the last batch of material temperature is 65 ℃, obtains yellow thickness oar liquid.Slurries are cooled to 10 ℃, stir 2 hours, filter on B.With cold toluene (100ml) filter wash cake,, obtain 120g (89.3%) lark crystalloid title compound at 40 ℃ to 45 ℃ vacuum oven inner dryings.mp156.2-157.9℃. 1H?NMRS(δ,CDCl 3,200MHz):8.41-8.37(m,2H,
Aromatics H), 7.78-7.74 (m, 2H, aromatics H), 7.60-7.51 (m, 2H, aromatics H), 7.43-7.34 (m, 2H, aromatics H).
Embodiment 2
[S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid
In 1 liter of 3 neck round-bottomed flask that mechanical stirrer, thermometer and band nitrogen inlet reflux exchanger are housed, add 2,2 '-the two Benzoyl chlorides (75.0g) of dithio-, L-Isoleucine (62.8g), sodium bicarbonate (55.3g) and THF (750ml).Along with being stirred under the nitrogen in 54 ℃ to 58 ℃ heating slurries 20 minutes, then in 60 ℃ to 65 ℃ heating 8 hours.Reaction mixture is cooled to room temperature, and slowly pour into mechanical stirrer is housed contain concentrated hydrochloric acid (60g), in the 3 liter of 3 neck round-bottomed flask that stirs the mixture fast of water (550ml) and methyl tertiary butyl ether (800ml).Order THF (150ml), methyl tertiary butyl ether (150ml) and 1 liter of flask of water (150ml) rinsing, rinsing liquid is added in 3 liters of flasks.Two-phase mixture is in stirring at room 30 minutes, allows its sedimentation then.Water below separating, (2 * 375ml 180ml) washes top organic phase with 3 parts of water.In the organic phase of quick stirring, add hexane (1060ml), obtain white thick slurry, in stirring at room 2 hours.Cross filter solid on B, (150ml) washes with hexane, at 65 ℃ of vacuum chamber inner dryings, obtains 105.9g (91.0%) title compound crude product.
To mechanical stirrer is housed, add title compound crude product (95.2g) and THF (1620ml) in 2 liter of 3 neck round-bottomed flask of still head and thermometer.Reflux lark solution steams flask with THF (1140ml).THF solution in the flask is cooled to room temperature, along with quick stirring, to wherein adding hexane (760ml), obtain white thick slurry, stirring at room 2 hours, on B, cross filter solid, (375ml) washes with hexane, at 67 ℃ of vacuum chamber inner dryings, obtains the solid shape title compound of 93.8g (98.5%) white, HPLC99.1% (according to area), fusing point 207-210 ℃.
1H?NMRS(δ,DMSO,200MHz):12.8-12.4(brs,2H,
CO 2H),8.72(d,J=8.3Hz,2H,NH),7.68-7.64(m,4H,
Aromatics H), 7.50-7.41 (m, 2H, aromatics H), 7.35-7.27
(m, 2H, aromatics H), and 4.39-4.32 (m, 2H, NCH), 1.95 (m, 2H, CH 3CH), 1.53 (m, 2H, CH 2), 1.31 (m, 2H, CH 2), 0.97 (d, J=6.9Hz, 6H, CH 3CH), 0.89 (m, 6H, CH 3CH 2).Embodiment 3
[S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid
In the 500ml of the application of sample funnel that mechanical stirrer, thermometer and band nitrogen inlet are housed three neck round-bottomed flasks, add [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid (25g) and acetate (163ml).The solution of dripping bromine (7.9g) in acetate (12ml) in the slurries that nitrogen stirs down lasts 15 minutes.Orange-yellow slurries stirred 4 hours under room temperature under nitrogen, filtered on B, and (2 * 50ml) wash, and obtain the title compound crude product of 31.7g wet with solvent with heptane.
In the 500ml separating funnel, load onto title compound crude product (31.7g), methyl tertiary butyl ether (69ml) and water (66ml).After the extraction, the water below separating is with methyl tertiary butyl ether (19ml) extraction.Merge the methyl tertiary butyl ether phase, water (50ml) is washed, and with rotatory evaporator it is condensed into oil under the vacuum (25mmHg), and last temperature of charge is 50 ℃.Oil kept 1 hour under 50 ℃ of vacuum, was dissolved in methyl tertiary butyl ether (80ml), filtered on B.Filtrate being transferred to is equipped with mechanical stirrer, in the 250ml three neck round-bottomed flasks of the addition funnel of thermometer and band nitrogen inlet.In heat (45 ℃) the methyl tertbutyl ethereal solution that stirs, add heptane (40ml).Muddy liquid adds heptane (19ml) again in 45 ℃ of maintenances 1 hour.Slurries are cooled to room temperature, are chilled to 10 ℃ then, stir 1 hour.Cross filter solid on B, (40ml) washes with heptane, 45 ℃ of vacuum chamber inner dryings 16 hours, obtains 19.3g (77.5%) title compound white crystals.Mp122-123 ℃; 1H NMRS (δ, CDCl 3, 200MHz): 8.81 (brs, 1H, CO 2H), 8.07 (d, J=7.8Hz, 1H, aromatics H), 7.67-7.54 (m, 2H,
Aromatics H), 7.44-7.36 (m, 1H, aromatics H), 5.28 (d,
J=9.4Hz,1H,CHN),2.28-2.23(m,1H,CHCH 3),
1.42-1.38(m,1H,CH 2),1.29-1.20(m,1H,CH 2),1.11
(d,J=6.8Hz,3H,CHCH 3),0.91(t,J=7.3Hz,3H,
CH 2CH 3)。
Embodiment 4
[S-(R *, R *)]-antiviral activity of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid
[S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid (embodiment 3) extrudes zinc from the nucleocapsid protein (NCp7) of HIV-1.NC albumen be kept to heavens in all retrovirus (South T., Blake P. waits the people, biological chemistry, 1990, be essential (Aldovini A. and Young R., Journal of Virology, 1990 29:7786) and to virus infection; 64:1920 and Gorelick R., Nigida S. waits the people, Journal of Virology, 1990; 64:3207).Zinc generally is fixed in the NC albumen by 1 or 2 zinc-finger.For HIV-1, exist 2 zinc finger pieces (Summers M., South T. waits the people, biological chemistry, 1990; 29:329) also specifically relevant with the PSI position on the viral RNA of controlling the viral RNA packing.Disturb this packing will cause non-infectious virion formation (Dannull J., Surovoy A. waits the people, EMBO, 1994; 13:1525).Shown already cause compound that zinc extrudes to the many cells cording have strong anti-HIV active and resist all retrovirus (Rice W., Schaeffer C. waits the people, nature, 1993; 361:473).
Developed the zinc of discharging from the HIV-1 NCp7 of purifying in order to monitoring based on the assay method of fluorescence.Fluorophore, N-(6-methoxyl group-8-quinolyl)-right-toluol sulfonamide (TSQ) increases fluorescent signal during the bonding zine ion in solution.With causing that drug incubation that zine ion is extruded contains the NCp7 protein of 2 zinc-dactylitic textures and 2 zine ions.The zinc that discharges with the TSQ chelating is monitored the fluorescence intensity that increases with respect to control group then.Mensuration is by following described carrying out: 10 μ m compounds are added among the 2.8 μ M NCp7 and 47 μ M TSQ in the buffered soln of 26 ℃ of 20 μ lpH7.4, last 90 minutes.Monitoring fluorescence time curve (excites: 355nm, emission: 400nm).Control group is the apo-NCp7 (no zinc) of the NCp7 of pastille and pastille not under condition determination.The fluorescence intensity (5.6 μ m) * 100 of extruding zinc divided by whole theoretical amount according to the actual fluorescence intensity that records is calculated zinc and is extruded percentage ratio.
For establishing [S-(R *, R *)]-the active pilot system of using of the cell anti-virus of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid is generally accepted and be generally used for this purpose in this area.For example, be used for people such as the active Weislow of the being determined as O.S. of assessing compound anti HIV-1 virus at J.Natl.Cancer Inst 1989; Adopted by American National cancer association described in the 81:577-586, document content is incorporated this paper into for referencial use at this.
The reagent of designing program and working in each stage in viral proliferation cycle to detect.Test relates generally to HIV killer T 4 lymphocytes.Small amount of H IV is added in the cell, needs two complete viral proliferation cycles at least so that obtain required cell killing effect.Interacting the active medicament of viral interference will protect cell to avoid born of the same parents with virion, cell or virogene product separates.System aspect several automatization with regulate a large amount of candidate agents and by common design to detect HIV (human immunodeficiency virus)-resistant activity.Yet, sex change or under culture condition rapid metabolic compound under this background, do not show activity.
Another pilot system that is used to estimate The compounds of this invention is called as the HIVH9 test.HIV H9 test cell line is measured and is suppressed the required inhibitor concentration of HIV-1 virus replication.In this system, viral growth is through a plurality of life cycles.Any inhibition of duplicating dynamics causes that all the propagation of virus descends with geometric ways.As a result, this test is to measure the sensitive method that compound suppresses HIV-1 virus replication ability.
H9 T-clone infects with HIV virus down in 0.01 superinfection (MOI) in batches, absorb after 2 hours, wash cell, be resuspended in RPMI-1640 (substratum that obtains easily well known to those skilled in the art)/10% foetal calf serum, and on 96 orifice plates, inoculate every hole 5 * 10 -3Individual cell.For cytotoxicity test, the double test board of the H9 cell that preparation is not infected.Serial dilution 1/3.16 medicine is transferred in the medium with 8 * concentration in methyl-sulphoxide (DMSO), is added in the substratum 3 parts of the same form then.Last DMSO concentration is 0.002 (0.2%).
Measure the propagation of virus with the room temperature measuring method, measure cytotoxicity with the XTT assay method back 7 days of infection.The XTT assay method is well known in the art, for example sees .J.Natl.Cancer lnst.1989; 81:577-586.By improved Borroto-Esoda and Boone, J.Virol, 1991; 65:1952-1959 carries out room temperature measuring and quantitative with the Molecular Dynamics Phosphoimager of band Imagequant software.By people such as improved Roehm at J.Immuno.Methods, 1991; The described method of 142:257-265 is carried out XTT mensuration, and quantitative with the MolecularDevices Thermomax plate reader of band Softmax software.
Data are transferred on the Microsoft Excel distribution plain film of analyzing usefulness by electronics.Calculate the room temperature measuring value that is equivalent to suppress 50% and 90% virus multiplication according to untreated control group.These are worth needed inhibitor concentration (IC to calculate generation from the data point of these room temperature activity avris with interpolation technique 50And IC 90).Be equivalent to 50% Cytotoxic XTT test value from untreated control group calculating.This is worth needed inhibitor concentration to calculate generation from the data point of these XTT value avris with interpolation technique.
Another pilot system that is used to measure antiviral activity claims to be the cem cell assay method.
Ratio with viral pair cell about 0.05 is exposed to HIV with T4 lymphocyte (cem cell system), and is placed on the 96 hole microtiter plates with the cellular control unit that does not infect.
Candidate's medicament is dissolved in methyl-sulphoxide (have in addition explanation except), in cell culture medium, dilutes 1:200 then.Other diluent of preparation before being added to the equal volume amounts substratum that contains infection or non-infected cells (half-log 10).
In 5% carbon dioxide atmosphere, hatch culture 6 or 7 hours in 37 ℃.With tetrazolium salts, XTT be added to institute porose in, hatch culture, allow viable cell dyeing first color, J.Natimal Cancer Institute., 1989; 81:577-586.The single hole of spectroscopic analysis is with the generation of quantitative first , and microscopic examination determines that to detect viable cell protection is active in addition.
The non-infected cells that the cell of the virus infection of trial drug and medication are handled on same flat board and other suitable control group (untreated cells infected and untreated non-infected cells, not celliferous pastille hole etc.) contrast.Compare observed data with other test of being done simultaneously, measure active.
The result of compound in the above-mentioned zinc extrusion test of following table 1 expression.Assessing compound causes the ability (being expressed as the % with respect to control group) that zinc is extruded from nucleocapsid protein NCp7.Table 1 is from the zinc-finger of HIV-1 nucleocapsid protein (NCp7)
In extrude zinc situation embodiment compound extrude rate with respect to the zinc of control group
3 30
Following table 2 shows the data sheet 2 anti-HIV activity (cem cell assay method) of embodiment 3 compounds when H9 and the evaluation of cem cell assay method
The compd E C of embodiment 50(μ m) aTC 50(μ m) b
3 14＞100a. protection cell is avoided the effective concentration of pathological changes caused by virus effect.B. the poisoning concentration that suppresses cell 50% growth with respect to control group.

Claims (12)

1. one kind prepares [S-(R *, R *)]-method of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid, this method comprises
A. make 2,2 '-dithio-Whitfield's ointment and halogenating agent reaction, preparation 2,2 '-the two benzoyl halogen of dithio-;
B. make 2,2 '-the two benzoyl halogen of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with the reaction of L-Isoleucine, preparation [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid, and
C. with [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid and halogen oxidant reaction, preparation [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.
2. the process of claim 1 wherein that halogenating agent is a thionyl chloride.
3. the process of claim 1 wherein 2,2 '-the two benzoyl halogen of dithio-are 2,2 '-the two Benzoyl chlorides of dithio-.
4. the process of claim 1 wherein that alkali is sodium bicarbonate.
5. the process of claim 1 wherein that halogen oxidant is a chlorine, bromine or iodine.
6. the method for claim 5, wherein halogen oxidant is a bromine.
7. one kind prepares [S-(R *, R *)]-method of 3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid, this method comprises
A. make 2,2 '-dithio-Whitfield's ointment and thionyl chloride prepared in reaction 2,2 '-the two Benzoyl chlorides of dithio-;
B. make 2,2 '-the two Benzoyl chlorides of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with at least two equivalent L-Isoleucine prepared in reaction [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid;
C. make [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid and bromine and acetic acidreaction preparation [S-(R *, R *)]-3-methyl-2-(3-oxo-3H-benzo [d] isothiazole-2-yl) valeric acid.
8. one kind prepares [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid, this method comprises:
A. make 2,2 '-dithio-Whitfield's ointment and halogenating agent prepared in reaction 2,2 '-the two benzoyl halogen of dithio-;
B. make 2,2 '-the two benzoyl halogen of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with the reaction of L-Isoleucine, preparation [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid.
9. the method for claim 8, wherein halogenating agent is a thionyl chloride.
10. the method for claim 8, wherein 2,2 '-the two benzoyl halogen of dithio-are 2,2 '-the two Benzoyl chlorides of dithio-.
11. the method for claim 8, wherein alkali is sodium bicarbonate.
12. one kind prepares [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-method of 3 methylvaleric acid, this method comprises:
A. make 2,2 '-dithio-Whitfield's ointment and thionyl chloride reaction, preparation 2,2 '-the two Benzoyl chlorides of dithio-;
B. make 2,2 '-the two Benzoyl chlorides of dithio-in tetrahydrofuran (THF) or tetrahydrofuran (THF) and alkali with the reaction of L-Isoleucine, preparation [S-(R *, R *)], L-2-{2-[2-(1-carboxyl-2-methyl butyl formamyl) phenyl connection alkylsulfonyl]-benzamido }-3 methylvaleric acid.
CN96199009A 1995-12-15 1996-11-27 [S-(R*,R*)]-3-methyl-2-(3-oxo-3h-benzo[d] isothiazol-2-yl] pentanoic acid and [S-(R*,R*)], L-2-(2-(2-(1-carboxy-2-methylbutyl carbamoyl) phenyldisulfonyl) benzoyl-amino)-3-methylpentanic acid Pending CN1208407A (en)

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US60/008,745 1995-12-15

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CN (1) CN1208407A (en)
AU (1) AU1083597A (en)
BG (1) BG102470A (en)
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CA (1) CA2233553A1 (en)
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IL (1) IL123903A0 (en)
NO (1) NO982709L (en)
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SK (1) SK81798A3 (en)
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GB9304903D0 (en) * 1993-03-10 1993-04-28 Zeneca Ltd Compounds,preparation and use
US5734081A (en) * 1994-08-05 1998-03-31 Warner-Lambert Company Arylthio compounds
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NO982709D0 (en) 1998-06-12
JP2000505067A (en) 2000-04-25
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HUP9902043A2 (en) 1999-11-29
SK81798A3 (en) 1998-11-04
NO982709L (en) 1998-06-12
BG102470A (en) 1999-08-31
KR20000064403A (en) 2000-11-06
HUP9902043A3 (en) 2000-03-28
AU1083597A (en) 1997-07-14
CA2233553A1 (en) 1997-06-26
PL327135A1 (en) 1998-11-23
NZ323803A (en) 1999-02-25
CZ182498A3 (en) 1998-09-16
EP0874835A1 (en) 1998-11-04
WO1997022598A1 (en) 1997-06-26
ZA9610479B (en) 1998-06-09
BR9612020A (en) 1999-06-29

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