SK81798A3 - Method of making [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]benzoyl- -amino}-3-methylpentanoic acid - Google Patents

Abstract

The present application discloses a synthesis of [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

Ob 1 a s t e c: h n 1 k y

The invention relates to the synthesis of the acid CS-CR **, R **)]

-3-methyl-2-C3-oxo-11-benzol-11 (isothiazol-2-yl) pentanoic acid and CS (CR ^x , R ^x )], L-2-C2-E 2-Cl (2-methylbutylcarbainoyl) phenyldisulphonylbenzoylamino] -3-methylpentanoic acid;

D o t e r i i Si. s t t e c h n i k v

No acid C-R ^*, R ^*)] -3-methyl-2- (3-oxo-3H-benzo honor izotiazol- • 2-yl 1) -pentanoic ..je useful as antiviral agents and can be used to treatment of patients infected with the virus I l-ITV. Acid C SC R ^x, R ^x) l, L-2-E2-E2-C-carboxy-2-methylbutylcarbamoyl carbamoyl) feriyldisulfony 1] -benzoylamino-3-methylpentanoic ... is an intermediate in the synthesis of ES <R ^x , R ^x )] - 3-methyl-2- [3- [3- (3 H) -benzo] -disole: i. azo 1 - 2 - y 1) pentan e. It may be useful in the treatment of bacterial and viral infections, including the treatment of patients infected with HIV. See, for example, U.S. Patent 5,463,122. Thus, it would be useful to have a simple, high-yielding process for the synthesis of acid S- (R **, R ^x )] -3-methyl-2-C3-oxo-3H-benzol] isothiazol-2-yl) pent ES - CR ^x , R ^x ) J, L - 2 - C 2 - E 2 - C 1 - (carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl] benzoylamino-3-methylpentanoic acid.

F ³ paragraph outside cut,

The present invention provides a process for the preparation of 1S- (R ^x , R ^x ) -3-methyl-2-C3-oxo-3 H -benzo [c] isothiazol-2-yl) pentanoic acid, which comprises contacting 2,2'- dithiosalicylic react with a halogenating agent to form 2,2'-dithiobisbenzoyl halide;

reacting 2,2'-dithiobisbenzoyl halide with

L-isoleucine in tetrahydrofuran or tetrahydrofuran and a base to form of the LS-CR ^*, R ^*>], L-2-I 2-L2-C-carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl] benzoylainino D - 3-methylpentanoic acid; and mentioning the acid (S-CR ^X , R ^x )], L-2- <2- (2-GL-carboxy-2-methylbutyl-carbonyl) 1.) phenyl disulfonyl-1-benzoyl, and in. amino} - 3-methyl-pent 1 and new J acid with a halogen oxidizing agent to form a C SC of R ^*, R ^*)] -3-methyl-2 <3-oxo - 3 H benzoĽdľl isothiazol-2 yl) pentane

In a preferred embodiment, the halogenating agent is a thiocyanate.

In a further preferred embodiment, it is 2,2 ' -dithiobisbenzoylhalogene, 2,2 ' -dithiobenzoyl halide.

At sodium. another i advantageous r e a 1 i c: i i. it is based on h y d r o g e n h h i n i t a n At another i advantageous is halogen oxidising agent chloro, b r č) m or Iodine. At another i advantageous r ~ e a 1 i z i j. J e h a 1 o g e n o m oxidising agent bromo.

The invention provides a particular aspect. pravykyse 1 iny ES - CR ^x , R ^x )] - 3-Methyl 1 - 2 - C 3 · - oxo - 3 H - benzo Cl Cl isothiazole 1 - 2 - y 1) pentanone J discloses the reaction of 2,2 ' -dithiosalicylic acid with thiones 1 c: h 1 ori to form 2, 2 ' -citiobisbenzoyl c: h 1 oride; introducing 2,2'-dithiobisbenzoylchloride by reacting with at least two equivalents of L-isoleucine in tetrahydrofuran or tetrahydrofuran and sodium bicarbonate to form L 1 R (R ^x , R ' ^x )], L-2-C2-E 2 -Cl -carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl-1-benzoylamino-3-methylpentane; and reacting the SC t R ^*, R ^*)], L-2-I 2 L-2-C-carboxy-2-methylbutylcarbamoyl) fenyldisulf sulfonyl] benzoylamino} -3-methylpentanoic acid with bromine and acetic acid formation of ES-CR ^x , R ^x )] - 3-methyl-2-C3-oxo-3H-benzo [eta] &lt; 1 &gt;

The invention also provides a method of preparing a polymer

E SC R ⁴ *, R ^x 3 3, 1- 2 -12- (2-C (1-carboxy-2-methylbutylcarbamoyl) phenyldis) phenoxy 1.3 henzoy 1. am j. 3 - 3 - ni t? ty 1 pentan e. j, which comprises 2, 2'-dithiosalicylic acid with a halogenating agent to form 2,2 &apos; auve cl en 1. e 2, 2 '- d i. of thiobisbenzoyl halide to react with L-isoleucine in tetrahydrofuran or tetrahydrofuran and base to form an acid

ES-CR ^x , R ^{x 13} , L.-2-C2-E2-C (1-carboxy-2-methylbutylcarbamoyl) feriyldisulfonylamine, 3-benzo-1-amino-3-methylpentanoic acid; .

R F ³ a further preferred aspect of the invention, the halogenating agent is thionyl chloride.

In a further preferred embodiment of the method according to the invention, it is 2, 2 ' - 1 ' d om 2,2 '- cl i t i o b 3. s b e n o y 1 c h 1. o r i d.

3. A further preferred embodiment of the process according to the invention is as described above.

The invention also provides a process for the preparation of C 5 - (R ^x , R ^x 3 3, L-2- <2-E 2 - (1-carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl-3-benzoylamino) -3-3-methylpentanoic acid, which comprising reacting 2,2'-dithiosalicylic acid with thionyl chloride to form 2,2'-dithiobisbenzoyl chloride; auve of 2,2'-cli-thiobisbenzoyl chloride by reaction with L-isoleucine in tetrahydrofuran or tetrahydrofuran and a base to form an acid

(S-CR ^M ', R **) 3, L-2- <2- E 2-C (1-carboxy-2-methylbutylcarbamoyl) phenyl] benzyl 13 benzoylamino 3-methyl-3-pentyl nove J.

1. ES-CR ^x , R ^x ) 3- (3-methyl-2-C3-oxo-3 H -benzo [d] isothiazol-2-yl) pentanoic acid and E SC R ^x , e. R ^x ) 3, L-2 (C 2 -C 2-C 1 -carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl 13 henzoyl amine No 3 - 3-methylpentanone J which occurs It is according to the following Reaction Scheme I.

Reaction Scheme I

co ₂ h co ₂ h sat ₂ or h

TI-IF- or

THF / NaHCO 3, v

Br _and acetic acid

In the first step of the synthesis, the acid 2, 2 ' - di-thiosalicylic acid from A1 company, 1 * 1 ilwaukee, U iscons in, convert 1 and to 1 h auride, 2,2 '- dithiobisbenzoy 1 ha 1. ogenide results in: n i ni c acid 1. iny

2, 2 &apos; -dithiosalicyl is reacted with a halogenating agent. The method for converting an acid to a halide is well known to those skilled in the art. Examples of suitable halogenating agents are, for example, thionyl chloride, phosgene, phosphorus trichloride, phosphorus trichloride and phosphorus tribromide.

In a preferred embodiment, the halogenating agent is thionyl chloride and the acid halide is acid chloride. The term halogen as used herein includes chlorine, bromine, fluorine and iodine.

The reaction is usually carried out under an inert atmosphere and an aprotic solvent.

Examples of suitable aprotic compounds are toluene, heptane, hexane and acetonitrile. In a preferred embodiment, the solvent is about 1%. 2, 2 '- · i. It can be used in the next step without prior purification, i.e. in a crude form, or can be purified by methods well known to those skilled in the art.

In the second step of the synthesis, the 2,2'-dithiobisbenzoyl halide is reacted with L-isoleucine in tetrahydrofuran or a mixture of tetrahydrofuran and a base to form L 2 -CR ^x , R **> 1, L-2 - <2-E 2 - C 1 -carboxy-2-methylbutylcarbamoyl) phenyl disulfonylphenylbenzoylamino} -3-methylpentane. The use of tetrahydrofuran (THF) or THF solution and a base gives higher yields and a higher purity of the product. Another advantage of tetrahydrofuran is that it is less toxic and carcinogenic than the other solvents used in the preparation of amides from chlorides and N-amino acids. The use of tetrahydrofuran as a solvent is not obvious since L-isoleucine has only limited solubility in THF and THF is usually decomposed by the hydrogen chloride formed during the reaction. In addition, the synthesis of the invention requires no steps to provide protection and deprotection of the .alpha.-amino acid and is less favorable for racemization, so that it provides products having excellent optical activity. [S- (R *, R ^M ) 3, L-2 - {2- [2- (2-Cl-carboxy-2-methylbutyl) carbamoyl] phenyldisulfonyl] benzoyl a. No 1-3-methylpentanone can be used in the next step as such or purified by methods well known to those skilled in the art. Examples of suitable c: hb are included. sodium citrate, sodium carbonate, potassium carbonate, and potassium bicarbonate. It heats the base with hydrogeoic acid.

characterized the third step of the synthesis, the acid C SC R ^x, R ^x), 3, L-2-C2-C 2-C 1 to carboxylic acid isopropyl-2-methylbutylcarbamoyl carbamoyl) ťenyldisulfony13benzoylamino 1-3-methylpentanoic duty shall reacted with a halogen oxidizing No INID 1. After the formation of the acid (S - (R ^x , R ^x ) -3-methyl-2-C 3 -oxo-3 H -benzo [d 3 isotlazol-2-yl) pentanoic acid. In this step, the disulfide bond of the acid [SC R ^x , R ^x ] 3, L-2- <2-E 2 -C (1-carboxy-2-methylbutylcarbamoyl) phenylene isobutyl 1.3 benzoyl amine i. No 3 - 3 - methylpentanoueoxide and sulfenyl bromide, formed as an intermediate, was cyclized to give SCSC R **, R ^M 3 -3-methyl-2-C 3 -oxo-3H-benzoE d3 isothiazol-2-y 1) pentanone J. C h a r i c h e c h e c h e c h e n i n t i n i n t i n i n i n t i n i n i n i n i n i b i n bromine, chlorine and iodine. At. In a preferred embodiment, the halogen oxidizing agent is bromine.

In this step of the synthesis, a slurry of the SC E R ^*, R ^*) J, L-2-I 2 - 2 E-C-carboxy-2-methylbutylcarbamoyl) phenyl those enylclisulf 1.3 benzoylamino 3-3-methylpentanoic acid and acetic acid, with stirring, adds a halogen oxidizing agent. After stirring, the precipitate formed is filtered off. The product precipitates directly from the reaction mixture in a high degree of purity.

Thereafter, the precipitate which is crude ES-CR ^x , R ^x ) 3-3-methyl-2-C3-oxo-3,1-1-benzo (d 3 isothiazol-2-yl) pentanoic acid is purified, by extraction with ether, preferably methyl t-butyl ether and water. The ether extracts were combined and concentrated to an oil. The oil was dissolved in ether and heptane was added to the resulting solution. The resulting precipitate was filtered and washed. and drying gives the acid SC E R ^*, R **) 3-3-metyl.-2 C-3-oxo-3H-benzo CL3 isothiazol-2-yl) pentanoic acid.

Ί

The following examples are illustrative only and are not to be construed as limiting the scope of the invention, which is clearly defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

2, 2 '- d i thi o>> s b e n z o y 1. c h 1 o r 1 d

Two-liter three-necked round-bottomed flask equipped with a mechanical stirrer, a thermometer and a reflux condenser with nitrogen inlet for 2, 2 ' Cl ml) and thionyl chloride (C128 g). The mixture was heated to 75 ° C with stirring and kept at this temperature under nitrogen for 21 hours to give a clear, yellow solution. The resulting solution was heated to 80 ° C and filtered through diatomaceous earth on a Buchner funnel. The filter cake was washed with toluene (100 mL) and the combined filtrates were transferred to a 2 L 3-neck round bottom flask equipped with a mechanical stirrer, thermometer, and distillation head. The hot solution was concentrated to a total volume of 000 ml under a vacuum of 0.5 mm Hg, the final batch temperature being 65 ° C. Fresh toluene (500 mL) was added to the mixture, which was reheated to 80 ° C. The hot solution was concentrated under vacuum (666.6 Pa) to a final volume of 300 mL and a fresh portion of toluene (500 mL) was added to the concentrated solution. The mixture was reheated to 80 ° C and the solution was concentrated to a final volume of 400 ml under vacuum (666.6 Pa) at a final batch temperature of 65 ° C. A thick yellow suspension was obtained, which was cooled to 10 ° C, stirred for 2 hours, and filtered through a Buchner funnel. The filter cake was washed with cold toluene (100 mL) and dried in a vacuum oven at 40 ° C to 45 ° C to give 120 g (89.3%) of the title compound as pale yellow crystals;

mp 156.2-257.9 ° C;

¹ H NMR (δ, CDCl ₃ , 200 ΓΙΗζ): 4.81 to 8.37 (m, 2H, aromatic hydrogen), 7.78 to 7.74 (m, 2H, aromatic hydrogen), 7, 60 to 7 51 (m,

2.Η, aromatic hydrogen), 7.43 to 7.34 (m, 2H, aromatic hydrogen).

Example 2

Β SC R ^M , R **) 11,1,1- 2- (2-β 2 (1-carboxy-2-methylbutylcarbamoyl) phenyl disulfonate 1, Z1 benzyl amino> -3-methyl pent á new á

One-liter three-necked round-bottomed flask equipped with a mechanical stirrer, thermometer and reflux condenser with inlet of nitrile cassanapine 1 and 2,2'-dithiobishenzoyl chloride (C 7 5,0 g), L-isoleucine C (52 , 8 g), sodium bicarbonate <55.3 g) and THF (750 ml). The suspension was heated to 54 ° C to 58 ° C under nitrogen for 20 minutes under continuous stirring, and then heated to 60 ° C to 65 ° C for 8 hours. The reaction mixture was cooled to room temperature and then poured into a 3 L round bottom flask equipped with a mechanical stirrer containing a rapidly stirred mixture of concentrated hydrochloric acid (60 g), water (550 ml) and methyl tert -butyl ether (800 ml). The one liter flask was successively rinsed with TI-IF (1.50 mL), methyl t -butyl ether (150 mL) and water (150 mL) and the rinses were added to a 3 L flask. The biphasic mixture was stirred for 30 minutes at room temperature and then allowed to settle. The lower aqueous phase was separated and the upper organic layer was washed with three portions of water (2 x 375 mL, 1.80 mL). Hexane (1060 mL) was added to the rapidly stirred organic organic layer to give a thick, white suspension which was stirred at room temperature for a further 2 hours. The solids were filtered on a Buchner funnel, washed with hexane (150 mL) and dried, in a vacuum oven at 65 to give 105.9 g (91.0%) of the crude title compound.

A 2 L round bottom flask equipped with a mechanical stirrer, a thermometer and a distillation head was charged with the crude title compound (95.2 g) and THF (1620 mL). The pale yellow solution was heated to reflux and THF (1140 mL) was distilled from the flask. The THF 'solution in the flask was cooled to room temperature and hexane (760 mL) was added to the cooled solution with vigorous stirring to give a thick white suspension which was stirred at room temperature: additional: 2 hours. The ace solids were filtered on a Buchner funnel, washed with hexane (375 mL) and dried in vacuum at 67 ° C to give 93.8 g ¢ 98.5%).

title: j i compounds in a: white ... and pe ex: J substances; l-IPLC 99 1% Cz area); temperature mp 207 to. 210 ° C; H 1 N 1 * 1 R C 8, DMSO, 200 MHz) .- 12, 8 until 12.4 (wide s, 21-1, CO _; : .H) 8, 72 C d, J = 8.3 Hz, 21-1, NH) 7, 68 : up to 7, 64 Cm, 4H, aromatic H), 7.50 to. 7.41 cm, 2H, aromatic hydrogen), 7, 35 up to 7.27 (m, 2. I I, a r o m a t i c: k o n o k) , 4, 39 to 4 , 32 Cm, 21-1, NCII) 95 C m 21-1 CH ₃ CH), 1.53 Cm, 2H, CH _a ), 1, 31 cm. 2H, CH3), 0.97 C d, J = 6.9 Hz, 6H, CH ₃ CH), 0.89 cm 3, 6H, ch ₃ c H- _a ).

Example 3

Ky se: 1 ina S - CR *, R ^K - 1 - 3 - methyl 1 - 2 - C 3 - oxo - 3 H - benzo d 1 isot, azo 1 - 2 - y 1) - pentane

In a five-liter three-necked three-necked round-bottomed flask equipped with a mechanical stirrer, a thermometer and a nitrogen inlet funnel was introduced Ľ SC R **, R **) J, L-2- <2-C 2 -C 1 -carboxy-2 - methylbutylcarbamoyl) phenyldisulfonylphenyl 11 benzoylamino amino-3-methylpentanoic acid (25 g) and acetic acid (163 ml). A solution of bromine (7.9 g) in acetic acid (12 ml) was added dropwise to the stirred suspension under nitrogen over 15 minutes. The orange suspension was stirred under nitrogen at room temperature for another 4 hours, filtered on a Buchner funnel and washed with heptane (2 x 50 mL) to give 31.7 g of crude solvent-moistened solution. J z 1 u n e e: n i n y.

A 500 mL separatory funnel was charged with the crude title compound (31.7 g), methyl t-butyl ether (69 mL) and water (66 mL). After extraction, the lower aqueous layer was separated to give 1: 1-methyl-1-methylbutyl ether.

Cl ml).

The layers were combined, washed with water (50 ml) and concentrated by rotary evaporation under vacuum (666.6 Pa) to a pH of 1, whereby The final charge was 50 ° C. The oil was kept under vacuum and at ° C for an additional 1 hour, dissolved in methyl t-butyl ether (80 mL) and filtered through a Buchner funnel. The filtrates were transferred to a 250 ml round bottom flask equipped with a mechanical stirrer, a thermometer, and a nitrogen inlet funnel.

To the warm (45 ° C) stirred methyl t-butyl ether solution was added heptane (40 mL). The liquid solution was maintained at 45 ° C for 1 hour, after which time another portion of heptane (19 mL) was added. The suspension was cooled to room temperature, then to 10 ° C and stirred for 1 hour. The solids were filtered on a Buchner funnel, washed with heptane (40 mL) and dried for 16 hours in a vacuum oven at 45 ^° C (19.3 g, 77%) of the title compound as white crystals;

m.p. Mp 2.30 ° C;

Ή NMR -C 6, CDC1 _3, 200 MHz); 8.81 (broad s, 1H, CO-JI), 8.07 (d, J = 7.8 Hz, 1H, aromatic hydrogen), 7.67 to 7.54 cm, 2.H, aromatic hydrogen), 7.44 to 7.36 m, 1H, aromatic H), 5.28 d, J = 9.4 LLZ, II-l, CHN), 2.28 to 2.23 m, 1H, CH CH _3), 1.42 to 1.38 m, 1 H, Chk), 1.29 to 1.20 m, III, CH.), 1.11 d, J = 6.8 Hz, 3H, CH CH _3),

0.91 t, J = 7.3 Hz, 31-1, CH _{and CH3).}

Example 4

A nt j. Russian act i. vitous acid 1 inv L S - CR ^x , R ^x )] - 3-methyl 1 -2 - C 3 - oxo - 3 H

- benzol (1 isothiazol-2-yl) pentanoic acid i

C SC R ^x , R ^x ) 1 -3-Methyl-2-C 3 -oxo-3H-benzo [d] isothiazol-2-yl) pentanoic acid C Example 3) causes zinc extrusion from the nucleocapsid protein CNCp7) HIV- first MC protein It is highly conserved among all retroviruses CSouth T., Blake P. et al., Bococ. 29: 7786) and is essential for CAldovini A. and Young R., J. Virology, 1990; 64: 1920 and Gorelick R., Nigida S. et al., J. Virology, 1990; 64: 3207). Zinc is usually held in MC proteins by using one or two zinc fingers. In the case of HIV-1, two zinc fingers CSummers M., South T. et al., B1ochemistrv, 1990; 29: 329), which specifically bind to the PSI site on the viral RNA that controls the packaging of the viral RNA. The interference of this wrapping causes. vzni l <ne i. cf. cv: hvironov CD annu 1.1 J., S urovoy A. et al., EMBO, 1994; 13: 1525). As has been shown in the past, compounds that cause zinc extrusion have potential anti-HIV activity in a number of cell lines against all retroviruses (Rice N., Schaeffer C. et al., Mature, 1993; 361: 473).

To monitor the zinc ejection from purified HIV-1 NCp7, a f r u r e c e r e c t e c t e e was developed. F1. N-C 6 -tethox y -8-c-hin-1-y 1) ρ-toluenesulfonamide (CTSO), another increased fluorescent signal after binding of zinc ion in solution. NCp7 protein, containing two zinc fingers, and two zinc ions were incubated with the zinc ion extrusion active ingredient. Zinc release was then sequestered by TSQ and increased fluorescence was monitored and compared to a control fluorescence signal. The assay described was performed as follows: 10 µL compound was added to 2.8 µM NCp7 and 47 µM TSQ in 20 µl buffer (pH 7.4) at 26 ° C for ninety minutes. Fluorescence Citation of 355 nm (400 nm emission) was monitored over time. Control measurements were performed using NC? 7, under test conditions without drug and apo NC? 7 (Zn) without drug. The percent Z n extrusion was calculated by dividing the currently measured fluorescence by the fluorescence of all theoretically extruded zinc (5, 6 at 1 * 1) x 100.

Assay systems that have been used to determine the cellular antiviral activity of CS- (R ^M , R ^M ) -3-Methyl-2- (C3-oxo-3 H -benzothiazolothiazol-2-yl) pentanoic acid are well known in the art and commonly used for these purposes. For example, the system used by IJ.S. was used to determine the activity of the HIV virus compound. National Cancer Institute and described by Weislow C). S. et al. J. Mati. Cancer Inst., 1989; 81: 577-586.

This procedure is designed to detect agents acting at any stage of the viral reproductive cycle. The assay involves essentially killing T4 lymphocytes with HIV. Small amounts of I-IIV are added to the cells and at least two complete viral reproductive cycles are necessary to achieve the desired cell death. Agents that interact with virions, cells or viral gene products to disrupt virus activity will protect cells from cytdysis. The system is automated to accommodate a greater number of candidate agents, and is typically designed to detect an anti-HIV agent. However, compounds that degenerate or are rapidly metabolized under the culture conditions used will not show activity in this assay.

Another assay system used to test the compounds of the invention is referred to as the HIV H9 assay. The HIV H9 cell assay measures the inhibitory concentration required to suppress ‘HIV-1 viral replication. In this system, viral growth takes place over a repeated life cycle. Any suppression of replication kinetics results in a geometric reduction in virus production. This assay is a sensitive means of measuring the ability of a compound to inhibit HIV-1 viral replication.

The H9 T cell line represents a virus infected with a multiple CM (JI) virus infection of 0.01. After two hours of absorption, the cells were washed and resuspended in RPMI-1640

readily available which is an expert in the field-:

well known) / 10% calf embryo era and inoculated (b cells / well per well).

another plate with uninfected H9 cells was prepared for the cytotoxicity assay. The active ingredients were sequentially: diluted 1: 3.16 in dimethylsulfoxide (CDMSO), transferred to medium at 8x concentration in three culture samples. The final DMSO concentration was 0.002

CO, 2%).

Viral production was measured by room temperature assay and cytotoxicity was measured by XTT assay 7 days post infection. XTT Assay It is known to those skilled in the art. See, for example, J. Natl. Cancer Inst. 1989; 81: 577-586. The room temperature test was performed as a modification of the Horroto-Esody and Boona method, J. Virol., 1991; 65: 1952-1959 and qualified by Molecular

Dynamics Phosphoimager with software was performed as a modification of the method

Methods., 1991; 142 = 257-265 and

Imageguant. XTT test up

Roehni et al., J. Immuno.

it was quantified by using a reader molecule

Deviees

Thermomax with software

Softmax.

The data was electronically transferred to a spreadsheet

Microsoft Excel where they were analyzed. Room temperature test values corresponding to 50% and 90% inhibition of viral production.

were calculated from untreated control samples. Concentration of inhibitor required to

1C < _{? a),} the interpolation 1 ova izd s 1 t, to obtain these values (IC 's and _H adjacent to these activities, at room temperature. The XTT assay values equivalent to 50% cytotoxicity are calculated from the untreated controls.

The inhibitory activity required to obtain this value was interpolated from data adjacent to these XTT values.

Another test system.

used to determine

P r o t i v r u s o e. The system is referred to as C EM cell

T4 lymphocytes (CEM cell line) were exposed to H1 in the virus, at approximately the ratio of: 0.05 virus per cell, and placed with them. n f i. k o n t m o k o n t r o 1 n m. b u n i d i d i d i d i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n i n t.

The candidate reagent was dissolved in dlmethyl.sul.foxl.de (unless otherwise specified) and then diluted at a ratio of: 1 = 200 in the cell culture medium. Further dilutions were made (half-log ₁₀ ) before addition of an equal volume of medium containing the contents infected or uninfected.

The cultures were incubated at 37 ° C in an atmosphere of 5% carbon dioxide for 6 or 7 days. XTT tetrazolium salt was then added to all wells and the cultures were incubated to allow the surviving cells to develop a formazan color, J. Naional Cancer

Institute. 1989 81: 577 to. 586. Individual; Wells were analyzed spectrometrically to determine the amount of formazan production and examined microscopically to delegate surviving cells. This detection was a confirmation of protective activity.

Virus-infected cells treated; The active substance was compared with uninfected cells, the active substance treated and other suitable control C cells (untreated and untreated uninfected cells, wells containing the active substance without cells, etc.) on the same plate. The data obtained were compared to data obtained from other tests performed simultaneously and the approximate activity was determined based on these data.

Table 1 shows the results of the zinc extrusion test of the compound of the invention. This compound was evaluated for its ability to cause zinc extrusion from the nucleocapsid protein MCp7 (expressed as a percentage and is based on the control).

TABLE 1

Extrusion of zinc from zinc fingers of HTV-1 nucleocapsid protein (NCp7)

Example% Zn extrusion relative to control

Table 2 below contains data for the compound of Example 3 obtained in the practice. H9 and CEI * I cell assay.

TABLE 2

C E 1 * 1 cell test for anti-HIV activity

Example compound

C u 1 * 1) ^b

> 100 ^{, and an} effective control that protects cells from the cytopathological effects of the virus ^t 'toxic concentration that inhibits cell growth by 50% relative to the control sample

Claims (10)

F ³ ATTENTION IS YEARS 1. Where's the right acid ± S? CF? ^x , R ^x ) 3 - 3 - methyl 1 - 2 - C 3 - oxo - 3 FI - -benzoCd 3 -isothiazol-2-ylpentanoic acid. s and t ý τη, that includes s a. u e d e n i e l <y s e 1. i n y 2,2'- · d 11 i. o s a 1 i. a halogenated reagent to form a 2,2'-dithiobisbenzoyl halide; b. not mentioned 2,2'-dithiobistenesenoyl 1 ha 1 oge: nidudore: Actions of L-iso leucine in tetrahydrofuran or tetrahydrofuran and a base to form acid Γ. SC R ^*, R *) 3, L-2-12-E 2- (l-carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl onylľl J benzoylamino-3-methylpentanoic; and c. reacting L 5 - (R ^x , R ^x ) 3, L-2- (2-β-C 1 -carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl-13-benzoylamino-1-3-methylpentanoic acid J with a halogen with an oxidizing agent to give LS - CR 2, R ^x ) -3 -3-ethyl-2- (3-oxo-3 H -benzo [d] isothiazol-2-yl) pentanoic acid. 2. The method of claim 1 wherein the halogenating agent is thionyl chloride. 3. A process according to claim 1, wherein the 2,2 ' -dithiobisbenzoyl halide is 2,2 &apos; -clithiobisbenzoyl chloride. 4. A process according to claim 1, wherein the base is sodium bicarbonate. 5. Method according to claim 1, you from n and no u J ú c i sa T ý ni, that halogen with an oxidizing agent. Is a chloro. bromine or Iodine. 6. Method according to claim 5; from n and no u J ú c i s; and T ý m, that halogen with an oxidizing agent. is a bromo. 7. P o sot:> pr prykyse 1 i. ny Γ S - CR ^x , R **) 1 - 3 - methyl 1 - 2 - C 3 - oxo - 3 H - benzo. d] isothiazol-2-yl) pentanoic acid, characterized in that it comprises: a. reacting 2,2'-dithiosalicylic acid with thionyl chloride to form '2,2'-dithiobisbehzoyl chloride; b. reacting 2,2'-dithiobisbenzoyl chloride with at least two equivalents of L-isoleucine in tetrahydrofuran or tetrahydrofuran and hydrogen carbonate and sodium bicarbonate to form L (R ^x , R ^x ) l, L-2- <2- 1'-C 1 -carboxy-2-methylbutylcarbanioyl) phenyl disulfonyl 1'l benzoyl amino 1 '-3-methylpentanoic J; and c. introduction of L S - CR ^X , R ^x 1,1,1-L-2-C 2 - 11 - 2-Cl-carboxy-2-methylbutylcarbamoyl) phenylenesulfonic acid 1'l benzoyl amino J-3 ..... methyll pentanoic acid reacted with bromine and acetic acid to give 11 'SC R ^x , R ^x >1' -3-ethyl-2-C 3 -oxo-3H-benzol (1 'isothiazol-2-yl) pentane. 8. S p is 1 sobpripravykyse ynyl Ll - CR ^*, R ^*) l, L. - 2 - <2 - 11 '2-C 1 -carboxy-2-methylbutylcarbamoyl) phenyl Idisulfones 11' benzoyl.anii.no 1-3-methylpentane, referred to as J It includes: a. reacting 2,2'-dithiosalicylic acid with a halogenating agent to form 2,2'-dithiobisbenzoyl halide; ab. entry 2, 2 '- 1 HA 1 ditiobisbenzoy ogenidudoreakcies L - iso leucínoin in tetrahydrofuran or tetrahydrofuran and a base to form a C SC of R ^x, R ^x) l, L-2 <2 - C2-l- carboxy-2-methylbutylcarbamoyl) phenyldisulfonyl-1-benzoylamino 1-3-methylpentanoic acid. 9. A process according to claim 8, wherein the halogenating agent is thionyl chloride. 10. The process of claim 8 wherein the 2,2 ' -dithiobisbenzoyl halide is zyol chloride. and J J Li c. s a 2,2 ditiobisben- 11. A method according to claim 8, wherein the base is sodium hydrogen carbonate. 12. S pecialproduction 1 i. ny S S - CR ^x , R ^x ) 1, L - 2 - € 2 - [2 - C1 - carboxy - 2-ethyl 1-butyl 1 carb in oy 1) beny 1 disu 1 f- ones 1. ll benzoy 1 and ino} - 3 - 1-pentanoic methylenes, containing 1 a. reacting 2,2 ' -dithiosalicylic acid with thiocyloxycarbonyl to form 2,2 ' -dithiocarboxylic acid; and b. reacting 2,2'-dithiobisbenzoyl chloride with L-isoleucine in tetrahydrofuran or tetrahydrofuran and a base to form CS- (R ^X , R ^x ) 1 ', L-2-C 2 -C 12 -Cl-carboxy-2-methylbutylcarbonyl oy 1) phenyl disu 1. phenyl 1] benzoyl et al. no J-3-methylpent; á new J.