Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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W097/22598 PCT~S96/18929
METHO~ 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
FIELD OF THE INVENTION
The present invention provides 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.
BACKGROUND OF THE INVENTION
The compound [S-(R*,R*)]-3-methyl-2-(3-oxo-3H-
benzo[d]isothiazol-2-yl)pentanoic acid is useful as an
antiviral agent and can be used to treat patients
infected with the ~IV virus~ The compound [S-(R*,R*)],
L-2-{2-[2-~1-carboxy-2-methylbutylcarbamoyl)-
phenyldisulfonyl]benzoylamino}-3-methylpentanoic acid
is an intermediate in the synthesis of [S-~R*,R*)]-
3-methyl-2-~3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic
acid and can be used to treat bacterial and viral
infections, including patients infected with the HIV
virus. See, ~or example, United States Patent Number
5,463,122, which is hereby incorporated by reference.
Thus, it would be useful to have a simple, high yield
method for 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-methylbutyl-
carbamoyl)phenyldisulfonyl]benzoylamino}-3-methyl-
pentanoic acid.
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W097/22598 PCT~S96118929
SUMMARY OF THE INVENTION
The present invention provides a method of making
[S-(R*,R*)]-3-methyl-2-(3-oxo-3H-benzo[d]isothiazol-
2-yl)pentanoic acid that comprises reacting 2,2'-
dithiosalicylic acid with a halogenating a~ent to make
2,2'-dithiobisbenzoyl halide; reacting the 2,2'-
dithiobisbenzoyl halide with L-isoleucine in
tetrahydrofuran or tetrahydrofuran and a base to make
[S-(R*,R*)],L-2-{2-[2-(l-carboxy-2-methylbutyl-
carbamoyl)phenyldisulfonyl]benzoylamino}-3-
methylpentanoic acid; and reacting the [S-(R*,R~)],
L-2-{2-[2-~l-carboxy-2-methylbutylcarbamoyl)-
phenyldisulfonyl]benzoylamino}-3-methylpentanoic acid
with a halogen oxidizing agent to make [S-(R*,R*)]-
3-methyl-2-(3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic
acid~
In a preferred embodiment, the halogenating agent
is thionyl chloride.
In another preferred embodiment, the 2,2'-
dithiobisbenzoyl halide is 2,2'-dithiobisbenzoyl
chloride.
In another preferred embodiment, the base is
sodium bicarbonate
In another preferred embodiment, the halogen
oxidizing agent is chlorine, bromine, or iodine.
In more preferred embodiment, the halogen
oxidizing agent is bromine.
Also provided by the present invention is a method
of making [S-(R*,R*)]-3-methyl-2-(3-oxo-3H-benzo[d]-
isothiazol-2-yl)pentanoic acid that comprises reacting
2,2'-dithiosalicylic acid with thionyl chloride to make
2,2'-dithiobisbenzoyl chloridei reacting the 2,2'-
dithiobisbenzoyl chloride with at least two equivalents
of L-isoleucine in tetrahydrofuran or tetrahydro~uran
and sodium bicarbonate to make [S-(R*,R*)],L-2-{2-[2-
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W097/22598 PCT~S96/18929
--3--
~1-carboxy-2-methylbutylcarbamoyl)phenyldisulfonyl~-
benzoylamino}-3-methylpentanoic acid; and reacting the
[S-(R*,R*)], L-2-{2-[2-(1-carboxy-2-methylbutyl-
earbamoyl~phenyldisulfonyl]benzoylamino}-3-
~ 5 methylpentanoic acid with bromine and acetic acid to
make [S-(R*,R*)]-3-methyl-2-(3-oxo-3H-benzo[d]-
isothiazol-2-yl)pentanoic aeid.
Also provided is a method of making [S-(R*,R*)],
L-2-{2-[2-(1-carboxy-2-methylbutylcarbamoyl)-
phenyldisulfonyl]benzoylamino}-3-methylpentanoic acid
that comprises reacting 2,2'-dithiosalicylic acid with
a halogenating agent to make 2,2'-dithiobisbenzoyl
halidei and reaeting the 2,2'-dithiobisbenzoyl halide
with L-isoleucine in tetrahydrofuran or tetrahydrofuran
and a base to make [S-(R*,R*)],L-2-l2-[2-(1-earboxy-
2-methylbutylcarbamoyl)phenyldisulfonyl]benzoylamino}-
3-methylpentanoic acid.
In another preferred embodiment of the method, the
halogenating agent is thionyl chloride.
In another preferred embodiment of the method, the
2,2'-dithiobisbenzoyl halide is 2,2'-dithiobisbenzoyl
chloride.
In another preferred embodiment, the base is
sodium bicarbonate~
Also provided is a method of making [S-(R*,R*)],
L-2-{2-[2-(1-carboxy-2-methylbutyl-carbamoyl)-
phenyldisulfonyl~benzoylamino}-3-methylpentanoic acid
that comprises reacting 2,2'-dithiosalicylic acid with
thionyl chloride to make 2,2'-dithiobisbenzoyl
chloride; and reacting the 2,2'-dithiobisbenzoyl
chloride with L-isoluecine in tetrahydrofuran or
tetrahydrofuran and a base to make [S-(R*,R*)],L-2-
{2-[2-(1-carboxy-2-methylbutylcarbamoyl)phenyl-
disulfonyl]benzoylamino}-3-methylpentanoic acid.
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W097/22598 PCT~S96/18929
--4--
DETAILE~ DESCRIPTION OF THE INVENTION
The present invention provides a 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-(l-
carboxyl-2-methylbutylcarbamoyl)phenyldisulfonyl~-
benzoylamino}-3-methylpentanoic acid in accordance with
Scheme I below.
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WO 97t22598 PCT/US96/18929
S cheme
~S-S~
C02H C02H
S02Cl l
[~s sJ~ --~C02H
COCl COCl
THF or
THF/NaHC03
~S-SJ~
HN NH
~C~2H ~--C02H
Br2
Acetic Acid
3 0 ~N~
O
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W097122598 PCT~S96118929
In the first step of the synthesis, 2,2'-
dithiosalicylic acid, which can be purchased from
Aldrich, Milwaukee, Wisconsin, is converted to its
corresponding acid halide, 2,2'-dithiobisbenzoyl
halide, by reacting the 2,2'-dithiosalicylic acid with
a halogenating agent. Those skilled in the art are
familiar with the conversion of an acid to an acid
halide~ Examples of suitable halogenating agents
include, but are not limited to, thionyl chloride,
phosgene, phosphorus trichloride, phosphorus
pentachloride, and phosphorus tribromide.
In a preferred embodiment, the halogenating agent
is thionyl chloride, and the acid halide is an acid
chloride~ The term "halogen" includes chlorine,
bromine, fluorine, and iodine.
In general, the reaction is carried out under an
inert atmosphere in an aprotic solvent. Examples of
suitable aprotic solvents include, but are not limited
to, toluene, heptane, hexane, and acetonitrile. In a
preferred embodiment, the solvent is toluene. The
2,2'-dithiobisbenzoyl halide can be used in the next
step as obtained, i.e. in crude form, or can be
purified by methods well-known to those skilled in the
art.
In the second step of the synthesis,
2,2'-dithiobisbenzoyl halide is reacted with
L-isoleucine in tetrahydrofuran or a mixture of
tetrahydrofuran and a base to form [S-(R*,R*)],
L-2-{2-~2-~l-carboxy-2-methylbutylcarbamoyl)-
phenyldisulfonyl]benzoylamino}-3-methylpentanoic acid.
Use of tetrahydrofuran (THF) or a solution of THF and a
base provides for higher yields and higher purities and
is less toxic and carcinogenic than other solvents used
in the formation of amides from chlorides and ~-amino
acids. Moreover, the use of THF as a solvent is not
obvious, as L-isoleucine has only limited solubi-ity in
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WO97/22598 PCT~S96/18929
THF, and THF is generally decomposed by hydrogen
chloride, which is generated in the reaction.
Moreover, the present synthesis does not require any
protection/deprotection steps for the ~-amino acid and
is less prone to racemization, resulting in products
having superior optical activity. The [S-(R*,R*)],
L-2-{2-[2~ carboxy-2-methylbutylcarbamoyl)-
phenyldisulfonyl]benzoylamino}-3-methylpentanoic acid
can be used in the next step as obtained or can be
further purified by methods well-known to those skilled
in the art. Examples of suitable bases include, but
are not limited to, sodium bicarbonate, sodium
carbonate, potassium car~onate, and potassium
bicarbonate. Preferably, the base is sodium
bicarbonate.
In the third step of the synthesis, [S-(R*,R*)~,
L-2-{2-[2-(l-carboxy-2-methylbutylcarbamoyl~-
phenyldisulfonyl3benzoylamino}-3-methylpentanoic acid
is reacted with a halogen oxidizing agent to make
[S-(R*,R~)]-3-methyl-2-(3-oxo-3H-benzo[d]isothiazol-
2-yl)pentanoic acid. In this step, the disulfide bond
of the [S-(R*,R*)],~-2-{2-[2-(l-carboxy-2-
methylbutylcarbamoyl)phenyldisulfonyl]benzoylamino}-
3-methylpentanoic acid is oxidized and the intermediate
sulfenyl bromide cyclized to form ~S-(R*,R*)]-3-methyl-
2-(3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic acid.
Suitable halogen oxidizing agents include, but are not
limited to bromine, chlorine, and iodine. In a
preferred embodiment, the halogen oxidizing agent is
bromine.
; In general, in this step of the synthesis, a
slurry of [S-(R*,R*)],L-2-{2-[2-(l-carboxy-2-
methylbutylcarbamoyl)phenyldisulfonyl]benzoylamino}-
3-methylpentanoic acid in acetic acid is stirred with
the addition of a halogen oxidizing agent. After
stirring, the precipitate formed is filtered. The
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W097/22598 PCT~S96/18929
product precipitates directly from the reaction mixture
in a high state of purity.
Next, the precipitate, which is crude [S-(R*~R*)3-
3-methyl-2-(3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic
acid is purified by extraction with an ether,
preferably methyl-t-butyl ether, and water. The ether
extracts are combined and concentrated to afford an
oil~ The oil is dissolved in an ether and then heptane
is added. The resulting precipitate is filtered,
washed and dried to yield [S-(R*,R*)]-3-methyl-2-
(3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic acid.
The following examples are intended to illustrate
specific embodiments of the present invention and are
not intended to limit the specification, including the
claims, in any way.
EXAMPLE 1
~ 2 r -D;th;ohishe~7.oyl chlori~e
To a 2 L three-necked round-bottomed flask fitted
with a mechanical stirrer, a thermometer, and a reflux
condenser with a nitrogen inlet are charged
2,2'-dithiosalicylic acid (120 g), toluene (600 mL),
dimethylformamide (1 mL~, and thionyl chloride (128 g).
With stirring, the mixture is heated at 75~C for
21 hours under nitrogen to give a clear yellow
solution. The solution is heated to 80~C and filtered
through diatomaceous earth on a Buchner funnel. The
filter cake is washed with toluene (100 mL), and the
combined filtrates are transferred to a 2 L three-
necked round-bottomed flask fitted with a mechanical
stirrer, a thermometer, and a distillation head. The
hot solution is concentrated to a total volume of
300 mL under vacuum ~5 mm Hg) and to a final batch
temperature of 65~C Fresh toluene (500 mL) is added
to the mixture which is reheated to 80~C. The hot
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solution is concentrated to a total volume of 300 mL
under vacuum (5 mm Hg), and a further charge of fresh
~ toluene (500 mL) is made. The mixture is reheated to
80~C and the solution concentrated to a final volume of
400 mL under vacuum (5 mm Hg) and to a final batch
temperature of 65~C to give a thick yellow slurry~ The
slurry is cooled to 10~C, stirred for 2 hours, and
filtered on a Buchner filter. The filter cake is
washed with cold toluene (100 mL) and dried in a vacuum
oven at 40~C to -45~C ~o give 120 g (89.3%) of the
title compound as pale yellow crystals
mp 156.2-157.9~C
1H NMRS (~, CDCl3, 200 MHz): 8.41-8.37 (m, 2H,
aromatic H), 7.78-7.74 (m, 2H, aromatic H), 7.60-7.51
(m, 2H, aromatic H), 7.43-7.34 (m, 2H, aromatic H).
EXAMPLE 2
rs- (R* R~)l,T,-2-r2-r2-(1-c~rboxy-2-methylhutyl-
c~rh~moyl~phenyldisulfonyllhenzoyl~mino~-
3-~ethylpe~t~oic ~c;d
To a 1 L three-necked round-bottomed flask fitted
with a mechanical stirrer, a thermometer, and a reflux
condenser with a nitrogen inlet are charged
2,2'-dithiobisbenzoyl chloride (75.0 g), L-isoleucine
(62.8 g), sodium bicarbonate (55.3 g) and THF (750 mL).
With stirring, the slurry is heated at 54~C to 58~C
under nitrogen for 20 minutes and then at 60~C to 65~C
for 8 hours. The reaction mixture is cooled to room
temperature and is slowly poured into a 3 L
three-necked round-bottomed flask fitted with a
mechanical stirrer containing a rapidly stirred mixture
of concentrated hydrochloric acid (60 g), water
(550 mL), and methyl t-butyl ether (800 mL). The 1 L
flask is rinsed successively with THF (150 mL), methyl
t-butyl ether (150 mL), and water (150 mL), and the
rinses are added to the 3 L flask. The two-phase
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-10-
mixture is stirred at room temperature for 30 minutes
and then allowed to settle. The lower aqueous layer is
separated, and the upper organic layer is washed with
three portions of water (2 x 375 mL, 180 mL). To the
rapidly stirred organic layer is added hexane (1060 mL)
to give a thick white slurry which is stirred at room
temperature for 2 hours. The solids are filtered on a
Buchner filter, washed with hexane (150 mL), and dried
in a vacuum oven at 65~C to give 105.9 g (91.0~) of the
crude title compound.
To a 2 L three-necked round-bottomed flask fitted
with a mechanical stirrer, a distillation head, and a
thermometer are charged the crude title compound
(95.2 g) and THF (1620 mL). The pale yellow solution
is heated under reflux, and THF (1140 mL) is distilled
out of the flask. The THF solution in the flask is
cooled to room temperature and to this, with rapid
agitation, is added hexane ~760 mL) to give a thick
white slurry which is stirred at room temperature for
2 hours. The solids are filtered on a Buchner filter,
washed with hexane (375 mL~ and dried in a vacuum oven
at 67~C to give 93.8 g (98.5%) of the title compound as
a white solid; HPLC 99.1~ (by area)i mp 207-210~C;
1H NMRS (~, DMSO, 200 MHz): 12.8-12.4 (br s, 2H,
CO2H), 8.72 (d, J = 8.3 Hz, 2H, NH), 7.68-7.64 (m, 4H,
aromatic H), 7.50-7.41 (m, 2H, aromatic H), 7.35-7.27
(mr 2H, aromatic H), 4.39-4.32 (m, 2H, NCH), 1.95 (m,
2H, CH3C~), 1.53 (m, 2H, CH2), 1.31 (m, 2H, CH2), 0.97
(d, J = 6.9 Hz, 6H, C~3CH~, 0.89 (m, 6H, C~3CH2).
EXAMPLE 3
rs- (R*,R~ 3-~et~yl-2-(3-o~o-3~-ben~or~lisothi~zo~-
2-yl)pent~noic ac;~
To a 500 mL three-necked round-bottomed flask
fitted with a mechanical stirrer, a thermometer, and an
addition funnel with a nitrogen inlet are charged
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W097t22598 PCT~S96/18929
[S-(R*,R*)~,L-2-{2-[2-(1-carboxy-2-methylbutyl-
carbamoyl)phenyldisulfonyl~benzoylamino}-3-
methylpentanoic acid (25 g~ and acetic acid (163 mL~.
To the stirred slurry under nitrogen is added dropwise
r 5 over 15 minutes a solution of bromine (7~9 g) in acetic
acid (12 mL). The orange slurry is stirred at room
temperature for 4 hours under nitrogen, filtered on a
Buchner funnel, and washed with heptane (2 x 50 mL) to
give 31 7 g of the crude solvent-wet title compound.
To a 500 mL separatory funnel are charged the
crude title compound (31.7 g), methyl t-butyl ether
(69 mL) and water (66 mL)~ After extraction, the lower
water layer is separated and extracted with methyl
t-butyl ether (19 mL). The methyl t-butyl ether layers
are combined, washed with water (50 mL), and
concentrated to an oil using a rotary evaporator under
vacuum (25 mm Hg) and to a final batch temperature of
50~C. The oil is held under vacuum at 50~C for 1 hour,
dissolved in methyl t-butyl ether (80 mL), and filtered
on a Buchner filter. The filtrates are transferred to
a 250 mL three-necked round-bottomed flask fitted with
a mechanical stirrer, a thermometer, and an addition
funnel with a nitrogen inlet. To the warm (45~C)
stirred methyl t-butyl ether solution is added heptane
(40~mL). The turbid solution is held at 45~C for
1 hour and a further charge of heptane (19 mL) is made.
The slurry is cooled to room temperature, then to 10~C
and stirred for 1 hour. The solids are filtered on a
Buchner funnel, washed with heptane (40 mL), and dried
in a vacuum oven at 45~C for 16 hours to give 19.3 g
(77.5%~ of the title compound as white crystals;
mp 122-1230C;
H NMRS (~, CDCl3, 200 MHz~: 8.81 (br s, lH, CO2H),
8.07 (d, J = 7.8 Hz, lH, aromatic H), 7.67-7.54 (m, 2H,
aromatic H), 7.44-7.36 (m, lH, aromatic H), 5.28 (d,
J = 9.4 Hz, lH, CHN), 2.28-2.23 ~m, lH, C~CH3),
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WO97/22598 PCT~S96/18929
-12-
1.42-1.38 (m, lH, CH2), 1.29-1.20 (m, lH, CH2), 1.11
~d, J = 6.8 Hz, 3H, CHC~3), 0.91 (t, J = 7.3 Hz, 3H,
CH2CE[3 ) .
~XAMPLE 4 5
Ant 3 V; r~l Activity of ~S-(R*,R*)l-3-~e~hyl-2-(3-oxo-
3H-hen7or~l;soth; ~7.01 -2-yl)pe~t~no;c ac;~
[S-(R*,R*~-3-methyl-2-(3-oxo 3H-benzord]-
isothiazol-2-yl)pentanoic acid (Example 3) causes the
extrusion of zinc from the nucleocapsid protein (NCp7)
of HIV-l. The NC protein is highly conserved among all
retroviruses (south T., Blake P., et al., R;oche~;stry,
1990j29:7786) and is essential for viral infectivity
(Aldovini A. and Young R., ~. V;rology, 1990j64:1920
and Gorelick R., Nigida S., et al., J. Virology,
1990;64:3207). The zinc is normally held in NC
proteins by 1 or 2 zinc fingers. In the case of HIV-1,
2 zinc fingers are present (Summers M., South T.,
et al , R; ochem;stry, 1990i29:329) and are involved
specifically with the PSI site on viral RNA which
controls the packaging of viral RNA. Interference of
this packaging causes the formation of non-infectious
virions (Dannull J., Surovoy A., et al., EMBO,
1994i13:1525). It has previously been shown that
compounds that cause zinc extrusion have potent
anti-HIV activity in multiple cell lines and against
all retroviruses (Rice W., Schaeffer C., et al.,
N~ture, 1993;361:473)~
A fluorescence-based assay has been developed to
monitor the ejection of zinc from purified HIV-1 NCp7.
The fluorophore, N-(6-methoxy-8-quinol~l)-p-
toluenesulfonamide (TSQ), has an increased fluorescent
signal upon bonding zinc ion in solution. The NCp7
protein containing 2 Zn-fingers and 2 Zn ions is
incubated with drug causing the extrusion of Zn ions.
The released Zn is then sequestered by the TSQ and the
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. -13-
increased fluorescence monitored relative to control
The assay was performed as follows: lO ~uM compound was
added to 2.8 ~M NCp7 and 47 ~uM TSQ in 20 ,uL of pH 7.4
buffer at 26~C for 9O minutes. Fluorescence
(excitation 355 nM emission 400 nM) was monitored
versus time. Controls were the NCp7 under assay
conditions without drug, and apo NCp7 (no Zn~ with
drug. The ~ Zn extrusion was calculated based on the
actual fluorescence measured divided by the
fluorescence of all theoretical Zn extruded
5.6 ~M) x lOO.
The test systems utilized to establish the
cellular antiviral activity of [S-(R*,R*)]-3-methyl-
2-(3-oxo-3H-benzo[d]isothiazol-2-yl)pentanoic acid are
well recognized in the art and are routinely employed
for such purpose. For example, the assay utilized to
evaluate the compound's activity against the HIV virus
is that employed by the U.S. National Cancer Institute
as described by Weislow O.S., et al., J. N~tl. C~ncer
Inst., 1989i81:577-586, incorporated herein by
reference.
The procedure is designed to detect agents acting
at any stage of the virus reproductive cycle. The
assay basically involves the killing of T4 lymphocytes
by HIV. Small amounts of HIV are added to cells, and
at least two complete cycles of virus reproduction are
necessary to obtain the required cell killing. Agents
which interact with virions, cells, or virus gene-
products to interfere with viral activities will
protect cells from cytolysis. The system is automated
in several features to accommodate large numbers of
candidate agents, and is generally designed to detect
anti-HIV activity. However, compounds which degenerate
or are rapidly metabolized in the culture conditions
may not show activity in this screen.
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Another test system utilized to evaluate the
invention compounds is called HIV H9 assay. The HIV H9
cell assay measures the inhibitor concentration
required to suppress HIV-1 virus replication. In this
system, viral growth occurs through multiple rounds of
the life-cycle. Any suppression of the replication
kinetics results in a geometric decrease in virus
production. As a result, this assay is a sensitive
means of measuring the ability of a compound to inhibit
HIV-l viral replication~
The H9 T-cell line is batch infected with HIV
virus at a multiple of infection (MOI) of 0.01. After
2 hours absorption, the cells are washed, resuspended
in RPMI-1640 (a readily available medium well-known to
those skilled in the art)/10% fetal calf serum, and
seeded at 5 x 10-3 cells/well o~ a 96-well plate. A
duplicate plate of uninfected H9 cells is prepared for
the cytotoxicity assay. Drugs are serially diluted
1/3.16 in dimethylsulfoxide (DMSO~, transferred to
media at an 8X concentration, and then added to the
cultures in triplicate. The final DMSO concentration
of 0.002 (0.2%).
Viral production is measured by room temperature
assay and cytotoxicity is measured by XTT assay at
7 days post-infection. The XTT assay is well-known to
those skilled in the art. See, for example, J. N~tl.
~ncer Inst., 1989;81:577-586. The room temperature
assay is per~ormed as a modification of Borroto-Esoda
and Boone, ~. V;rol., l991j65:1952-1959 and quantitated
using a Molecular Dynamics Phosphoimager with
Imagequant software. The XTT assay is performed as a
modification of Roehm, et al., J. Tmml~no Methods.,
1991;142:257-265 and quantitated using a molecular
Devices Thermomax plate reader with Softmax software.
Data is electronically transferred to a Microsoft
Excel spreadsheet for analysis. The room temperature
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W097/22598 PCT~S96/18929
assay values equivalent to 50~ and 90~ inhibition of
virus production are calculated from the untreated
controls. The concentrations of inhibitor required to
produce these values ~IC50 and ICgo) are interpolated
from data points flanking these room temperature
activities. The XTT assay values equivalent to 50
cytotoxicity are calculated from the untreated
controls The concentrations of inhibitor required to
produce this value are interpolated from data points
flanking these XTT values.
Yet another test system employed to determine
antiviral activity is called the CEM cell as~ay.
T4 lymphocytes (CEM cell line) are exposed to HIV
at a virus to cell ratio approximately 0 05, and plated
along with noninfected control cells in 96-well
microliter plates.
Candidate agent is dissolved in dimethylsulfoxide
(unless otherwise noted), then diluted 1:200 in cell
culture medium. Further dilutions (half-log10) are
prepared before adding to an equal volume of medium
containing either infected or noninfected cells
Cultures are incubated at 37~C in a 5% carbon
dioxide atmosphere for 6 or 7 days. The tetrazolium
salt, XTT, is added to all wells, and cultures are
incubated to allow formazan color development by viable
cells, ~. NAt;on~l CAncer T~st;tute, 1989j81:577-586.
Individual wells are analyzed spectrophotometrically to
quantitate formazan production, and in addition are
viewed microscopically for detection of viable cells
confirmation of protective activity.
Drug-tested virus-infected cells are compared with
drug-treated noninfected cells and with other
appropriate controls (untreated infected and untreated
noninfected cells, drug-contA;n;ng wells without cells,
etc.~ on the same plate. Data are reviewed in
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-16-
comparison with other tests done at the same time and a
determination about activity is made.
Table l below presents the results of the compound
in the zinc extrusion assay described above. The
compound was evaluated for its ability to cause the
extrusion of zinc from nucleocapsid protein NCp7
(expressed as % relative to control).
TABLE l. Zn Extrusion From the Zn Fingers
of ~IV-l Nucleocapsid Protein
(NCp7)
Compound of~ Zn Extrusion Relative
Example to Control
3 30
Table 2 below presents data for the compound of
Example 3 when evaluated in the H9 and the CEM cell
assays.
TABLE 2. Anti-EIV Activity CEM Cell Assay
Compound of a b
ECso tUM) TCso t~UM)
Example
. 3 14 >lOO
a Effective concentration that protects cells
from viral cytopathic effects
b Toxic concentration that inhibits the growth
of cells 50% relative to control
