Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3895
NMR (DMSO-d6): δ 2.31 (s, 6H), 4.01 (m, 2H), 7.27 (s, 1H),
7.30-7.41 (m, 2H, on treatment with D2O showed dd, J ) 1.8
and 8.8 Hz, 1H), 7.47 (br s, 1H, disappeared on treatment with
D2O), 7.57 (d, J ) 8.8 Hz, 1H), 7.74 (s, 2H), 8.00 (d, J ) 1.8
Hz, 1H), 9.39 (br t, J ) 5.5 Hz, 1H, disappeared on treatment
with D2O), 13.06 ppm (br s, 1H, disappeared on treatment with
D2O). IR (Nujol): ν 1640, 1680, 3280, 3300, 3400 cm-1. Anal.
(C19H18ClN3O4S (419.88)), C, H, N, Cl, S.
and K103N-Y181C stock solutions had titers of 3.0 × 105, 1.3
× 106, and 2.5 × 105 CCID50/mL, respectively.
HIV Titr a tion . Titration of HIV was performed in C8166
cells by the standard limiting dilution method (dilution 1:2,
four replica wells per dilution) in 96-well plates. The infectious
virus titer was determined by light microscope scoring of
syncytia after 4 days of incubation. Virus titers were expressed
as CCID50/mL.
An ti-HIV Assa ys. The activity of test compounds against
multiplication of WT HIV-1, Y181C, and K103N-Y181C in
acutely infected cells was based on inhibition of virus-induced
cytopathicity in MT-4 cells. The activity of the compounds
against the K103R multiplication in acutely infected cells was
based on inhibition of p24 antigen in C8166 cells. Briefly, an
amount of 50 µL of culture medium containing 1 × 104 cells
was added to each well of flat-bottom microtiter trays contain-
ing 50 µL of culture medium with or without various concen-
trations of test compounds. Then an amount of 20 µL of HIV
suspensions (containing the appropriate amount of CCID50 to
cause complete cytopathicity at day 4) was added. After
incubation at 37 °C, cell viability was determined by the 3-(4,5-
dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT)
method.13 Alternatively, p24 levels were determined by an
immunoenzymatic kit (Abbott). The cytotoxicity of test com-
pounds was evaluated in parallel with their antiviral activity
and was based on the viability of mock-infected cells, as
monitored by the MTT method.
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd r a zid es
7, 9, 11, 14, a n d 16. Exa m p le. N-{3-[(3,5-Dim eth ylp h en yl)-
su lfon yl]-5-ch lor o-1H-in d ole-2-ca r bon yl}glycin e h yd r a -
zid e (7). A mixture of 20 (0.50 g, 0.0011 mol), ethanol (5 mL),
and hydrazine hydrate (2.5 mL) was stirred at room temper-
ature for 3 h. After dilution with water, the crude product was
filtered and dried to afford 0.36 (75%) of pure 7, mp 224-227
°C (from ethanol). 1H NMR (DMSO-d6): δ 2.33 (s, 6H), 4.03
(m, 2H), 4.38 (br s, 2H, disappeared on treatment with D2O),
7.28 (s, 1H), 7.37 (dd, J ) 1.8 and 8.7 Hz, 1H), 7.58 (d, J ) 8.7
Hz, 1H), 7.75 (s, 2H), 8.00 (d, J ) 1.8 Hz, 1H), 9.16 (br s, 1H,
disappeared on treatment with D2O), 9.39 (br m, 1H, disap-
peared on treatment with D2O), 13.00 ppm (very br, 1H,
disappeared on treatment with D2O). IR (Nujol): ν 1640, 3300
cm-1. Anal. (C19H19ClN4O4S (434.89)), C, H, N, Cl, S.
Gen er a l P r oced u r e for th e P r ep a r a tion of Acid s 19,
22, 23, a n d 27. Exa m p le. 3-[(3,5-Dim eth ylp h en yl)su lfo-
n yl]-1H-in d ole-2-ca r boxylic a cid (19). Lithium hydroxide
monohydrate (0.24 g, 0.0057 mol) was added to a solution of
18 (0.70 g, 0.0019 mol) in THF (20 mL) and water (20 mL).
Then the reaction mixture was stirred at room temperature
for 24 h. After dilution with water, the mixture was acidified
with 1 N HCl until pH 2 was reached. The acid was extracted
with ethyl acetate, washed with brine, and dried. Removal of
the solvent furnished 0.63 g (94%) of satisfactorily pure 19,
mp 277-278 °C (from ethanol). Anal. (C17H14ClNO4S (363.81)),
C, H, N, Cl, S.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of E t h yl
E st er s 20, 21, 24-26, a n d 28. E xa m p le. N-{3-[(3,5-Di-
m eth ylp h en yl)su lfon yl]-5-ch lor o-1H-in d ole-2-ca r bon yl}-
glycin e Eth yl Ester (20). Benzotriazol-1-yloxytris(dimeth-
ylamino)phosphonium hexafluorophosphate (BOP reagent)
(0.61 g, 0.0014 mol) was added to a solution of 19 (0.50 g,
0.0014 mol), glycine ethyl ester hydrochloride (0.39 g, 0.0027
mol), and triethylamine (0.42 g, 0.0041 mol) in anhydrous DMF
(25 mL). Then the reaction mixture was stirred at room
temperature for 72 h. After dilution with water, the solid that
formed was filtered, washed with water, and dried to afford
0.50 g (80%) of pure 20, mp 209-211 °C (from ethanol). 1H
NMR (DMSO-d6): δ 1.25 (t, J ) 7.1 Hz, 3H), 2.31 (s, 6H), 4.12-
4.30 (m, 4H), 7.27 (s, 1H), 7.37 (dd, J ) 1.7 and 8.8 Hz, 1H),
7.57 (d, J ) 8.8 Hz, 1H), 7.72 (s, 2H), 8.03 (d, J ) 1.7 Hz, 1H),
9.49 (t, J ) 5.6 Hz, 1H, disappeared on treatment with D2O),
13.13 ppm (br, 1H, disappeared on treatment with D2O). IR
(Nujol): ν 1635, 1740, 3200 cm-1. Anal. (C21H21ClN2O5S
(448.92)), C, H, N, Cl, S.
Ack n ow led gm en t. Authors thank Italian Ministero
della Salute, Istituto Superiore di Sanita`, Fourth Na-
tional Research Program on AIDS (Grant Nos. 40C.8
and 40D.46) for financial support. Acknowledgment is
due to Italian MIUR (cofin 2002).
Su ppor tin g In for m ation Available: Chemical and physi-
cal data, elemental analysis results, and IR and 1H NMR
spectra for 8-16 and 21-28. This material is available free
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An tivir a l Assa y P r oced u r es. Com p ou n d s. Compounds
were solubilized in DMSO at 200 mM and then diluted in
culture medium.
Cells a n d Vir u ses. MT-4, C8166, and H9/IIIB cells were
grown at 37 °C in a 5% CO2 atmosphere in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS), 100 IU/mL
penicillin G, and 100 µg/mL streptomycin. Cell cultures were
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derived from an IIIB strain passaged in C8166 cells in the
presence of efavirenz (up to 2 µM). The Y181C mutant (NIH
N119) is derived from an AZT-sensitive clinical isolate pas-
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