Studies on quinones. Part 37. Synthesis and biological activity of o-aminoester functionalised benzo- and naphtho[2,3-b]-thiophenequinones

Article abstract of DOI:10.1248/cpb.50.1215

The synthesis of 3-amino-2-methoxycarbonyl-4,7-dimethoxybenzo[b]thiophene (5) and benzothieno[3,2-d][1,3]oxazin 15 from 3,6-dimethoxy-2-nitrobenzaldehyde (1) is reported. Benzo[b]thiophene-4,7-quinones 9 and 10 were prepared in good yields by oxidative de

Full text of DOI:10.1248/cpb.50.1215

September 2002
Chem. Pharm. Bull. 50(9) 1215—1218 (2002)
1215
Studies on Quinones. Part 37.1) Synthesis and Biological Activity of
o-Aminoester Functionalised Benzo- and Naphtho[2,3-b]-
thiophenequinones
Jaime A. VALDERRAMA, ,a Claudio ASTUDILLO,a Ricardo A. TAPIA,a Eric PRINA,b Emilie ESTRABAUD,c
*
d
Renaud MAHIEUX,c and Alain FOURNET
a Facultad de Química, Pontificia Universidad Católica de Chile; Casilla 306, Santiago-22, Chile: b Groupe de Recherche
sur les interactions macrophage-Leishmania, Unite d’Immunophysiologie et Parasitisme Intracellulaire. Institut Pasteur;
c
rue du Dr. Roux 75724, Paris Cedex, France: Unité d’Epidémiologie et Physiopathologie des Virus Oncogènes; 28, rue
du Dr Roux-Institut Pasteur 75724 Paris Cedex 15, France: and d Institut de Recherche pour le Développement (IRD); 213
rue La Fayette 75480, Paris Cedex, France. Received April 1, 2002; accepted June 24, 2002
The synthesis of 3-amino-2-methoxycarbonyl-4,7-dimethoxybenzo[b]thiophene (5) and benzothieno[3,2-
d][1,3]oxazin 15 from 3,6-dimethoxy-2-nitrobenzaldehyde (1) is reported. Benzo[b]thiophene-4,7-quinones 9 and
10 were prepared in good yields by oxidative deprotection of the corresponding dimethoxybenzothiophenes 8 and
7. Cycloaddition reaction of quinone 8 with 1-(E)-trimethylsilyloxy-1,3-butadiene followed by acid-induced arom-
atization provides access to naphtho[2,3-b]thiophene-4,9-quinone 13 and 14. The in vitro activity of the new
quinones against Leishmania amazonensis and human-T-cell leukemia virus type 1 (HTLV-1) is reported.
Key words benzo[b]thiophene-4,7-quinone; oxidative demethylation; Diels–Alder reaction; benzothieno[3,2-d][1,3]oxazin-4-
one; Leishmania amazonensis; HTLV-1
Natural and synthetic quinonoid compounds are well- dimethoxybenzo[b]thiophene with ceric ammonium nitrate
known substances which possess a variety of biological prop- (CAN).16) On the basis of these results we investigated the
erties such as antibacterial, antifungal, antiprotozoal, inhibi- synthesis of ortho-aminoesters 5 via cyclisation of 3,6-
tion of the human immunodeficiency virus (HIV)-1 reverse dimethoxy-2-nitrobenzonitrile (3) with methyl thioglycolate.
transcriptase and antitumor activity.2—7) Some of these phar- Compound 5 could be a valuable intermediate in the synthe-
macological effects have been attributed to the formation of sis of angular tetracyclic thiophene-containing quinones due
DNA-damaging anion-radical intermediates formed by to the possibility to extend the bicyclic system through either
bioreduction of the quinone nucleus.8) Among heterocyclic the thiophene and/or the quinone rings.
quinones with cytotoxic activity, those containing a thio-
3,6-Dimethoxy-2-nitrobenzaldehyde (1), prepared as re-
phene nucleus fused to a quinone system have received little ported,17) was converted into 2,5-dimethoxy-6-nitrobenzoni-
attention9,10) despite the antitumoral activity of thiophene trile (3) in 54% total yield, by reaction with hydroxylamine
analogues of daunomycin and mitoxantrone.11,12)
followed by dehydration of 3,6-dimethoxy-2-nitrobenzalde-
As part of a program directed towards the design and syn- hyde oxime (2) with acetic anhydride. The thiophene ring
thesis of carbo- and heterocyclic quinones as potential an- formation on 3 was attempted by reaction with methyl thio-
tiprotozoal agents, we have reported the synthesis and an- glycolate in N,N-dimethylformamide (DMF) using potassium
tiprotozoal activity of benzopyrano- and benzo[b]thio- carbonate however, methyl (2-cyano-3,6-dimethoxyphenyl-
phenequinones.1,13,14) Since the thiophene ring-containing sulfanyl)acetate (4) was isolated as the main product along
quinones showed promising antiprotozoal activity against with small amounts of the methyl 3-amino-4,7-dimethoxy-
trypanosome cruzi and strains of Leishmania spp.1,14) we de- benzo[b]thiophene-2-carboxylate (5) (1H-NMR). Better re-
cided to continue the studies on synthesis and biological sults were obtained when the reaction of 3 with methyl thio-
evaluation of new members of this series containing func- glycolate was performed using potassium hydroxide in DMF
tional groups on C-2 and C-3 positions of the thiophene ring. which afforded 5 in 80% yield (Chart 1). Isolation of com-
This paper describes the preparation of functionalised pound 4 provides evidence for the pathway in which the for-
benzo[b]thiophenes and their application to the synthesis mation of the thiophene ring is initiated by displacement of
of benzo- and naphthothiophenequinones with ortho- the nitro group followed by base-induced aldol condensation.
aminoester functionality on the thiophene ring. Moreover we
In order to prepare methyl 3-amino-4,7-dioxo-4,7-dihy-
report the cytotoxic activity of the new quinones against drobenzo[b]thiophen-2-carboxylate (6) from 5, oxidative
Leishmania amazonensis and human-T-cell leukemia virus demethylation with CAN16) was attempted; nevertheless, the
type 1 (HTLV-I), the causative agent of two well-defined dis- treatment gave a complex reaction mixture. This result was
eases: adult T-cell leukemia/lymphoma (ATLL) and tropical attributed to intermolecular amination reactions between sub-
spastic paraparesis/HTLV-1 associated myelopathy (TSP/ strate 5 and the nascent quinone 6.
HAM).15)
In view of this result we decided to protect the amino
In a previous work we have described the synthesis of group in 5 before attempting the release of the corresponding
a variety of 2-substituted benzo[b]thiophene-4,7-quinones quinone with CAN. Protection of the amino group in 5, as
using a two step sequence which involves the reaction of the corresponding amide, was tried by acetylation of 5 with
dimethoxy-o-acylnitroarenes with methyl thioglycolate fol- acetic anhydride at room temperature. This treatment gave
lowed by oxidative deprotection of the corresponding a mixture of methyl 3-acetylamino-4,7-dimethoxybenzo[b]-
To whom correspondence should be addressed. e-mail: jvalderr@puc.cl
© 2002 Pharmaceutical Society of Japan

1216
Vol. 50, No. 9
Reagents: (a) 1-TMSO-1,3-butadiene, CH2Cl2 ; (b) THF-HCl aq
Chart 3
Reagents: (a) NH2OH.HCl, NaOH, EtOH, 67%; (b) Ac2O, 80%; (c) HSCH2CO2Me,
K2CO3, DMF, 43%; (d) HSCH2CO2Me, KOH, DMF, 80%
Chart 1
Reagents: (a) NaOH aq; (b) Ac2O; (c) CAN, MaCN, H2O
Chart 4
Table 1. IC50 and TC50 Values of Quinones 9, 10, 13 and 14
Quinone
IC50 (mM)
TC50 (mM)
9
10
13
14
15
Ͼ75
Ͼ80
1.56
0.1
15
8
Ͼ80
4
Amphotericin B
5
[1]benzothieno[3,2-d][1,3]oxazin-4-one (15) was attempted
by reaction with sodium hydroxide, followed by reaction of
the resulting sodium carboxylate with acetic anhydride using
a reported methodology.18) The treatment provided heterocy-
cle (15) and all attempts to obtain quinone 16 by oxidative
demethylation of 15 with CAN were unsuccessful and the
starting material was recovered in these experiments (Chart
4).
Compounds 9, 10, 13 and 14 were evaluated in vitro
against intracellular Leishmania amazonensis amastigotes
stage in mouse peritoneal macrophages. The IC50 and TC50
were determined using amphotericin B as the reference drug,
and the results are summarized in Table 1. The results indi-
cate that the thiophene-containing quinones 9, 10 and 14
have cytotoxic activity which is probably due in part to the
quinone nucleus. Comparison of the activity between
quinones 13 and 14 suggest that the electron-donating effect
of the amino group decreases the biological effect probably
by lowering the oxidant capacity of the quinone system.
Compounds 9, 10, 13 and 14 were evaluated on HTLV-1
transformed HUT-102 cell lines infected with 250000
cells/ml. These compounds displayed a reduction in cellular
proliferation by 75% (see Table 2) but induced a weak cellu-
lar viability after the quinone treatment.
Reagents: (a) CAN, MeCN, H2O; (b) Ac2O; (c) HCl, MeOH, H2O
Chart 2
thiophene-2-carboxylate (7) together with methyl 3-diacetyl-
amino-4,7-dimethoxybenzo[b]thiophene-2-carboxylate (8),
and all attempts to obtain 7 as the main product were unsuc-
cessful. Preparation of 7 was achieved in 58% total yield by
reaction of 5 with acetic anhydride at reflux followed by se-
lective deacylation of 8 (Chart 2).
Compounds 7 and 8 were then submitted to oxidative de-
protection with CAN, providing the corresponding stable
quinones, methyl 3-acetylamino-4,7-dioxo-4,7-dihydrobenzo-
[b]thiophene-2-carboxylate (10) and methyl 3-diacetylamino-
4,7-dioxo-4,7-dihydrobenzo[b]thiophene-2-carboxylate (9) in
82 and 86% yield, respectively (Chart 2).
The synthesis of naphtho[2,3-b]thiophenquinones from
quinone 9 and 1-(E)-trimethylsilyloxybuta-1,3-diene was at-
tempted. The reaction of 9 and the diene at room temperature
gave a mixture of regioisomers, methyl 3-diacetylamino-5-
trimethylsilyloxy-4,9-dioxo-4,4a,5,8,8a,9-hexahydronaph-
tho[2,3-b]thiophene-2-carboxylate (11) and methyl 3-di-
acetylamino-8-trimethylsilyloxy-4,9-dioxo-4,4a,5,8,8a,9-
hexahydronaphtho[2,3-b]thiophene-2-carboxylate (12) which,
by reaction with 5% hydrochloric acid in THF gave a mix-
ture of methyl 3-amino-4,9-dioxo-4,4a,5,8,8a,9-hexahydro-
naphtho[2,3-b]thiophene-2-carboxylate (13) and methyl 3-
acetylamino-4,9-dioxo-4,4a,5,8,8a,9-hexahydronaphtho[2,3-
b]thiophene-2-carboxylate (14) in 74 and 26% yields, respec-
tively (Chart 3).
In conclusion, we have reported the synthesis and the in
vitro leishmanicidal and anti-HTLV-1 activity of benzo- and
naphtho[2,3-b]thiophene-4,7-quinones containing the ortho-
aminoester functionality on the thiophen ring. The biological
activity of these highly functionalised heterocyclic quinones
and the possibility to construct nitrogen heterocycles fused to
the thiophene ring represents an advance in the search for
novel antiprotozoal and antiviral agents.
Heterocyclisation of compound 5 to 6,9-dimethoxy-4H-

September 2002
1217
Table 2. Inhibition of HTLV-1 Transformed Cell Lines (HUT-102) by chromatography on silica gel (chloroform) gave pure 8 as a pale yellow solid
Quinones 9, 10, 13 and 14
(202 mg, 77%). mp 157—159 °C (ethanol); IR: 3372, 1719, 1699; 1H-NMR
d: 2.30 (s, 6H), 3.82 (s, 3H), 3.89 (s, 3H), 3.95 (s, 3H), 6.69 (d, 1H, Jϭ8.5),
6.81 (d, 1H, Jϭ8.5); 13C-NMR d: 172.4, 161.5, 150.4, 148.4, 136.6, 130.1,
127.4, 126.5, 107.4, 106.1, 56.1, 56.0, 52.6, 26.0; Anal. Calcd for
C16H17NO6S: C, 54.69; H, 4.88; N, 3.99; S, 9.12. Found: C. 54.68; H, 4.86;
N, 4.03; S, 9.26.
Quinone
% Inhibition
9
10
13
14
10 mM : 75
1 mM : 5
10 mM : 84
1 mM : 0
Methyl 3-Acetylamino-4,7-dimethoxybenzo[b]thiophene-2-carboxy-
late (7) A solution of 8 (110 mg, 0.31 mmol), hydrochloric acid (1 ml,
18%) and methanol (5 ml) was refluxed for 1 h. The mixture was diluted
with water and the solid was column chromatographed on silica gel
(dichloromethane) to yield pure 7 as a white solid (67 mg, 70%). mp 185—
10 mM : 41
1 mM : 0
10 mM : 86
1 mM : 0
1
187 °C (ethanol); IR: 3254, 1700 and 1667; H-NMR d: 2.20 (s, 3H), 3.88
(s, 3H), 3.89 (s, 3H), 3.91 (s, 3H), 6.65 (d, 1H, Jϭ8.5), 6.72 (d, 1H, Jϭ8.5),
8.62 (s, 1H); 13C-NMR d: 168.4, 163.2, 150.6, 148.3, 134.4, 129.7, 125.1,
119.1, 106.7, 105.8, 56.1, 23.8; Anal. Calcd for C14H15NO5S: C, 54.36; H,
4.89; N, 4.53; S, 10.36. Found: C, 54.24; H, 4.91; N, 4.50; S, 10.35.
Experimental
All reagents were of commercial quality, reagent grade, and were used
Methyl 3-Diacetylamino-4,7-dioxo-4,7-dihydrobenzo[b]thiophene-2-
without further purification. Mps were determined on a Köfler hot-stage ap- carboxylate (9) To a solution of compound 8 (200 mg, 0.57 mmol) in ace-
paratus and are uncorrected. The IR spectra were recorded on a FT Bruker tonitrile (6 ml) was added with stirring a solution of CAN (1.25 g, 2.28
spectrophotometer using KBr discs and the wave numbers are given in cmϪ1
.
mmol) in water (6 ml). The mixture was stirred for 30 min at room tempera-
ture, diluted with water, and extracted with chloroform (3ϫ10 ml). The or-
The 1H- and 13C-NMR spectra were acquired at 200 and 50 MHz, respec-
tively, on a Bruker AC-200P in deuteriochloroform. Chemical shifts are re- ganic layer was dried over MgSO4 and evaporated under reduced pressure.
The residue was chromatographed on silica gel (chloroform) to yield pure 9
ported in d ppm downfield to tetramethylsilane (TMS), and J- values are
given in Hertz. Silica gel Merck 60 (70—230 mesh), and TLC aluminiun as yellow crystals (157 mg, 86%). mp 128—130 °C (hexane); IR: 1720,
1
foil 60 F254 were used for preparative column and analytical TLC, respec- 1598, 1587; H-NMR d: 2.32 (s, 6H), 3.94 (s, 3H), 6.83 (d, 1H, Jϭ10.5),
tively. Compound
dimethoxybenzaldehyde as previously reported.19)
2,5-Dimethoxy-6-nitrobenzaldehyde Oxime (2) A suspension of 2,5- 52.33; H, 3.45; N, 4.36; S, 9.98. Found: C, 52.21; H, 3.48; N, 4.41; S, 9.94.
dimethoxy-6-nitrobenzaldehyde (1) (300 mg, 1.42 mmol), hydroxylamine Methyl 3-Acetylamino-4,7-dioxo-4,7-dihydrobenzo[b]thiophene-2-car-
1
was prepared from commercially available 2,5- 6.97, (d, 1H, Jϭ10.5); 13C-NMR d: 179.9, 179.3, 171.7, 159.8, 144.8, 139.7,
139.0, 137.5, 135.0, 134.6, 53.3, 26.0; Anal. Calcd for C14H11NO6S: C,
hydrochloride (117.5 mg, 1.69 mmol), aqueous sodium hydroxide (2 ml, boxylate (10) A solution of compound 7 (100 mg, 0.32 mmol) in acetoni-
10%) and ethanol (10 ml) was stirred for 30 min at 60 °C. The mixture was trile (6 ml) was added with stirring to a solution of CAN (702 mg, 1.28
poured into acetic acid (15 ml, 5%) and the precipitate was recrystallized mmol) in water (6 ml) and the mixture was stirred for 30 min at room tem-
from ethanol to afford 2 as a pale yellow solid (214 mg, 67%). mp 115— perature. Work-up of the mixture followed by purification of the crude by
1
117 °C (ethanol); H-NMR d: 3.86 (s, 6H, OMe), 6.96 (d, 1H, Jϭ9.2), 7.04 column chromatography over silica gel (chloroform) afforded 10 (73 mg,
(d, 1H, Jϭ9.2), 7.59 (br s, 1H), 8.34 (s, 1H); 13C-NMR d: 186.1, 155.3,
82%). mp 122—124 °C (hexane); IR; 3325, 1727, 1583; 1H-NMR d: 2.27 (s,
144.5, 120.1, 116.2, 114.2, 57.3, 56.8; Anal. Calcd for C9H10N2O5: C, 47.79; 3H), 3.94 (s, 3H), 6.83 (d, 1H, Jϭ10), 6.91 (d, 1H, Jϭ10), 9.11 (s, 1H); 13C-
H, 4.46; N, 12.38. Found: C, 47.66; H, 4.51; N, 12.32.
3,6-Dimethoxy-2-nitrobenzonitrile (3) solution of
NMR d: 181.2, 179.5, 168.0, 162.1, 143.5, 139.0, 138.1, 137.5, 131.7,
(200 mg, 125.1, 52.9, 23.9; Anal. Calcd for C12H9NO5S: C, 51.61; H, 3.25; N, 5.02; S,
A
2
0.88 mmol) in acetic anhydride (20 ml) was heated at reflux for 3 h. The 11.48. Found: C, 51.23; H, 3.19; N, 5.01; S, 11.05.
mixture was poured into ice-water and the precipitate was filtered and
3-Acetylamino- and 3-Amino-2-methoxycarbonynaphtho[b]thiophene-
washed with water. The solid was purified by column chromatography over 4,9-quinone (13) and (14) A solution of 9 (100 mg, 0.31 mmol) and (E)-
silica gel with chloroform to give compound 3 as yellow solid (147 mg, 1-trimethylsilyloxy-1,3-butadiene (0.811 g, 5.7 mmol) in dichloromethane
1
80%). mp 150—152 °C (hexane); IR: 2231 and 1534; H-NMR d: 3.91 (s, (3 ml) was stirred for 2 d at room temperature. After the solvent was re-
3H), 3.96 (s, 3H), 7.11 (d, 1H, Jϭ9.4), 7.29 (d, 1H, Jϭ9.4); 13C-NMR d: moved under reduced pressure, the residue was dissolved in THF-hydrochlo-
155.3, 143.2, 119.4, 114.7 111.0, 97.0, 57.5, 57.1; Anal. Calcd for ric acid (20 ml, 5%), and the solution was left at room temperature for 1 h.
C9H8N2O4: C, 51.93; H, 3.87; N, 13.46. Found: C, 51.73; H, 3.89; N, 13.29.
The mixture was diluted with water, extracted with chloroform (3ϫ10 ml)
(2-Cyano-3,6-dimethoxyphenylsulfanyl)acetate (4) A suspension of 3 and the organic extract was dried over Na2SO4. Evaporation of the solvent
(200 mg, 0.96 mmol), methyl thioglycolate (110 mg, 0.95 mmol), and potas- followed by column chromatography (chloroform) of the residue afforded 13
sium carbonate (158 mg) in DMF (5 ml) was stirred for 2 h at 60 °C. The (less polar compound) as a deep red solid (66 mg, 74%). mp 179—181 °C
mixture was poured into cooled diluted hydrochloric acid (50 ml, 10%) The
(petroleum ether); IR: 3463, 3346, 1657; 1H-NMR d: 3.91 (s, 3H), 7.07
precipitate was filtered, washed with water and the solid was recrystallized (br s, 2H), 7.77 (m, 2H), 8.22 (m, 2H); 13C-NMR d: 180.2, 178.4, 164.3,
from ethanol to afford pure 4 as a pale pink solid (110 mg, 43%). mp 139— 153.5, 146.9, 134.3, 133.7, 133.6, 133.3, 127.7, 127.1, 127.0, 107.1, 51.9;
140 °C (hexane); IR: 2224, 1740; 1H-NMR d: 3.65 (s, 3H), 3.70 (s, 2H), Anal. Calcd for C14H9NO4S: C, 58.53; H, 3.16; N, 4.88; S, 11.16. Found: C,
3.88 (s, 6H), 6.92 (d, 1H, Jϭ9.2), 7.06 (d, 1H, Jϭ9.2); 13C-NMR d: 169.6,
156.0, 154.1 125.9, 116.2, 114.5, 112.6, 108.5, 56.6, 56.5, 52.5, 35.7; Anal.
Calcd for C12H13NO4S: C, 53.92; H, 4.90; N, 5.24; S, 11.99. Found: C,
53.78; H, 4.95; N, 5.39: S, 11.86.
58.32; H, 3.24; N, 4.76; S, 10.86.
Further elution with chloroform afforded pure 14 (25 mg, 26%) as yellow
solid. mp 163—165 °C (1 : 1 hexane–benzene); IR: 3380, 1753, 1595; 1H-
NMR d: 2.31 (s, 3H), 3.95 (s, 3H), 7.81 (m, 2H), 8.23 (m, 2H), 9.45 (br s,
Methyl 3-Amino-4,7-dimethoxybenzo[b]thiophene-2-carboxylate (5) 1H); 13C-NMR d: 179.2, 176.3, 165.2, 160.6, 156.9, 147.7, 136.9, 133.0,
solution of 3 (200 mg, 0.96 mmol), methyl thioglycolate (110 mg, 132.6, 130.5, 129.1, 127.9, 127.5, 118.9, 50.9, 24.1; Anal. Calcd for
A
0.95 mmol), and potassium hydroxide (65 mg) in DMF (5.0 ml) was stirred C16H11N5O5S: C, 58.35; H, 3.37; N, 4.25; S. 9.74. Found: C, 58.30; H, 3.33;
for 2 h at 80 °C. The solution was diluted with water and the precipitate was N, 4.21; S, 9.80.
filtered and washed with water. The solid was purified by flash chromatogra-
6,9-Dimethoxy-4H-[1]benzothieno[3,2-d][1,3]oxazin-4-one (15) A so-
phy with chloroform to afford 5 as a white solid (205 mg, 80%). mp 146— lution of 5 (300 mg, 1.12 mmol), sodium hydroxide (70 mg, 1.75 mmol) and
1
147 °C (ethanol); IR: 3479, 3372, 1674; H-NMR d: 3.86 (s, 3H), 3.89 (s,
ethanol (30 ml) was refluxed for 2 h. The mixture was cooled and the precip-
3H), 3.90 (s, 3H), 6.56 (d, 1H, Jϭ8.5), 6.71 (d, 1H, Jϭ8.5), 6.71 (br s, 2H, itate was filtered, washed with ethanol and dried. A suspension of the
exchangeable with D2O); 13C-NMR d: 165.9, 151.8, 151.1, 148.6, 131.3; sodium salt (200 mg, 0.6 mmol) in acetic anhydride (10 ml) was refluxed for
122.1, 107.6, 104.3, 95.7, 56.0, 55.8, 51.2; Anal. Calcd for C12H13NO4S: C, 3 h and the mixture was cooled overnight. The precipitate was filtered,
53.92; H, 4.90; N, 5.24; S, 11.99. Found: C, 53.81: H, 4.92; N, 5.21; S, washed with ethanol, and purified by column chromatography over silica gel
12.02.
(chloroform) to give pure 15 as a pale green solid (170 mg, 55%), mp 293—
1
Methyl 3-Diacetylamino-4,7-dimethoxybenzo[b]thiophene-2-carboxy- 294 °C (methanol); IR: 1740; H-NMR d: 2.53 (s, 3H), 3.91 and 3.94 (2s,
late (8) A solution of 5 (200 mg, 0.75 mmol) in acetic anhydride (20 ml) 6H), 6.76 (d, 1H, Jϭ8.5), 6.86 (d, 1H, Jϭ8.5); 13C-NMR d: 194.1, 163.9,
was refluxed for 3 h. The mixture was poured into ice-water and the precipi- 151.9, 150.8, 148.6, 118.2, 109.2, 107.0, 100.5, 56.6, 56.2, 21.7. Anal. Calcd
tate was filtered and washed with water. Purification of the solid by column for C13H11NO4S: C, 56.31; H, 4.00; N, 5.05; S, 11.56. Found: C, 56.21; H,

1218
Vol. 50, No. 9
3.98; N, 4.98: S, 11.44.
(1999).
Bioassays. In Vitro Activity against Leishmania amazonensis The
isolation of macrophages and parasites (L. amazonensis, strains LV79) was
described previously in full details.19) For all drugs, stock solutions were pre-
pared in DMSO at a concentration of 100 mg/ml. Two fold serial dilutions
were made from 500 mg/ml in culture medium supplemented with 0.5%
DMSO final. Twenty-four hours after infection, freshly prepared drugs were
added to the infected cultures decreasing both the first final drug concentra-
tion to 100 mg/ml and the final DMSO concentration to 0.1%. This DMSO
concentration was proven to have no effect on control cultures. Thirty hours
after drug addition, infected cultures were examined using an inverted phase
contrast Zeiss microscope (magnification of 400). Note was made of toxic
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Cell Lines and Cell Proliferation Assay20) HUT-102 are HLTV-1-
transformed cell lines and were grown in RPMI-1640 medium supplemented
with 10% fetal calf serum, L-Gln, and penicillin–streptomycin. To measure
cellular proliferation or viability, a cell proloferation-viability kit (XTT;
Roche Molecular Biochemicals) was used. In this assay, tetrazolium salt
XTT is cleaved to form an orange formazan dye by metabolic active cells. 14) Valderrama J. A., Fournet A., Valderrama C., Bastias S., Astudillo C.,
This dye is directly quantified using an enzyme-linked immunosorbent
reader at 492 nm.
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(2000).
Acknowledgements Financial support is gratefully acknowledged to
FONDECYT (Grant 8980003 and 2970082).
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