Synthesis and antiprotozoal evaluation of benzothiazolopyrroloquinoxalinones, analogues of kuanoniamine A

Article abstract of DOI:10.1016/S0968-0896(03)00311-0

Boc-aminoethylindoloquinone 8, a key intermediate for the building of pentacyclic quinoneimines, analogues of kuanoniamine A, was synthesized by alkylation of 4,7-dimethoxyindole 3 with 1,2-dibromoethane followed by transformation into azide, reduction of the latter with trimethylphosphine in the presence of 2-(tert-butoxycarbonyloximino)-2-phenylacetonitrile and oxydative demethylation of the Boc-amine 6 with silver(II) oxide. Quinone 8 was then treated in situ with the thiazole o-quinodimethane 10 to afford a regioisomeric mixture of the tetracyclic quinones 11. Treatment of the mixture with trifluoroacetic acid and molecular sieves 4-A provided the corresponding quinoneimines 12. Separation of the regioisomers was performed by preparative thin-layer chromatography on silica gel. The structural assignment was made by 2D ¹H-¹³C HMBC correlations performed on the less polar regioisomer 12b. In vitro anti-leishmanial assays showed that the tested compounds possess a good potency towards two Leishmania sp. as well as against a virulent strain of Toxoplasma gondii and without any cytotoxicity against THP-1 cells.

Full text of DOI:10.1016/S0968-0896(03)00311-0

Bioorganic & Medicinal Chemistry 11 (2003) 3407–3412
Synthesis and Antiprotozoal Evaluation of
Benzothiazolopyrroloquinoxalinones, Analogues of
Kuanoniamine A
a,
a
b
b
Ricardo A. Tapia, * Yolanda Prieto, Fe
´
b,
lix Pautet, Nadia Walchshofer,
d
c
Houda Fillion, * Bernard Fenet and Marie-Elizabeth Sarciron
aFacultad de Quı´mica, Pontificia Universidad Cato´lica de Chile, Correo 22, Santiago, Chile
bLaboratoire de Chimie Organique, EA 635, Faculte´ de Pharmacie, Universite´ Claude Bernard Lyon 1, 8 Avenue Rockefeller,
F-69373 Lyon Cedex 08, France
c
Centre de RMN, Universite´ Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France
Laboratoire de Parasitologie EA 1887, Facult e´ de Pharmacie, Universite´ Claude Bernard Lyon 1, 8 Avenue Rockefeller,
F-69373 Lyon Cedex 08, France
d
Received 21 March 2003; accepted 8 May 2003
Abstract—Boc-aminoethylindoloquinone 8, a key intermediate for the building of pentacyclic quinoneimines, analogues of kuano-
niamine A, was synthesized by alkylation of 4,7-dimethoxyindole 3 with 1,2-dibromoethane followed by transformation into azide,
reduction of the latter with trimethylphosphine in the presence of 2-(tert-butoxycarbonyloximino)-2-phenylacetonitrile and oxyda-
tive demethylation of the Boc-amine 6 with silver(II) oxide. Quinone 8 was then treated in situ with the thiazole o-quinodimethane
1
sieves 4-A provided the corresponding quinoneimines 12. Separation of the regioisomers was performed by preparative thin-layer
0 to afford a regioisomeric mixture of the tetracyclic quinones 11. Treatment of the mixture with trifluoroacetic acid and molecular
˚
1
13
chromatography on silica gel. The structural assignment was made by 2D H- C HMBC correlations performed on the less polar
regioisomer 12b. In vitro anti-leishmanial assays showed that the tested compounds possess a good potency towards two Leishmania
sp. as well as against a virulent strain of Toxoplasma gondii and without any cytotoxicity against THP-1 cells.
#
2003 Elsevier Ltd. All rights reserved.
Introduction
related pyrrolothiazolobenzoquinolinones was repor-
4
b
ted. However, evaluation of their biological activities
was limited by their low solubility in usual solvents.
During the last two decades, many polycyclic and aro-
matic alkaloids, isolated from plants or marine sources,
show various biological properties like antifungal, anti-
microbial, antiviral or cytotoxic activities. Most of these
derivatives possess in common a benzonaphthyridinone
skeleton fused to a benzene and (or) an heterocyclic
ring. Among them, kuanoniamine A (Scheme 1), a
marine metabolite containing a thiazole ring inhibits in
vitro the proliferation of KB cells and possesses anti-
In a previous paper, we described the synthesis and
5
a
antiprotozoal activity of pyrazinoindoloquinones,
naphthofuranquinones and naphthothiophenquinones
5
b
containing a fused benzothiazole ring. In continuation
of our work, we aimed to synthesize new pentacyclic
analogues of kuanoniamine A having in their structure
a pyrroloquinoxalinone moiety. A retrosynthetic path-
way for these quinoneimines is described in Scheme 1.
The Diels–Alder trapping of a thiazole o-quinodi-
methane (o-QDM) with an appropriate indoloquinone
would lead to the tetracyclic quinones which could be
cyclized into the target quinoneimines for estimation of
their antiprotozoal activities against virulent strains of
Leishmania sp. and Toxoplasma gondii.
1
2
3
viral properties. Recently, the synthesis of structurally
*Corresponding authors. Tel.: +56-2-686-4429; fax: +56-2-686-4744;
e-mail: rtapia@puc.cl (R. A. Tapia); tel.: +33-478-77-7138; fax: +33-
78-77-7158; e-mail: fillion@univ-lyon1.fr (H. Fillion).
4
0
968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00311-0

3408
R. A. Tapia et al. / Bioorg. Med. Chem. 11 (2003) 3407–3412
˚
sieves (4A) at reflux of absolute ethanol provided the
Results and Discussion
corresponding quinoneimines 12a and 12b with the
same ratio. Separation of the regioisomers was effi-
ciently performed by preparative thin layer chromato-
graphy on silica gel using several successive elutions
with a mixture of ethylacetate/chloroform (1:1).
Chemistry
The Boc-aminoethylindoloquinone 8 is the key inter-
mediate for the building of the pentacyclic framework.
Its synthesis is described in Scheme 2. The preparation
of 4,7-dimethoxyindole
3
from 2,5-dimethoxy-
benzaldehyde in 26–46% overall yields was previously
To explain the weak regioselectivity observed for the
trapping of o-QDM 10 with indoloquinone 8, we calcu-
late for the latter the LUMO molecular orbital coeffi-
cients by the density functional theory (DFT) method
using the GAUSSIAN 98 package with a 3-21G* basis
set. The calculations indicate that the larger orbital
coefficient is located at 5-C (0.3066 for 5-C and 0.2743
for 6-C). On the other hand, the larger HOMO orbital
coefficient for o-QDM 10 is situated at CHBr (0.305 for
4
reported. Starting from the same aldehyde, we describe
an efficient procedure which affords 3 in an improved
yield (Scheme 2). Thus, treatment of 2,5-dimethoxy-
benzaldehyde with ethyl azidoacetate according to the
6
Hemetberger–Knittel reaction gave the intermediate
2-azido-3-(2,5-dimethoxyphenyl)-2-propenoate7
ethyl
which was cyclized into ethyl dimethoxyindole-2-car-
5
a,7
boxylate 1.
Hydrolysis of 1 with 10% sodium
hydroxide followed by neutralisation with 6 Mhydro-
chloric acid provided dimethoxyindole-2 carboxylic acid
CHBr and 0.289 for CH ). Therefore, the calculations
2
predict a regioselective Diels–Alder reaction with 11a as
the major regioisomer. The fact that the cycloaddition
reaction is weakly regioselective cannot be explained by
FMO considerations.
2
and quinoline at 150–160 C gave 3 in 63% overall yield
. Then, decarboxylation of 2, performed with copper
ꢀ
calculated from 2,5-dimethoxybenzaldehyde.
8
Treatment of 3 with 1,2-dibromoethane in the presence
of benzyltributylammonium chloride (BTBAC) at reflux
for 10 h afforded 4 which was transformed into 5 by the
mean of sodium azide and BTBAC. Reaction of 5 with
trimethylphosphine followed by addition of 2-(tert-butoxy-
9
carbonyloximino)-2-phenylacetonitrile (Boc-ON)
at
ꢀ
À10 C and stirring at room temperature for 2 h led to
the desired Boc-amine 6. Oxidative demethylation of the
azide 5 and Boc-amine 6 with silver(II) oxide in the
presence of 6 N nitric acid gave the corresponding qui-
nones 7 and 8 in 81 and 82% yields, respectively.
Then, treatment of 4-(bromomethyl)-5-(dibromo-
1
0
methyl)-1,3-thiazole 9 with sodium iodide in DMF
generated 5-(bromomethylene)-4-methylene-4,5-dihy-
dro-1,3-thiazole 10 which was trapped in situ with
indoloquinone 8 (Scheme 3). The Diels–Alder reaction
afforded directly the aromatized tetracyclic quinones
1
1a and 11b as an unseparable mixture of regioisomers
with a ratio 11a/11b: 52/48. Treatment of 11a+11b with
a mixture of dichloromethane/trifluoroacetic acid (4:1)
at room temperature for 2 h and then, with molecular
Scheme 2. Reagents and conditions: (a) Na, EtOH, N
ꢀ
3
CH
À10 C, rt, 2 h, 75%; (b) toluene, reflux, 5 h, 99%; (c) 10% NaOH,
2
CO Et,
2
ꢀ
ꢀ
reflux, 1 h, 6 MHCl, 0 C, 90%; (d) Cu, quinoline, N
2
, 150–160 C, 3
h, 95%; (e) Br–CH
f) NaN
h, rt, 2 h, 76%; (h) AgO, HNO
À10 C, 1 min, 82%.
2
–CH
2
–Br, NaOH, BTBAC, reflux, 10 h, 74%;
ꢀ
(
1
3
, BTBAC, reflux, 20 h, 93%; (g) Me
3
P, Boc-ON, À10 to 0 C,
, rt, 15 min, 81%; (i) AgO, HNO
3
,
Scheme 1.
3
ꢀ

R. A. Tapia et al. / Bioorg. Med. Chem. 11 (2003) 3407–3412
3409
The structural assignment was made by 2D 1H– C
HMBC correlations performed on the less polar regio-
isomer of the quinonimine 12b (Table 1). The use of the
13
three J couplings: 4-H with 12a-C and 4b-C and 6-H
with 4b-C allowed the structure of the regioisomer
3
3
12bto be established. Finally, the two J couplings: 12-H
with 3a-C and 11-C confirmed this assignment.
3
characteristic long-range J coupling of the benzothi-
3
azole nucleus let us to assign first 3a-C and 12a-C.
3
Indeed, the J coupling between 2-H and 12a-C appears
as a doublet typical of a cross-peak through the nitro-
Biology
3
gen atom of the thiazole ring ( J1 =15 Hz), while
that between 2-H and 3a-C gives an unresolved singlet
2aCÀ2H
The in vitro antiprotozoal activities of compounds 1–8,
11a+11b, 12a and 12b were evaluated against promas-
tigote forms of Leishmania donovani and Leishmania
major, and against T. gondii (Table 2). The potential
toxicities of these derivatives were also determined against
an infected human myelomonocytic THP-1 cell line. Pen-
3
indicating a coupling of <5 Hz for J
. Then,
3
aCÀ2H
Table 1. 2D H–13C HMBC correlations for 12b (CDCl
1
3
, 500.13 and
1
25.78 MHz, d ppm)
Atom
2
13C
1H
HMBC [J(C,H)]
1J
2J
3J
4J
157.1
138.1
117.8
130.8
151.8
49.2
9.16
8.90
2-H
3
4
4
4
6
7
8
9
1
1
1
1
1
a
2-H, 12-H
4-H
2-H
a
b
4-H
12-H
4-H, 6-H
12-H
4.40
4.19
6-H
7-H
7-H
6-H
42.5
a
129.0
126.0
108.0
179.8
133.7
123.5
155.4
7-H, 9-H, 10-H
7-H
6-H
4-H
6.90
6.81
9-H
10-H
10-H
9-H
0
1
1a
2
2a
12-H
4-H
12-H
9.06
12-H
2-H, 4-H
Scheme 3. Regioisomers ratio: 11a/11b=52/48; 12a/12b=52/48.
Table 2. In vitro inhibitory activity against Leishmania sp. and Toxoplasma gondii, and cytotoxicity against THP-1 cells of the synthetized com-
poundsa
Compd
Leishmania donovani
IC50 (mM)
Leishmania major
IC50 (mM)
Toxoplasma gondii
IC50 (mM)
THP-1 cells
IC50 (mM)
1
2
3
4
5
6
7
8
0.004
0.062
0.076
0.250
0.045
0.064
0.008
0.043
0.006
0.006
0.008
0.007
0.0018
0.0011
0.0007
0.0007
0.0021
0.0034
0.0012
0.0031
0.0021
0.0008
0.0051
0.0027
0.096
0.011
0.392
0.011
0.099
0.110
0.817
0.081
0.075
0.615
0.083
0.0043
0.010
0.013
0.012
0.0093
0.0026
0.0044
0.0051
0.007
0.005
0.0075
0.0080
0.210
0.0060
0.0012
1
1
1
1a+11b
2a
2b
Pentamidine
Pyrimethamine
Spiramycin
0.0034
0.0042
0.0034
Sulfadiazine
a
The values are the meansÆSD of triplicate experiments

3410
R. A. Tapia et al. / Bioorg. Med. Chem. 11 (2003) 3407–3412
tamidine for the anti-leishmanial assays and pyr-
imethamine, spiramycin, and sulfadiazine for anti-
toxoplasmal tests, were used as the reference drugs.
4,7-Dimethoxy-1H-indole-2-carboxylic acid (2). An aqu-
eous solution of 10% sodium hydroxyde (45 mL) was
added to indole 1 (3.0 g, 1.20 mmol). The mixture was
heated to reflux for 1 h then, cooled in an ice-bath at
ꢀ
Almost all the tested compounds are more active
against T. gondii than the reference drugs and less toxic
towards THP-1 cells excepted 2 and 4. Moreover, com-
pounds 1, 2, 4–8, 11a+11b, and 12b exhibit significant
activities against both strains of Leishmania sp. and T.
gondii. Concerning the inhibitory activity towards L.
major, it is noteworthy that the best compounds are
those bearing a Boc-moiety 6, 8, 11a+11b and the qui-
noneimine function (12a and 12b). It seems, in these
cases, that lipophilicity plays an important role in the
antileishmanial activity. Among the quinoneimine
regioisomers, we observed a large difference for their
IC50 towards L. donovani and TPH-1 cells.
0 C and neutralized with 6 MHCl. The precipitate was
filtered, washed with water and dried. Compound 2 was
ꢀ
obtained as a white solid (2.4 g, 90%). Mp 204–205 C.
1
À1
IR (KBr) n cm 3370, 1550, 1525. H NMR (DMSO-
d , 200 MHz) d: 3.82 (s, 3H, OCH ), 3.84 (s, 3H, OCH ),
6.38 (d, 1H, J=8.3 Hz, 5-H or 6-H), 6.64 (d, 1H, J=8.3
6
3
3
Hz, 6-H or 5-H), 7.02 (d, 1H, J=2.2 Hz, 3-H), 11.70 (s,
1
3
1H, COOH), 12.80 (br s, 1H, NH). C NMR (DMSO-
d6, 50 MHz) d: 55.0, 55.5, 98.6, 104.3, 105.4, 119.4,
127.6, 129.0, 141.1, 147.6, 162.3. Anal. calcd for
C H NO : C 59.73, H 5.01, N 6.33. Found: C 59.49,
1
1
11
4
H 4.89, N 6.06.
4,7-Dimethoxy-1H-indole (3). A mixture of compound 2
(
1.0 g, 4.52 mmol), quinoline (2.5 mL, 2.10 mmol) and
ꢀ
copper (0.2 g) was heated under nitrogen at 150–160 C
for 3 h. After evaporation of the solvent, the residue was
purified by column chromatography on silica gel with
dichloromethane as eluent to give compound 3 as a
Conclusion
We have described an efficient procedure to obtain 4,7-
dimethoxyindole in an improved yield. Then, alkylation
of the latter followed by transformation into azide,
reduction and oxidative demethylation afforded the new
Boc-aminoethylindoloquinone 8. A Diels–Alder trap-
ping of the thiazole o-QDM 10 with quinone 8 gave the
regioisomeric tetracyclic quinones 11. Treatment of 11
with trifluoroacetic acid followed by addition of molec-
ular sieves in refluxing ethanol provided the pentacyclic
quinoneimines 12a and 12b containing benzothiazole
and pyrroloquinoxaline rings as part of their structures.
All the prepared compounds were evaluated in vitro
against virulent strains of Leishmania sp. and T. gondii.
Almost all of them show significant inhibitory activities
against L. donovani and T. gondii with lower cytotoxi-
cities against TPH-1 cells than the reference drugs. The
best results towards L. major are observed with com-
pounds bearing a Boc-moiety 6, 8, 11a+11b and the
pentacyclic quinoneimine 12b.
ꢀ
white solid (0.76 g, 95%). Mp 118–119 C (ref 11
ꢀ
À1
1
119 C). IR (KBr) n cm 3370, 1525, 1500. H NM R
(CDCl , 200 MHz) d: 3.95 (s, 3H, OCH ), 3.96 (s, 3H,
3
3
OCH ), 6.42 (d, 1H, J=8.3 Hz, 5-H or 6-H), 6.55 (d,
1H, J=8.3 Hz, 6-H or 5-H), 6.66 (dd, 1H, J=2.4 and
3.1 Hz, 3-H), 7.11 (t, 1H, J=2.4 Hz, 2-H), 8.48 (br s,
3
1
3
1H, NH). C NMR (CDCl , 50 MHz) d: 55.7, 98.8,
3
100.4, 101.5, 120.0, 122.6, 127.7, 141.2, 147.8.
1-(2-Bromoethyl)-4,7-dimethoxy-1H-indole (4). A mix-
ture of indole 3 (100 mg, 0.56 mmol), 1,2-dibromo-
ethane (1 mL), sodium hydroxide (70 mg, 1.75 mmol),
and BTBAC (20 mg, 0.06 mmol) in dichloromethane (2
mL) was heated to reflux for 10 h. After filtration, the
solid was washed with dichloromethane. The combined
organic layers were evaporated and the residue was
recrystallized from ethanol. Compound 4 was obtained
ꢀ
as a white solid (118 mg, 74%). Mp 55–55.5 C. IR
À1
1
(
KBr) n cm
2950, 1525, 1500. H NMR (CDCl3,
2
3
00 MHz) d: 3.69 (t, 2H, J=6.9 Hz, N-CH –CH –Br),
2
2
.92 (s, 6H, OCH ), 4.70 (t, 2H, J=6.9 Hz, N-CH –
3
2
Experimental
CH –Br), 6.39 (d, 1H, J=8.3 Hz, 5-H or 6-H), 6.53 (d,
2
Chemical synthesis
1H, J=8.3 Hz, 6-H or 5-H), 6.57 (d, 1H, J=3.1, 3-H),
1
3
6
.97 (d, 1H, J=3.1 Hz, 2-H). C NMR (CDCl3,
Melting points were measured with a Stuart Scientific
SMP3 apparatus and are uncorrected. IR spectra were
recorded on a Bruker Vector 22 spectrophotometer
50 MHz) d: 31.9, 50.8, 55.6, 55.8, 98.9, 99.1, 102.2,
121.9, 126.2, 128.2, 142.1, 147.8. Anal. calcd for
C H BrNO : C 50.72, H 4.97, N 4.93. Found: C
1
2
14
2
1
13
using KBr discs. H and C NMR spectra were
obtained on Bruker AM-200, AM-300 and DRX-500
spectrometers using tetramethylsilane as an internal
reference. Column chromatography was performed on
silica gel Merck 60 (70–230 mesh). Thin layer chroma-
tography separations were performed on Merck Kie-
selgel 60 (70–230 mesh). Elemental analyses were
carried out on a Fisons EA 1108 CHNS-O analyzer.
50.57, H 5.17, N 4.94.
1-(2-Azidoethyl)-4-,7-dimethoxy-1H-indole (5). A mix-
ture of indole 4 (250 mg, 0.88 mmol), sodium azide (4
eqiv), BTBAC (30 mg, 0.09 mmol) in toluene (5 mL)
was heated to reflux for 20 h. After filtration, the solid
was washed with dichloromethane. The combined
organic layers were evaporated and the residue was
purified by column chromatography on silica gel with
dichloromethane as eluent. Compound 5 was obtained
Compound 1 was prepared according to ref 5a. 4-(Bro-
momethyl)-5-(dibromomethyl)thiazole 9 was obtained
by selective bromination of the commercially available
À1
as a liquid (200 mg, 93%). IR (KBr) n cm 2100, 1525,
1
1500. H NMR (CDCl , 200 MHz) d: 3.65 (t, 2H, J=6.1
3
1
0
4
,5-dimethylthiazole as previously reported.
Hz, N-CH –CH –N ), 3.93 (s, 3H, OCH ), 3.94 (s, 3H,
2
2
3
3

R. A. Tapia et al. / Bioorg. Med. Chem. 11 (2003) 3407–3412
3411
OCH ), 4.53 (t, 2H, J=6.1 Hz, N–CH –CH -N ), 6.41
3
silica gel with a mixture of dichloromethane/ethylace-
tate (3:1) as eluent. Compund 8 was obtained as a yel-
2
2
3
(
6
d, 1H, J=8.3 Hz, 5-H or 6-H), 6.56 (d, 1H, J=8.3 Hz,
-H or 5-H), 6.63 (d, 1H, J=3.1 Hz, 3-H), 6.96 (d, 1H,
ꢀ
À1
low solid (37 mg, 82%). Mp 145 C. IR (KBr) n cm
1
3
1
J=3.1 Hz, 2-H). C NMR (CDCl , 50 MHz) d: 48.3,
3384, 1687, 1665, 1652. H NMR (CDCl , 300 MHz) d:
3
3
5
1
2
2.7, 55.6, 98.8, 99.4, 102.2, 122.0, 126.3, 128.1, 142.2,
47.8. Anal. calcd for C H N O : C 58.53, H 5.73, N
2.75. Found: C 58.27, H 5.57, N 22.48.
1.42 (s, 9H, CH ), 3.51 (m, 2H, J=6.0 Hz, N–CH –
CH –NHCOO–), 4.47 (t, 2H, J=6.0 Hz, N–CH –CH –
3
2
1
2
14
4
2
2
2
2
NHCOO–), 4.70 (br s, 1H, NH), 6.58 (dd, 2H, J=10.2
Hz, 5-H and 6-H), 6.61 (d, 1H, J=2.8 Hz, 3-H), 6.88 (d,
1H, J=2.8 Hz, 2-H). HRMS Calcd. for C H N O :
1
-(2-tert-Butylcarbamoylethyl)-4-,7-dimethoxy-1H-indole
1
5
19
2
4
+
(
6). Trimethylphosphine (0.84 mL, 1.05 equiv) was
added under nitrogen to a stirred solution of indole 5
200 mg, 0.813 mmol) in dry THF (8 mL). Stirring was
291.13448. Found: 291.13407 (MH ).
(
6-(2-tert-Butylcarbamoylethyl)-5,9-dioxo-6,9-dihydro-
5H-indolo[6,5-f][1,3]benzothiazole (11a) and 6-(2-tert-bu-
tylcarbamoylethyl)-5,9-dioxo-6,9-dihydro-5H-indolo[5,6-
f][1,3]benzothiazole (11b). A solution of 4-(bromo-
continued at room temperature for 2 h. Then, a solution
of Boc-ON (208 mg, 0.84 mmol) in dry THF (8 mL) was
ꢀ
was maintained at 0 C for 1 h and then, at room tem-
added to the reaction mixture cooled at À10 C. Stirring
ꢀ
perature for 5 h. The mixture was extracted with ether
9
methyl)-5-(dibromomethyl)thiazole 9 (0.216 g, 0.6
mmol) in dry DMF (2.0 mL) was slowly added to a
ꢀ
(
200 mL), washed with 2 Msodium hydroxide (3 Â50
stirred and heated solution, at 60 C, of quinone 8 (145
mL), then, with water (2Â50 mL) and dried over mag-
nesium sulfate. Removing of the solvent left a residue
which was purified by column chromatography on silica
gel with dichloromethane as eluent. Compound 6 was
mg, 0.5 mmol) and NaI (5 equiv) in DMF (3 mL). Stir-
ring and heating were maintained for 2 h. After cooling,
water (50 mL) and 10% aqueous solution of sodium
bisulfite were added to eliminate excess of iodine. Then,
the mixture was extracted with ethyl acetate (2Â30 mL),
washed with water (2Â30 mL) and dried over magne-
sium sulfate. Removing of the solvent left a residue
which was purified by column chromatography on silica
gel with dichloromethane/ethyl acetate (1:1) as eluent to
afford an unseparable mixture of 11a+11b as a yellow
ꢀ
obtained as an orange solid (197 mg, 76%). Mp 80 C.
À1
1
IR (KBr) n cm 3442, 1714, 1697. H NMR (CDCl ,
3
3
N–CH –CH –NHCOO–), 3.90 (s, 3H, OCH ), 3.91 (s,
3
00 MHz) d: 1.41 (s, 9H, CH ), 3.49 (m, 2H, J=5.8 Hz,
3
2
2
3
H, OCH ), 4.47 (t, 2H, J=5.8 Hz, N-CH –CH –
3 2 2
NHCOO–), 4.60 (br s, 1H, NH), 6.37 (d, 1H, J=8.4 Hz,
-H or 6-H), 6.51 (d, 1H, J=8.4 Hz, 6-H or 5-H), 6.54
d, 1H, J=2.9 Hz, 3-H), 6.89 (d, 1H, J=2.9 Hz, 2-H).
Anal. calcd for C H N O : C 63.73, H 7.55, N 8.74.
ꢀ
5
(
solid (119 mg, 61%; ratio 11a/11b: 52/48). Mp 122 C.
À1
1
IR (KBr) n cm 3367, 1720, 1681, 1650. H NM R
(CDCl , 300 MHz): 11a d: 1.42 (s, 9H, CH ), 3.47–3.64
1
7
24
2
4
3
3
Found: C 63.70, H 7.62, N 8.42.
(m, 2H, N–CH –CH –NHCOO–), 4.50–4.70 (m, 2H, N-
CH –CH –NHCOO–), 4.93 (br s, 1H, NH), 6.76 (d, 1H,
2 2
2
2
1
(
-(2-Azidoethyl)-1H-indole-4,7-dione (7). Silver(II) oxide
100 mg) and nitric acid (6 N, 0.25 mL) were added at
J=2.7 Hz, 8-H), 6.97 (d, 1H, J=2.7 Hz, 7-H), 8.73 (s,
1H, 4-H), 8.77 (s, 1H, 10-H), 9.17 (d, 1H, 2-H). 11b d:
1.42 (s, 9H, CH ), 3.47–3.64 (m, 2H, N–CH –CH –
room temperature to a solution of indole 5 (70 mg, 0.28
mmol) in THF (3 mL), and the reaction mixture was
stirred for 15 min, quenched with water (0.5 mL), and
extracted with dichloromethane. The organic phase was
washed with 10% aqueous solution of sodium bicarbo-
nate, the solvent was removed and the residue was
purified by column chromatography on silica gel with
dichloromethane as eluent. Compound 7 was obtained
as a yellow liquid (50 mg, 81%). IR (KBr) n cm 2100,
1
(t, 2H, J=5.4 Hz, N–CH –CH –N ), 4.43 (t, 2H, J=5.4
Hz, N–CH –CH –N ), 6.49 (d, 1H, J=10.2 Hz, 5-H or
3
2
2
NHCOO–), 4.50–4.70 (m, 2H, N–CH –CH –NHCOO–),
2
2
5.07 (br s, 1H, NH), 6.77 (d, 1H, J=2.7 Hz, 8-H), 6.98
(d, 1H, J=2.7 Hz, 7-H), 8.71 (s, 1H, 4-H), 8.83 (s, 1H,
10-H), 9.19 (d, 1H, 2-H). Anal. calcd for C H N O S:
2
0
19
3
4
C 60.44, H 4.82, N 10.57, S 8.07. Found: C 60.43, H
4.80, N 10.60, S 8.08.
À1
6,7-Dihydrobenzothiazolo[6,5-h]pyrrolo[1,2,3-de]quinoxa-
line-11-one (12a) and 6,7-dihydrobenzothiazolo[5,6-h]pyr-
rolo[1,2,3-de]quinoxaline-11-one (12b). A mixture of
quinones 11a+11b (100 mg, 0.25 mmol) was added
under stirring at room temperature to a solution of di-
chloromethane/trifluoroacetic acid (4:1) (10 mL). Stir-
ring was maintained for 2 h. The reaction mixture was
concentrated under vacuum, absolute ethanol (10 mL)
1
700, 1650, 1590. H NMR (CDCl , 200 MHz) d: 3.66
3
2 2 3
2
2
3
6
J=2.7 Hz, 3-H), 6.93 (d, 1H, J=2.7 Hz, 2-H).
NMR (CDCl , 50 MHz) d: 48.3, 51.2, 107.5,127.4,
-H), 6.55 (d, 1H, J=10.2 Hz, 6-H or 5-H), 6.56 (d, 1H,
1
3
C
3
128.3, 130.6, 137.0, 137.1, 177.9, 183.0. Anal. calcd for
C H N O : C 55.55, H 3.73, N 25.91. Found: C 55.61,
˚
and molecular sieves (4 A) were added. Then, the mix-
1
0
8
4
2
H 3.78, N 25.89.
ture was heated to reflux under nitrogen for 8 h.
Removing of the solvent afforded a mixture of quino-
neimines 12a+12b as a yellow solid (22 mg, 31%; ratio
1-(2-tert-Butylcarbamoylethyl)-1H-indole-4,7-dione (8).
Silver(II) oxide (55 mg) and nitric acid (6 M, 1 mL)
1
12a/12b: 52/48 from the H NMR spectrum of the mix-
ꢀ
.156 mmol) in THF (33 mL), and the reaction mixture
were added at À10 C to a solution of indole 6 (50 mg,
ture). Separation of the regioisomers was realized by
preparative thin-layer chromatography on silica gel with
10 successive elutions with ethyl acetate: chloroform
0
was stirred for 1 min, quenched with water (0.5 mL) at
ꢀ
À10 C, and extracted with dichloromethane. The
(1:1) as eluent. 12a: R =0.43 (chloroform/ethanol,
f
ꢀ
À1
1
organic phase was washed with 10% aqueous solution
of sodium bicarbonate, the solvent was removed and the
residue was purified by column chromatography on
19:1). Mp >300 C. IR (KBr) n cm 1649. H NM R
(CDCl , 300 MHz) d: 4.18 (t, 2H, J=6.7 Hz, 7-H), 4.41
3
(t, 2H, 6.7 Hz, 6-H), 6.79 (d, 1H, J=2.8 Hz, 10-H), 6.89

3412
R. A. Tapia et al. / Bioorg. Med. Chem. 11 (2003) 3407–3412
(
4
d, 1H, J=2.8 Hz, 9-H), 8.94 (s, 1H, 12-H), 9.0 (s, 1H,
-H), 9.18 (s, 1H, 2-H).12b: R =0.49 (chloroform/etha-
Acknowledgements
f
ꢀ
À1
1
nol, 19:1). Mp >300 C. IR (KBr) n cm 1656. H
NMR (CDCl , 300 MHz) d: 4.17 (t, 2H, J=6.8 Hz, 7-
H), 4.38 (t, 2H, 6.8 Hz, 6-H), 6.79 (d, 1H, J=2.8 Hz, 10-
H), 6.88 (d, 1H, J=2.8 Hz, 9-H), 8.87 (s, 1H, 4-H), 9.03
The authors acknowledge FONDECYT (Research
grant 1020874) and ECOS-CONICYT C00B07 for
financial support of this work.
3
(
s, 1H, 12-H), 9.14 (s, 1H, 2-H). HRMS Calcd. for
C H N OS: 279.04663. Found: 279.04664.
1
5
9
3
References and Notes
Anti-leishmanial activity against promastigotes of L.
donovani MHOM/ET/67/L82 and L. major MHOM/
PT/92/CRE26: LV9 was assessed in 96-well plates (Fal-
1. Molinski, T. F. Chem. Rev. 1993, 93, 1825.
2. Faulkner, J. Nat. Prod. Rep. 1999, 16, 155.
ꢀ
1
3
4
. Carrol, A. R.; Scheuer, P. J. J. Org. Chem. 1990, 55, 4426.
. (a) The synthesis of 3 was performed through nitration of
con) at 27 C using CellTiter 96 Aqueous Non-Radio-
active Cell Proliferation Assay (Promega), colorimetric
5
2,5-dimethoxybenzaldehyde followed by a Henry reaction
with nitromethane and a reductive cyclization as described by:
Hollis Showalter, H. D.; Pohlmann, G. Org. Proc. Int. 1992,
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ium/well. The compound was dissolved in DMSO and
then diluted at the appropriate concentration in the
standard culture medium [RPMI 1640 medium (sigma)
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2
4
4, 484. (b) Be
neteau, V.; Besson, T. Tetrahedron Lett. 2001,
´ ´
2, 2673. For the reductive cyclization of the intermediate o,b-
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ciron, M.-E.; Walchshofer, N.; Fillion, H. Eur. J. Org. Chem
50
culture, the drug being tested as a serial 4-fold dilution
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at each concentration.
2
002, 4005. (b) Tapia, R. A.; Alegria, L.; Pessoa, C. D.; Salas,
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Growth inhibition of T. gondii: the virulent RH strain of
T. gondii was maintained in culture with the human
myelomonocytic cell line THP-1 (ECACC number
2
6
175.
. Hemetsberger, H.; Knittel, D.; Weidman, H. Monatsh.
8
8081201, Sophia-Antipolis, France) as previously
2
Chem. 1969, 100, 1599.
. Tani, M.; Ikegami, H.; Tashiro, M.; Hiura, T.; Tsukiota,
1
described. Three different experiments were performed
in quadruplicate.
7
C.; Kaneko, C.; Shimizu, M.; Uchida, M.; Aida, Y.;
Yokoyama, Y.; Murakami, Y. Heterocycles 1992, 34, 2349.
8. An analogous synthesis of 3 in 28% overall yield was men-
Assays of cytotoxicity of the drugs were conducted on a
human myelomonocytic cell line THP-1 (European col-
lection of animal cell culture number 88081201: Sophia-
Antipolis, France). These non-adherent cells were sus-
pended in RPMI 1640 medium (DAP, Vogelgrun,
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mg/mL of streptomycin and 10% fetal calf serum
tioned in: Roue
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. Ariza, X.; Urpi, F.; Viladomat, C.; Vilarrasa, J. Tetra-
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, N. Doctorat de l’Universite de Reims Cham-
´ ´
9
1
1
1
1. Marshall-Aubart, K.; Heathcock, C. H. J. Org. Chem.
999, 64, 16.
´
12. Sarciron, M.-E.; Walchshofer, N.; Paris, J.; Petavy, A. F.;
(
DAP). The growth of THP-1 cells was assessed in 96-
ꢀ
well plates at 37 C using the method described above
for parasites.
Peyron, F. Parasite 1998, 5, 359.