Structure-activity relationships of indole compounds derived from combretastatin A4: Synthesis and biological screening of 5-phenylpyrrolo[3,4-a] carbazole-1,3-diones as potential antivascular agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.022

A series of 5-(3′,4′,5′-trimethoxyphenyl)pyrrolo[3,4-a] carbazole-1,3(2H,10H)-diones was designed as cis-restricted analogues of 3-aroylindoles, arylthioindoles and 3-benzylidoneindolin-2-ones derived from combretastatin A4 (CA-4). Starting from various indoles, compounds were synthesized by means of a convenient two-step procedure involving a one-pot multicomponent reaction as key step. Intermediate tetrahydro[3,4-a]carbazoles and their corresponding carbazoles were submitted to biological screening tests involved in antivascular action, including the cytotoxicity against murine B16 melanoma cells, the rounding up of endothelial cells (EA.hy 926) and the inhibition of tubulin polymerization. Of the 31 compounds screened, those bearing a methoxy group at the 8-position endowed significant biological activities. A carbazole compound 30 was identified as a promising candidate for further development of novel vascular targeting agents.

Full text of DOI:10.1016/j.ejmech.2010.05.022

European Journal of Medicinal Chemistry 45 (2010) 3726e3739
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships of indole compounds derived from
combretastatin A4: Synthesis and biological screening of 5-phenylpyrrolo[3,4-a]
carbazole-1,3-diones as potential antivascular agents
,
b
b
c
Nancy Ty a, Grégory Dupeyre a, Guy G. Chabot b , Johanne Seguin , Lionel Quentin , Angèle Chiaroni ,
*
François Tillequin a, Daniel Scherman b, Sylvie Michel a, Xavier Cachet a
,
*
a Laboratoire de Pharmacognosie, Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, UMR 8638 CNRS, 4 avenue de l’Observatoire, 75006 Paris, France
b Laboratoire de Pharmacologie Chimique et Génétique, INSERM U 1022-CNRS UMR 8151, Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de
l’Observatoire, 75006 Paris, France
c Laboratoire de Cristallochimie, Institut de Chimie des Substances Naturelles, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
A series of 5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-a]carbazole-1,3(2H,10H)-diones was designed as cis-
restricted analogues of 3-aroylindoles, arylthioindoles and 3-benzylidoneindolin-2-ones derived from
combretastatin A4 (CA-4). Starting from various indoles, compounds were synthesized by means of
a convenient two-step procedure involving a one-pot multicomponent reaction as key step. Intermediate
tetrahydro[3,4-a]carbazoles and their corresponding carbazoles were submitted to biological screening
tests involved in antivascular action, including the cytotoxicity against murine B16 melanoma cells, the
rounding up of endothelial cells (EA.hy 926) and the inhibition of tubulin polymerization. Of the 31
compounds screened, those bearing a methoxy group at the 8-position endowed significant biological
activities. A carbazole compound 30 was identified as a promising candidate for further development of
novel vascular targeting agents.
Article history:
Received 15 March 2010
Received in revised form
9 May 2010
Accepted 10 May 2010
Available online 15 May 2010
Keywords:
Antitumor agents
Endothelial cells
Multicomponent reactions
Tubulin
Ó 2010 Elsevier Masson SAS. All rights reserved.
Vascular-disrupting agents
1. Introduction
Two different approaches have emerged to target tumor
vasculature: i) the antiangiogenic approach which aims to prevent
During the two last decades, one of the most significant
advances in cancer therapy has been likely the development of
targeted therapies including small molecules and monoclonal
antibodies, offering the theoretical advantages of limited adverse
effects on normal cells and of their use in combination with other
therapies without adversely affecting tolerability [1].
The tumor vasculature presents an attractive target in cancer
therapy because a functional network of blood vessels is an
essential requirement for tumor progression and metastasis, and
also because tumor vessels are markedly distinct from functional
normal vessels [2]. Furthermore, impairment of oxygen and nutri-
ents supply by a single vessel can lead to the destruction of
numerous tumor cells [2].
the formation of new vessels in tumors, and ii) the antivascular
approach that compromises established tumor vasculature, using
‘vascular disrupting agents’ (VDAs), which leads to hemorrhagic
necrosis within the tumors [2,3].
Apart from flavone acetic acid analogues, small molecule VDAs
[2,3c,4] also include antitubulin agents and particularly inhibitors
of tubulin polymerization interacting at the colchicine (1) binding-
site on tubulin such as combretastatin A4 (CA-4, 2), a stilbene
initially isolated from the bark of Combretum caffrum Kuntz (Fig. 1)
[5]. The corresponding disodium phosphate prodrug of CA-4 (fos-
bretabulin, 3) is currently in advanced stage clinical development
since it recently entered phase II/III studies in combination with
carboplatin or paclitaxel in patients with anaplastic thyroid cancer.
[4,6] Moreover, second-generation VDAs such as the CA-4P
analogue OXi4503 (CA-1P, 4) are under active development. The
OXi4503 is a compound presenting a dual mechanism of action, i.e.,
in addition to its vascular disrupting properties, it also displays
a direct cytotoxic activity against tumor cells, therefore offering an
improved antitumor efficacy [5b,6,7].
* Corresponding authors. Tel.: þ33 153731580; fax: þ33 140469658.
E-mail addresses: guy.chabot@parisdescartes.fr (G.G. Chabot), xavier.cachet@
parisdescartes.fr (X. Cachet).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.022

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3727
Fig. 1. Structures of colchicine, CA-4, fosbretabulin and Oxi4503.
CA-4 has attracted considerable research attention because of its
2. Chemistry
potent biological properties and also because of its relatively simple
chemical structure. Considerable efforts have been devoted to the
synthesis of non-isomerizable analogues of CA-4, since its corre-
sponding E-isomer (trans) is devoid of vascular disrupting activity.
In this context, the indole nucleus, frequently found in natural
tubulin polymerization inhibitors, has been advantageously
employed as core structure of potent synthetic tubulin inhibitors
[8]. Indeed, several recent studies have highlighted the potent
activities of the structurally related arylthioindoles (ATIs) [9], 3-
aroylindoles [10] and 1,3-dihydroindol-2-ones[11] (Fig. 2).
In these series, the most active compounds shared some
common structural features, i.e., two non-coplanar aryl groups
(including a 3,4,5-trimethoxyphenyl unit linked through a two-
atom bridge), and a methoxy group either at the 5- or 6- position on
the phenyl ring of indole nucleus. To our knowledge, there is no
prior report on the synthesis and biological evaluation of cis-locked
analogues of these indolic derivatives as potential antivascular
agents. Therefore, as an extension of our previous work on 3-
aroylindoles [10b] we focused our attention on the synthesis and
biological activities of a new series of 5-phenylpyrrolo[3,4-a]
carbazole-1,3-diones (5e35) [12]. In particular, we report here the
effects on the biological activities of the following modifications: i)
substitutions at the 7- and/or 8-positions in the A-ring (most of the
synthesized compounds are disubstituted, bearing a methoxy
group at the 7-position (R2) and a substituent of various electronic
character at the 8-position (R3)); ii) aromaticity of the C-ring; and
iii) pyrrole N-benzylation. The biological screening tests included
cytotoxicity against B16 melanoma cells, the analysis of the
morphological effects on immortalized HUVEC (EAhy 926), and the
effect on the inhibition of tubulin polymerization (ITP).
The structures of target 5-phenylpyrrolo[3,4-a]carbazoles are
depicted in Table 1. Tetrahydrocarbazoles (THCs) 5e10 and 12e17
were prepared by means of a convenient one-step, three compo-
nent reaction involving
a maleimide, the 3,4,5-trimethox-
yphenylacetophenone, and various indoles (Scheme 1) [13]. Each
THC is obtained as a unique racemic pair of diastereoisomers in a cis
relative configuration [14] (Fig. 3). Reaction conditions, originally
described by Noland et al. [13], were optimized to reduce the
formation of both corresponding carbazoles and undesired by-
products. However, in some cases, particularly when nitroindoles
and/or maleimide were engaged in the one-pot reaction, carbazoles
were obtained either concomitantly with tetrahydrocarbazoles (26,
28, 31, 32, 34) or as the sole reaction product (23). Finally, carba-
zoles 19e22, 24, 26, 29e32 and 34 were synthesized, in good to
excellent yield, by oxidation of the corresponding THCs with DDQ.
THCs 11, 18 and carbazoles 27, 35 (Scheme 2), bearing an amino
group at the 8-position, were prepared by reduction with Zn/AcOH
of respective 8-nitro derivatives 10, 17 and 26, 34. Attempts to
obtain 8-amino-7-methoxyTHCs from trifluoroacetamides 9 and 16
were unsuccessful, mainly due to the low stability of these
compounds under basic conditions. In contrast, under the same
conditions, carbazoles 24 and 32 could be easily deprotected to
afford compounds 25 and 33.
3. Biological evaluation
The biological activities of the 31 new 5-phenylpyrrolo[3,4-a]
carbazoles are summarized in Tables 2e4. The compounds were
tested for their potential vascular disrupting effects by means of
Fig. 2. General structures of selected 3-aroylindoles, 3-arylthioindoles, 1,3-indolin-2-ones, and of 5-phenylpyrrolo[3,4-a]carbazole-1,3-diones 5e35.

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N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
Table 1
Structures of 5-phenylpyrrolo[3,4-a]carbazole-1,3-diones.
Compounda
R1
R2
R3
THC
C
5
6
7
8
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
H
H
H
H
H
H
H
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
NO2
NH2
H
H
H
OCH3
Br
NO2
NHCOCF3
NH2
H
H
H
H
OCH3
e
9
e
10
11
12
13
14
15
16
e
OCH3
H
OCH3
OCH3
OCH3
NO2
NH2
Br
NHCOCF3
NH2
H
17
18
H
a
THC ¼ tetrahydrocarbazole; C ¼ carbazole.
three screening tests that have previously been employed
successfully to identify potent 3-aroylindoles [10b]. These tests
include cytotoxicity against B16 melanoma cells, induction of
a rapid morphological change of endothelial EA.hy 926 cells
(rounding up), considered predictive of a potential in vivo anti-
vascular activity [16], and the inhibition of tubulin polymerization
(ITP). Colchicine (1) and CA-4 (2) were routinely included in the
tests, because a good correlation is observed between their potent
inhibitory activities on cancer cells, their marked effects on the
morphology (rounding up) of endothelial cells, and their strong
inhibition of tubulin polymerization.
monosubstituted compounds bearing a methoxy (6, 13, 20, 29), an
amino (11, 18, 27, 35) or a nitro (10, 17, 26, 34) group; and the 7,8-
disubstituted (8, 9, 15, 16, 22e25, 31e33) compounds. As presented
in Tables 2 and 3, the nature and the position of the A-ring
substituents markedly influenced the biological effects.
Concerning the cytotoxic activity, compounds 5, 14, 19, 21, 27,
and 30 were the most active ones, with IC50 in the low micromolar
range (<10 mM) (Tables 2 and 3). For comparison purposes, refer-
ence compounds colchicine 1 and CA-4 2 exhibited cytotoxic
activity in the nanomolar range, as expected. For the tetrahy-
drocarbazoles (Table 2), cytotoxic activity was best for the N-ben-
zylated compound 5 bearing no substituent at positions R2 and R3;
whereas for the N-H series compound 14 bearing a methoxyl at R3
The biological activities were examined for the following
compounds: the non-substituted compounds (5, 12, 19, 28); the 7-
Scheme 1. Synthesis of Tetrahydrocarbazoles 5e10, 12e17 and Carbazoles 19e24, 26, 28e32, 34. Reagents and conditions: a) 35% aq. HCl (0.3 or 1.3 equiv), EtOH, reflux; b) DDQ
(2 equiv), 1,4-dioxane, reflux, 30 min.

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3729
Fig. 3. Ortep drawing of the molecule (drawn with program PLATON) [15] giving the atomic labelling. Displacement ellipsoids are shown at the 30% probability level.
Scheme 2. Synthesis of aminotetrahydrocarbazoles 11, 18 and aminocarbazoles 25, 27, 33, 35. Reagents and conditions: a) Zn (30 equiv), AcOH (20 equiv), THF, 20 ꢀC; b) K2CO3
(2 equiv), 1,4-dioxane-H2O, 20 ꢀC, 17 h.

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N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
Table 2
Biological activities of tetrahydrocarbazoles in the N-Bn and the N-H series.
Compound
R2
R3
Cytotoxicity IC50
[
m
M]a
Morphology[m
M]b
5
6
7
8
9
10
11
12
13
14
15
16
17
18
H
OCH3
H
OCH3
OCH3
NO2
NH2
H
OCH3
H
OCH3
OCH3
NO2
NH2
e
H
H
OCH3
Br
NHCOCF3
H
H
H
3.7 ꢁ 0.1
50
NDc (>80)
ND (>80)
ND (>40)
20.0 ꢁ 5.3
ND (>40)
16.6 ꢁ 2.8
ND (>40)
ND (>80)
2.6 ꢁ 1.5
NAd
2.5
NA
20e
NA
10
1.3
NA
0.3
50
NA
NA
100
0.003
0.008
H
OCH3
Br
60.9 ꢁ 4.3
23.1 ꢁ 4.2
ND (>50)
ND (>100)
0.031 ꢁ 0.003
0.003 ꢁ 0.001
NHCOCF3
H
H
e
e
Colchicine (1)
CA-4 (2)
e
a
Cytotoxicity on murine B16 melanoma cells. Results are expressed as the mean ꢁ SEM of the concentration that causes 50% cell kill (IC50).
The lowest active concentration that causes a rounding up of immortalized HUVEC (EA.hy 926) after a 2 h exposure time.
ND: IC50 not precisely determined.
NA: no rounding up observed at the maximum of solubility.
Endothelial cells showed a stellate morphology.
b
c
d
e
was the most cytotoxic. In the carbazole series (Table 3), N-ben-
indicating a selective antivascular activity with low intrinsic cyto-
toxicity. This may be a result of differences in cell permeability.
zylated analogues bearing no substituent at R2 and R3 (19), or
bearing a methoxyl at R3 (21) was linked to a significant cytotoxic
activity. Also of interest, compound 27 bearing a NH2 on R2 was also
markedly cytotoxic. In the N-H carbazole series (Table 3),
compound 30 bearing a methoxy group at R3 was the most cyto-
toxic with an IC50 value in the nanomolar range.
With regard to the morphological effects on endothelial cells,
only compounds 7, 12, 14, and 30 presented a rounding up at low
micromolar concentrations (<10 mM) (Tables 2 and 3). Fig. 4 shows
representative photographs of these morphologically active
compounds compared to control (1% DMSO) and CA-4 (2). Of these
compounds, 30 was the most active with a rounding up of endo-
thelial cells still significant in the nanomolar range (25 nM, Table 3).
With the exception of 12, this morphological activity on endothelial
cells was linked to the presence of a methoxy group at R3.
In terms of inhibition of tubulin polymerization (ITP) activity,
only the 8-methoxylated compounds 7, 14 and 30 presented
significant activity with IC50 in the low micromolar to sub-
micromolar concentrations (Table 4, Fig. 5). It is noteworthy that
these IC50 values were rather close to the ones of the reference
compounds colchicine (1) and CA-4 (2), especially regarding
compound 30.
4. Discussion
From the above results, some interesting structureeactivity
relationships can be disclosed. Several structural modifications led
to a marked decrease in biological potency of 30. Thus, the corre-
sponding tetrahydrocarbazole 14, which differs mainly in the
spatial orientation of the 3,4,5-trimethoxyphenyl ring, was less
active than 30 in the three biological tests (i.e. IC50 ¼ 1.13 and
2.60
mM for tubulin inhibition and cytotoxicity, respectively, and
a morphological effect observed at a 0.30
mM concentration).
Furthermore, N-alkylation of compounds 30 and 14 at the 2-posi-
tion on the maleimide ring, by a bulky benzyl group was clearly
deleterious for biological activities. Indeed, carbazole 21 displayed
weaker cytotoxicity (IC50 ¼ 3.9
mM) and was almost inactive in the
two other tests, whereas its corresponding tetrahydrocarbazole 7,
retained only poor activities in both the tubulin polymerization
assay (IC50 ¼ 3.49
mM) and the morphological test on endothelial
cells (2.5 M). Finally, and as already noted, shifting the methoxy
m
group from the 8 to the 7 position (tetrahydrocarbazoles 6, 13 and
carbazoles 20, 29) led to a dramatic loss of activities.
Taking into account the results of the three biological testing
procedures employed in this study, 30 emerged as a particularly
active compound. Indeed, in addition to its excellent inhibition of
tubulin polymerization activity (close to reference compounds 1
and 2), the concentration of 25 nM to cause the rounding up of
endothelial cells was also remarkable. However, the cytotoxic
activity of 30 was several-fold higher (200-fold) than CA-4 (2)
These results clearly demonstrate that the planar pyrrolo[3,4-a]
carbazole nucleus (designated herein as ‘carbazole’), bearing
a 3,4,5-trimethoxyphenyl ring at the 5-position, may represent
a novel scaffold in the development of new potential antivascular
agents. Beside the requirement of an aromatic C-ring to maintain an
optimal geometry between the A-ring and the 3,4,5-trimethox-
yphenyl ring, the main structural features that contribute to

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3731
Table 3
Biological activities of carbazoles in the N-Bn and the N-H series.
Compound
R2
R3
Cytotoxicity IC50
[
m
M]a
Morphology [m
M]b
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
NO2
NH2
H
H
H
OCH3
Br
NO2
NHCOCF3
NH2
H
H
H
H
OCH3
3.3 ꢁ 2.0
12.8 ꢁ 4.9
3.9 ꢁ 1.3
ND (>100)
ND (>50)
ND (>100)
ND (>50)
ND (>25)
4.1 ꢁ 0.8
ND (>100)
ND (>100)
0.6 ꢁ 0.1
13.5
43.9 ꢁ 4.0
16.8 ꢁ 0.2
>50
12.8 ꢁ 2.9
0.031 ꢁ 0.003
0.003 ꢁ 0.001
NAc
100
100
NA
NA
NA
NA
NA
NA
100
100
0.025
100
NA
100
NA
NA
OCH3
H
OCH3
OCH3
OCH3
NO2
NH2
e
Br
NHCOCF3
NH2
H
H
e
35
Colchicine (1)
CA-4 (2)
0.003
0.008
e
e
a
Cytotoxicity on murine B16 melanoma cells; ND: IC50 not precisely determined.
The lowest active concentration that causes a rounding up of immortalized HUVEC (EA.hy 926) after a 2 h exposure time.
NA, not active at maximum solubility in cell culture medium.
b
c
biological activity are the presence of a methoxy group at the 8-
position, and a free NH at the pyrrole N2 position.
position, emerged as new lead. This potent inhibitor of tubulin
polymerization induces a significant rounding up of endothelial
cells at nanomolar concentrations, and could be considered as a cis-
restricted analogues of 3-aroylindoles and 1,3-indolin-2-ones
derived from CA-4. Other substitution pattern on the A-ring led to
decrease in biological activities. These results deserve further
developments to fully ascertain the potential antivascular activity
of 30 and to better define the SAR of the new pyrrolo[3,4-a]
carbazole scaffold.
Interestingly, the lack of activity of the 7-methoxylated
compounds may suggest that 5-phenylpyrrolo[3,4-a]carbazole-1,3-
diones could rather be considered as cis-locked analogues of 3-
aroylindoles and 1,3-indolin-2-ones, instead of analogues of ATIs.
Indeed, in the first two series, and unlike ATIs, compounds bearing
a methoxyl at a position equivalent to the 8-position on the
carbazole scaffold (i.e., the 6-position, cf. Fig. 2) were found to be
the most active ones [9,10,11].
6. Experimental section
5. Conclusion
All reagents were used as purchased without further treatment
unless otherwise stated. All solvents were dried according to
standard procedures. Melting points were determined on a LEICA
VM microscope equipped with a heating stage and are uncorrected.
Infrared spectra were obtained with a Nicolet 510 FT-IR apparatus.
NMR spectra were recorded at 300 or 400 MHz (1H) and at 75 or
100 MHz (13C) with a AC 300 and an Advance 400 BRUKER spec-
By means of a systematic biological screening of 31 new 5-
phenylpyrrolo[3,4-a]carbazole-1,3-diones, based on three simple
tests, several cytoskeletal disrupting compounds were identified.
Of particular interest, the carbazole 30, bearing a methoxyl at the 8-
Table 4
Inhibition of tubulin polymerization of selected compounds.
trometers. Chemical shifts are given in parts per million (ppm,
d)
Compound
IC50
3.49
>30
1.13
0.82
0.27
0.37
[
m
M]a
relative to solvent peaks as internal standards ( : CDCl3: 7.27 ppm
d
(1H), 77.0 ppm (13C); DMSO-d6: 2.50 ppm (1H), 40.6 ppm (13C);
acetone-d6: 2.05 ppm (1H), 29.8 and 206.0 ppm (13C); coupling
constants are given in Hertz (Hz, J)). Mass spectra (MS) were
measured with a Nermag R10-10C mass spectrometer (CI/NH3) or
with a ZQ 2000 Waters mass spectrometer (ESI); High-resolution
mass spectrometry (HRMS) spectra were performed at the Labo-
ratoire de Spectrométrie de Masse (I.C.S.N./C.N.R.S., Gif sur Yvette,
7
12
14
30
Colchicine (1)
CA-4 (2)
a
Above a threshold value of 30 mM, IC50 were not precisely determined.

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N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
Fig. 4. Morphological effects of selected 5-phenylpyrrolo[3,4-a]carbazole-1,3-diones. Exponentially growing endothelial cells (EA.hy 926) were exposed to the test compounds at
the indicated concentrations and incubated for 2 h (37 ꢀC, 5% CO2). Representative photographs shown were recorded at a magnification of 100ꢃ for Control and CA-4 (2), and at
200ꢃ for compounds 7, 12, 14 and 30.
France). Elemental analysis were performed at the Laboratoire de
Microanalyses (I.C.S.N./C.N.R.S., Gif sur Yvette, France). Analyses
indicated by the symbols of the elements were within ꢂ0.4% of the
theorical values. Flash column chromatography was performed
with silica gel (SDS 60 ACC 35e70 mM). The reactions were moni-
tored by thin layer chromatography (TLC) using Merck Kieselgel 60
F254 silica gel; zones were detected visually under ultraviolet
irradiation (254 and 366 nm) and by spraying with sulfuric vanillin
Fig. 5. Representative inhibition curves of tubulin polymerization with various concentrations of compounds 2 (CA-4, open circles), 30 (solid circles), 14 (open squares), and 7 (solid
triangles).

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3733
followed by heating. OPLC (Optimum Performance Layer Chroma-
tography) was performed with a OPLC 50Ô Bionisis apparatus.
J ¼ 2.2 Hz, 1H), 6.63 (d, J ¼ 8.7 Hz, 1H), 7.02 (d, J ¼ 2.2 Hz, 1H),
7.32e7.23 (m, 5H),10.15 (br. s, D2O exch.,1H, NH); 13C NMR (75 MHz,
acetone-d6):
d
¼ 35.1, 38.9, 39.2, 40.3, 41.7, 54.7, 55.5 (x 2), 59.7, 94.6,
6.1. General procedure 1 for the synthesis of tetrahydrocarbazoles
with N-benzylmaleimide: preparation of compounds 5e10
105.6 (x 2),108.6,112.9,119.9,120.3,126.6,127.5,128.0 (x 2),128.4 (x
2), 136.4, 137.1, 138.1, 139.6, 153.3 (x 2), 156.2, 175.4, 178.0; IR (NaCl):
n
0 3372, 2930, 1774, 1702, 1590, 1455 cmꢂ1; MS (ZQ2000/ESIþ): m/z
To a mixture of indole, 3,4,5-trimethoxyacetophenone and N-
benzylmaleimide in equimolar quantities (0.86 mmol) were
successively added absolute ethanol (5 mL) and a 35% aqueous
solution of hydrochloric acid (from 0.33 to 1.3 equivalents,
depending on the case). The mixture was heated at reflux until the
disappearance of the indole (usually noted after a reaction time of
16e20 h) and, after being cooled in an ice bath, was filtered. The
precipitate was then washed with cold ethanol and dried over P2O5.
A silica gel chromatography of the precipitate could be performed
to isolate the desired tetrahydrocarbazole if needed.
549 [M þ Na]þ, 565 [M þ K]þ; Anal. C31H30N2O6 (C, H, N).
6.1.4. (þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-5-(30,40,50-trimethoxyphenyl)-
7-nitro-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-1,3(2H,3aH)-
dione (10)
General procedure
1 was performed with 5-nitroindole
(0.86 mmol) and 100 L (1,3 eq) of 35% HCl. After 3 days, the crude
m
precipitate (148 mg), consisting of carbazole 26 and tetrahy-
drocarbazole 10 in a 15:85 ratio (as estimated by 1H NMR analysis;
yields: 8 and 25%, respectively), was purified with difficulty by flash
chromatography on silica gel (cyclohexane/EtOAc 95/5), providing
pure compounds 26 and 10.
6.1.1. (þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-5-(30,40,50-trimethoxyphenyl)-
5,10,10 b-tetrahydropyrrolo[3,4-a]carbazole-1,3(2H,3aH)-dione (5)
Tetrahydrocarbazole 10 was obtained as a yellow fluffy solid. Mp
According to general procedure 1 (volume of 35% HCl: 25
eq), compound 5 was obtained as a pale pink solid (242 mg, 57%). Mp
(CH2Cl2/MeOH): 112e115 ꢀC; 1H NMR (400 MHz, CDCl3):
mL, 0.33
(acetone/MeOH): 228e229 ꢀC. 1H NMR (300 MHz, acetone-d6):
d
¼ 2.18 (ddd, J ¼ 13.1 Hz, J ¼ 10.1 Hz, J ¼ 9.0 Hz, 1H), 2.55 (ddd,
d
¼ 2.12 (m,
J ¼ 13.1 Hz, J ¼ 6.0 Hz, J ¼ 4.3 Hz,1H), 3.65 (ddd, J ¼ 10.1 Hz, J ¼ 8.5 Hz,
J ¼ 6.0 Hz, 1H), 3.68 (s, 6H, OCH3 ꢃ 2), 3.75 (s, 3H, OCH3), 4.36 (ddd,
J ¼ 9.0 Hz, J ¼ 4.3 Hz, J ¼ 1.6 Hz,1H), 4.53 (dd, J ¼ 8.5 Hz, J ¼ 1.6 Hz,1H),
4.53 (d, J ¼ 14.6 Hz, 1H), 4.61 (d, J ¼ 14.6 Hz, 1H), 6.55 (s, 2H),
7.32e7.23 (m, 5H), 7.65 (d, J ¼ 9.1 Hz,1H), 7.68 (d, J ¼ 2.2 Hz,1H), 8.00
(dd, J ¼ 9.1 Hz, J ¼ 2.2 Hz,1H),11.12 (br. s, D2O exch.,1H, NH); 13C NMR
1H), 2.47 (m, 1H), 3.54 (m, 1H), 3.65 (s, 6H, OCH3 ꢃ 2), 3.72 (s, 3H,
OCH3), 4.26 (m, 1H), 4.41 (d, J ¼ 7.5 Hz, 1H), 4.49 (d, J ¼ 14.6 Hz, 1H),
4.57 (d, J ¼ 14.6 Hz, 1H), 6.47 (s, 2H), 6.77 (d, J ¼ 7.5 Hz, 1H), 6.82 (t,
J ¼ 7.5 Hz, 1H), 7.05 (td, J ¼ 7.5 Hz, J ¼ 1.0 Hz, 1H), 7.29e7.23 (m, 5H),
7.47 (d, J ¼ 7.5 Hz, 1H), 10.35 (br. s, D2O exch., 1H, NH); 13C NMR
(75 MHz, CDCl3):
d
¼ 36.4, 40.3, 40.7, 41.7, 43.2, 56.9 (x 2), 61.1, 107.2
(75 MHz, acetone-d6):
d
¼ 34.6, 38.5, 39.2, 40.2, 41.9, 55.6 (x 2), 59.8,
(x 2),113.0,114.4,120.0,120.8,122.7,127.5,129.5 (x 6),137.9,138.7 (x
105.9 (x 2), 111.5, 115.8, 116.3, 116.7, 125.3, 127.6, 128.1 (x 2), 128.4 (x
2), 132.2, 136.3, 137.7, 138.5, 140.2, 141.1, 153.6 (x 2), 174.8, 177.6; IR
(NaCl): n0 3330, 2924, 1778, 1704, 1587, 1458 cmꢂ1; MS (ZQ2000/
ESIþ): m/z 564 [M þ Na]þ; Anal. C30H27N3O7 (C, H, N).
2),141.0,154.8 (x 2),177.1,179.4; IR (NaCl): n
0 3374, 3060, 2998, 2939,
2838, 1776, 1702, 1591, 1506, 1461, 1422 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 519 [M þ Na]þ, 535 [M þ K]þ; Anal. C30H28N2O5 (C, H, N).
6.1.2. (þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-7-methoxy-5-(30,40,50-
trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-
1,3(2H,3aH)-dione (6)
6.1.5. 2-Benzyl-5-(30,40,50-trimethoxyphenyl)-7-nitropyrrolo[3,4-a]
carba-zole-1,3(2H-10H)-dione (26)
Carbazole 26 was obtained as a bright yellow solid. Mp (acetone/
General procedure 1 was performed with 5-methoxyindole
n-hexane): 316e318 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.82 (s,
(0.86 mmol) and 100
mL (1.3 eq) of 35% HCl. After 22 h, tetrahy-
3H, OCH3), 3.83 (s, 6H, OCH3 ꢃ 2), 4.88 (s, 2H), 7.04 (s, 2H), 7.37e7.8
(m, 5H), 7.68 (s, 1H), 7.81 (d, J ¼ 9.1 Hz, 1H), 8.39 (dd, J ¼ 9.1 Hz,
J ¼ 2.3 Hz, 1H), 8.57 (d, J ¼ 2.3 Hz, 1H), 13.06 (br. s, D2O exch., 1H,
drocarbazole 6 was obtained pure as a white fluffy solid (171 mg,
38%). Mp (acetone/MeOH): 182e183 ꢀC; 1H NMR (300 MHz,
acetone-d6):
d
¼ 2.14 (ddd, J ¼ 13.1 Hz, J ¼ 9.6 Hz, J ¼ 8.8 Hz,1H), 2.47
NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 41.3, 56.6 (x 2), 60.8, 106.7
(ddd, J ¼ 13.1 Hz, J ¼ 5.9 Hz, J ¼ 4.5 Hz, 1H), 3.53 (m, 1H), 3.54 (s, 3H,
OCH3), 3.68 (s, 6H, OCH3 ꢃ 2), 3.73 (s, 3H, OCH3), 4.23 (ddd, J ¼ 8.8 Hz,
J ¼ 4.5 Hz, J ¼ 1.4 Hz,1H), 4.39 (dd, J ¼ 8.4 Hz, J ¼ 1.4 Hz,1H), 4.49 (d,
J ¼ 14.6 Hz,1H), 4.58 (d, J ¼ 14.6 Hz,1H), 6.22 (d, J ¼ 2.5 Hz,1H), 6.52
(s, 2H), 6.71 (dd, J ¼ 8.8 Hz, J ¼ 2.5 Hz,1H), 7.31e7.22 (m, 5H), 7.36 (d,
J ¼ 8.8 Hz, 1H), 10.20 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz,
(x 2), 113.1, 113.3, 116.2, 119.3, 121.0, 123.5, 126.5, 127.7 (x 2), 127.9,
129.1 (x 2), 130.9, 134.0, 135.6, 137.3, 138.6, 141.1, 144.1, 146.6, 153.9
(x 2), 167.8, 168.5; IR (NaCl): n0 3319, 2921, 2840, 1761, 1711, 1582,
1486, 1402 cmꢂ1; MS (ZQ2000/ESIþ): m/z 560 [M þ Na]þ, 576
[M þ K]þ; Anal. C30H23N3O7 (C, H, N).
acetone-d6):
d
¼ 34.8, 38.9, 39.3, 40.3, 41.8, 54.6, 55.5 (x 2), 59.6,
6.1.6. (þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-8-bromo-7-methoxy-5-
(30,40,50-trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]
carbazole-1,3(2H, 3aH)-dione (8)
General procedure 1 was performed with 6-bromo-5-methox-
yindole (0.86 mmol) and 25 mL (0.33 eq) of 35% HCl. After 18.5 h, the
crude product was purified by flash chromatography on silica gel
(cyclohexane/EtOAc 96/4), providing tetrahydrocarbazole 9 as
a light beige solid (270 mg, 52%). Mp (acetone/MeOH): 215e216 ꢀC;
101.2, 105.8 (x 2), 111.3, 111.8, 112.9, 126.3, 127.5, 128.4e128.0 (x 4),
128.5, 132.3, 136.4, 137.1, 139.4, 153.3 (x 2), 153.5, 175.3, 178.0; IR
(NaCl): n0 3380, 2929, 1778, 1702, 1590, 1459 cmꢂ1; MS (ZQ2000/
ESIþ): m/z 549 [M þ Na]þ, 565 [M þ K]þ; Anal. C31H30N2O6 (C, H, N).
6.1.3. (þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-8-methoxy-5-(30,40,50-
trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-
1,3(2H,3aH)-dione (7)
General procedure 1 was performed with 6-methoxyindole
(0.86 mmol) and 25 mL (0.33 eq) of 35% HCl. After 21 h, tetrahy-
1H NMR (300 MHz, acetone-d6):
d
¼ 2.13 (ddd, J ¼ 13.1 Hz, J ¼ 10.1 Hz,
J ¼ 9.2 Hz,1H), 2.49 (ddd, J ¼ 13.1 Hz, J ¼ 5.9 Hz, J ¼ 4.3 Hz,1H), 3.55 (s,
3H, OCH3), 3.58 (ddd, J ¼ 10.1 Hz, J ¼ 8.5 Hz, J ¼ 5.9 Hz,1H), 3.69 (s, 6H,
OCH3 ꢃ 2), 3.73 (s, 3H, OCH3), 4.23 (ddd, J ¼ 9.2 Hz, J ¼ 4.3 Hz,
J ¼ 1.6 Hz,1H), 4.41 (dd, J ¼ 8.5 Hz, J ¼ 1.6 Hz,1H), 4.51 (d, J ¼ 14.6 Hz,
1H), 4.60 (d, J ¼ 14.6 Hz, 1H), 6.25 (s, 1H), 6.54 (s, 2H), 7.31e7.24 (m,
5H), 7.69 (s, 1H), 10.33 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz,
drocarbazole 7 was obtainedpure as a pink fluffysolid (108 mg, 24%).
Mp (acetone/MeOH): 217e218 ꢀC. 1H NMR (300 MHz, acetone-d6):
d
¼ 2.08 (m,1H), 2.45 (ddd, J ¼ 13.1 Hz, J ¼ 5.9 Hz, J ¼ 4.5 Hz,1H), 3.52
(ddd, J ¼ 9.7 Hz, J ¼ 8.5 Hz, J ¼ 5.9 Hz,1H), 3.66 (s, 6H, OCH3 ꢃ 2), 3.73
(s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.21 (ddd, J ¼ 8.5 Hz, J ¼ 4.5 Hz,
J ¼ 1.5 Hz,1H), 4.36 (dd, J ¼ 8.4 Hz, J ¼ 1.5 Hz,1H), 4.50 (d, J ¼ 14.6 Hz,
1H), 4.59 (d, J ¼ 14.6 Hz, 1H), 6.48 (s, 2H), 6.51 (dd, J ¼ 8.7 Hz,
acetone-d6):
d
¼ 34.7, 39.0, 39.2, 40.3, 41.8, 55.6 (x 3), 59.7, 101.7,
105.9 (x 3), 113.3, 115.5, 125.8, 127.5, 128.0 (x 2), 128.4 (x 2), 129.3,
132.4, 136.4, 137.2, 139.0, 149.3, 153.5 (x 2), 175.1, 177.9; IR (NaCl): n0

3734
N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3372, 2925, 1778, 1702, 1590, 1457, 1419 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 629 [M þ Na]þ, 645 [M þ K]þ; Anal. C31H29BrN2O6 (C, H, N).
7.06 (ddd, J ¼ 8.1 Hz, J ¼ 6.4 Hz, J ¼ 1.8 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz,
1H), 10.06 (br. s, D2O exch., 1H, NH), 10.30 (br. s, D2O exch., 1H, NH);
13C NMR (75 MHz, acetone-d6):
d
¼ 34.4, 38.8, 40.4, 41.4, 55.4 (x 2),
6.1.7. N-[(þ/ꢂ)-(3aR,5R,10bR)-2-Benzyl-1,2,3,3a,4,5,10,10b-
octahydro-7-methoxy-5-(30,40,50-trimethoxyphenyl)-1,3-
dioxopyrrolo[3,4-a]carbazol-8-yl]-2,2,2-trifluoroacetamide (9)
General procedure 1 was performed with 2,2,2-trifluoro-N-(5-
59.6, 105.7 (x 2), 111.2, 112.8, 118.6, 119.2, 121.3, 126.1, 128.3, 137.0,
137.3, 139.8, 153.3 (x 2), 176.2, 178.8; IR (KBr): n0 3470, 3345, 3186,
3073, 2942, 1784, 1708, 1592, 1508, 1459 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 429 [M þ Na]þ; Anal. C23H22N2O5 (C, H, N).
methoxyindol-6-yl)acetamide (0.86 mmol) and 25 mL (0.33 eq) of
35% HCl. After 21 h, the crude product was purified by flash chro-
matography on silica gel (cyclohexane/EtOAc 95/5), providing tet-
rahydrocarbazole 9 as a white fluffy solid (229 mg, 42%). Mp
(acetone/MeOH): 215e216 ꢀC; 1H NMR (300 MHz, acetone-d6):
6.2.2. 5-(30,40,50-Trimethoxyphenyl)pyrrolo[3,4-a]carbazole-1,3
(2H,10H)-dione (28)
Carbazole 28 was obtained as an orange amorphous solid (58 mg,
8%). Mp (acetone/n-hexane): 342e344 ꢀC. 1H NMR (300 MHz,
d
¼ 2.14 (m, 1H), 2.48 (ddd, J ¼ 13.4 Hz, J ¼ 5.8 Hz, J ¼ 4.4 Hz, 1H),
acetone-d6):
(s, 1H), 7.47 (m, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.67 (d, J ¼ 8.0 Hz, 1H),
11.21 (br. s, D2O exch., 1H, NH), 12.29 (br. s, D2O exch., 1H, NH); 13
NMR (75 MHz, acetone-d6):
2), 114.5, 120.4, 121.2, 122.6, 126.7, 128.1, 130.4, 134.1, 135.2, 138.1,
143.1 (x 2), 153.6 (x 2), 169.9, 170.5; IR (KBr): n0 3352, 3180, 3050,
2927, 2829, 2742, 1763, 1710, 1581, 1509, 1456 cmꢂ1; MS (ZQ2000/
ESIþ): m/z 425 [M þ Na]þ, 441 [M þ K]þ; Anal. C23H18N2O5 (C, H, N).
d
¼ 3.80 (s, 9H, OCH3 ꢃ 3), 6.92 (s, 2H), 7.10 (m,1H), 7.42
3.57 (ddd, J ¼ 9.9 Hz, J ¼ 8.5 Hz, J ¼ 5.8 Hz, 1H), 3.61 (s, 3H, OCH3),
3.68 (s, 6H, OCH3 ꢃ 2), 3.72 (s, 3H, OCH3), 4.24 (ddd, J ¼ 9.1 Hz,
J ¼ 4.4 Hz, J ¼ 1.3 Hz,1H), 4.43 (dd, J ¼ 8.5 Hz, J ¼ 1.3 Hz,1H), 4.50 (d,
J ¼ 14.6 Hz, 1H), 4.58 (d, J ¼ 14.6 Hz, 1H), 6.28 (s, 1H), 6.53 (s, 2H),
7.29e7.23 (m, 5H), 8.30 (s, 1H), 9.19 (br. s, D2O exch., 1H, NH), 10.41
C
d
¼ 56.5 (x 2), 60.7, 106.6 (x 2), 113.0 (x
(br. s, D2O exch., 1H, NH); 13C NMR (75 MHz, acetone-d6):
d
¼ 34.7,
39.0, 39.3, 40.3, 41.8, 55.4, 55.6 (x 2), 59.7, 100.3, 104.7, 105.9 (x 2),
113.4, 120.4, 123.3, 127.5, 128.0 (x 2), 128.4 (x 2), 129.1, 131.1, 136.4,
137.2, 139.6, 144.2, 153.4 (x 2), 175.2, 177.9; IR (NaCl): n0 3403, 2926,
1785, 1705, 1591, 1463, 1435 cmꢂ1; MS (ZQ2000/ESIþ): m/z 660
[M þ Na]þ, 676 [M þ K]þ; Anal. C33H30F3N3O7 (C, H, N).
6.2.3. (þ/ꢂ)-(3aR,5R,10bR)-7-Methoxy-5-(30,40,50-
trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-
1,3(2H,3aH)-dione (13)
General procedure 2 was performed with 5-methoxyindole
(1.72 mmol) and 50 mL (0.33 eq) of 35% HCl. After a reaction time of
6.1.8. 2-Benzyl-7-methoxy-5-(30,40,50-trimethoxyphenyl)-8-
nitropyrrolo[3,4-a]carbazole-1,3(2H,10H)-dione (23)
21h,thecrudeproductwaspurified by flashchromatographyonsilica
gel (CH2Cl2/EtOAc 50/50), providing tetrahydrocarbazole 13 as
General procedure 1 was performed with 5-methoxy-6-nitro-
indole (0.86 mmol) and 75
m
L (1 eq) of 35% HCl. After 2 days, the
a
greenish fluffy solid (229 mg, 30%). Mp (acetone/MeOH):
crude product was purified by flash chromatography on silica gel
(cyclohexane/EtOAc 75/25), providing carbazole 23 as a bright
yellow solid (23 mg, 5%). Mp (acetone/n-hexane): 143e145 ꢀC; 1H
253e255 ꢀC; 1H NMR (300 MHz, acetone-d6):
d
¼ 2.23 (ddd,
J ¼ 13.2 Hz, J ¼ 8.9 Hz, J ¼ 8.3 Hz,1H), 2.45 (ddd, J ¼ 13.2 Hz, J ¼ 6.0 Hz,
J ¼ 4.6 Hz, 1H), 3.47 (td, J ¼ 8.9 Hz, J ¼ 6.0 Hz, 1H), 3.54 (s, 3H, OCH3),
3.69 (s, 6H, OCH3 ꢃ 2), 3.71 (s, 3H, OCH3), 4.22 (ddd, J ¼ 8.3 Hz,
J ¼ 4.6 Hz, J ¼ 1.4 Hz, 1H), 4.35 (dd, J ¼ 8.9 Hz, J ¼ 1.4 Hz,1H), 6.22 (d,
J ¼ 2.4 Hz,1H), 6.55 (s, 2H), 6.70 (dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz,1H), 7.36 (d,
J ¼ 8.8 Hz, 1H), 10.04 (br. s, D2O exch., 1H, NH), 10.12 (br. s, D2O exch.,
NMR (300 MHz, DMSO-d6):
d
¼ 3.63 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 3.82 (s, 6H, OCH3 ꢃ 2), 4.87 (s, 2H), 6.95 (s, 2H), 7.09 (s, 1H),
7.37e7.30 (m, 5H), 7.58 (s, 1H), 8.11 (s, 1H), 12.58 (br. s, D2O exch.,
1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 41.3, 56.6 (x 3), 60.7,
106.5 (x 3), 109.5, 112.5, 114.9, 125.0, 125.6, 127.7 (x 2), 128.8, 129.1
(x 2), 131.1, 134.1, 135.7, 136.0, 137.3, 138.4, 139.6, 144.7, 146.3, 153.8
(x 2), 167.9, 168.7; IR (NaCl): n0 3368, 2931, 2831, 1758, 1708, 1581,
1518, 1469, 1431, 1407 cmꢂ1; MS (ZQ2000/ESIþ): m/z 567 [M þ H]þ,
1H, NH); 13C NMR (75 MHz, acetone-d6):
d
¼ 34.3, 39.0, 40.4, 41.5,
54.6, 55.5 (x 2), 59.6, 101.2, 105.9 (x 2), 111.2, 111.8, 112.7, 126.4, 128.8,
132.3, 137.1, 139.6, 153.3 (x 2), 153.5, 176.2, 178.8; IR (KBr): n0 3408,
3335, 3199, 3076, 2946, 2833, 1783, 1709, 1589, 1463 cmꢂ1; MS
(ZQ2000/ESIþ): m/z 459 [M þ Na]þ; Anal. C24H24N2O6 (C, H, N).
590 [M
þ
Na]þ, 606 [M
þ
K]þ; HRMS (ESIþ): calcd for
C31H25N3O8Na [M þ Na]þ 590.1539, found 590.1564.
6.2.4. (þ/ꢂ)-(3aR,5R,10bR)-8-Methoxy-5-(30,40,50-
trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-
1,3(2H,3aH)-dione (14)
6.2. General procedure 2 for the synthesis of tetrahydrocarbazoles
with maleimide: preparation of compounds 12e17
General procedure 2 was performed with 6-methoxyindole
In these cases, the resulting ethanol solution of reactants was
degassed prior to the heating stage.
(0.86 mmol) and 25 mL (0.33 eq) of 35% HCl. After 16 h, the reaction
mixture was filtered and the mother liquors were thereafter purified
by flash chromatography on silica gel (CH2Cl2/EtOAc 95/5) and then
6.2.1. (þ/ꢂ)-(3aR,5R,10bR)-5-(30,40,50-Trimethoxyphenyl)-4,5,10,10b-
tetrahydropyrrolo-[3,4-a]carbazole-1,3(2H,3aH)-dione (12)
by OPLC (CH2Cl2/EtOAc 60/40, flash volume 750
750 L/min), thus providing 14 as a beige amorphous solid (36 mg,
10%). Mp (acetone/n-hexane): 261e263 ꢀC. 1H NMR (300 MHz,
acetone-d6):
mL, elution flow rate
m
General procedure 2 was applied to 1H-indole (1.72 mmol) in
presence of 50
mL (0.33 eq) of 35% HCl. After a reaction time of 2
d
¼ 2.19 (ddd, J ¼ 13.2 Hz, J ¼ 8.8 Hz, J ¼ 8.0 Hz,1H), 2.43
days, the reaction mixture was filtered. The crude product was then
washed with absolute ethanol, dried under vacuum and purified by
flash chromatography on silica gel, using a gradient of EtOAc in
CH2Cl2 (70/30 / 50/50) as eluent, in order to isolate carbazole 28
and tetrahydrocarbazole 12.
(ddd, J ¼ 13.2 Hz, J ¼ 6.1 Hz, J ¼ 4.8 Hz, 1H), 3.45 (td, J ¼ 8.8 Hz,
J ¼ 6.1 Hz, 1H), 3.67 (s, 6H, OCH3 ꢃ 2), 3.71 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 4.21 (ddd, J ¼ 8.0 Hz, J ¼ 4.8 Hz, J ¼ 1.7 Hz, 1H), 4.31 (dd,
J ¼ 8.8 Hz, J ¼ 1.7 Hz, 1H), 6.50 (dd, J ¼ 8.7 Hz, J ¼ 2.2 Hz, 1H), 6.51 (s,
2H), 6.65 (d, J ¼ 8.7 Hz, 1H), 7.02 (d, J ¼ 2.2 Hz, 1H), 10.04 (br s., D2O
exch., 1H, NH), 10.09 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz,
Tetrahydrocarbazole 12 was obtained as an amorphous orange
solid (272 mg, 39%). Mp (acetone/MeOH): 245e247 ꢀC; 1H NMR
acetone-d6):
d
¼ 34.4, 38.9, 40.4, 41.4, 54.7, 55.4 (x 2), 59.7, 94.6,105.6
(300 MHz, acetone-d6):
d
¼ 2.23 (ddd, J ¼ 13.3 Hz, J ¼ 8.7 Hz,
(x 2), 108.6, 112.8, 119.8, 120.4, 126.8, 137.0, 138.1, 139.9, 153.3 (x 2),
156.2, 176.5, 178.9; IR (NaCl): n0 3406, 3166, 3068, 2938, 2829, 1756,
1710,1628,1591, 1459 cmꢂ1; MS (ZQ2000/ESIþ): m/z 459 [M þ Na]þ,
475 [M þ K]þ; HRMS (ESIþ): calcd for C24H24N2O6Na [M þ Na]þ
459.1532, found 459.1526.
J ¼ 8.0 Hz, 1H), 2.46 (ddd, J ¼ 13.3 Hz, J ¼ 6.1 Hz, J ¼ 4.8 Hz, 1H), 3.48
(td, J ¼ 8.7 Hz, J ¼ 6.1 Hz, 1H), 3.67 (s, 6H, OCH3 ꢃ 2), 3.71 (s, 3H,
OCH3), 4.27 (ddd, J ¼ 8.0 Hz, J ¼ 4.8 Hz, J ¼ 1.5 Hz, 1H), 4.37 (dd,
J ¼ 8.7 Hz, J ¼ 1.5 Hz, 1H), 6.51 (s, 2H), 6.79 (m, 1H), 6.84 (m, 1H),

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3735
6.2.5. (þ/ꢂ)-(3aR,5R,10bR)-5-(30,40,50-Trimethoxyphenyl)-7-nitro-
4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-1,3(2H,3aH)-dione (17)
35% HCl. After 2.5 days, the reaction mixture was filtered to afford
pure carbazole 32 as an amorphous orange solid (12 mg, 3%).
Mother liquors were purified by chromatography on silica gel, using
a gradient of EtOAc in CH2Cl2 (95/5 / 70/30) as eluent, in order to
obtain further amounts of 32 (48 mg, 10%) and 16 (91 mg, 19%).
Tetrahydrocarbazole 16 is a tan amorphous solid. Mp (THF/n-
General procedure
2
was followed with 5-nitroindole
(0.86 mmol) and 100 L (1.3 eq) of 35% HCl. After 7 days, the crude
m
precipitate (89 mg) consisting of an inseparable mixture of carba-
zole 34 and tetrahydrocarbazole 17 in a 15:85 ratio (as estimated by
1H NMR analysis; yields: 4 and 25%, respectively). Purification of 17
was achieved by crystallization of the crude precipitate in a 1:1
mixture of DMSO/MeOH. In contrast, pure carbazole 34, was
obtained by oxidation of 17 (see below).
hexane):157e159 ꢀC; 1H NMR (300 MHz, acetone-d6):
d
¼ 2.23 (ddd,
J ¼ 13.1 Hz, J ¼ 9.6 Hz, J ¼ 8.6 Hz,1H), 2.47 (ddd, J ¼ 13.1 Hz, J ¼ 6.0 Hz,
J ¼ 4.4 Hz,1H), 3.49 (ddd, J ¼ 9.6 Hz, J ¼ 8.6 Hz, J ¼ 6.0 Hz,1H), 3.62 (s,
3H, OCH3), 3.72 (s, 3H, OCH3), 3.70 (s, 6H, OCH3 ꢃ 2), 4.23 (ddd,
J ¼ 8.6 Hz, J ¼ 4.4 Hz, J ¼ 1.5 Hz,1H), 4.38 (dd, J ¼ 8.6 Hz, J ¼ 1.5 Hz,1H),
6.28 (s, 1H), 6.57 (s, 2H), 8.30 (s, 1H), 9.21 (br. s, D2O exch., 1H, NH),
Tetrahydrocarbazole 17 is a bright yellow crystalline solid. Mp
(DMSO/MeOH): 320e322 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 2.16 (m, 1H), 2.31 (m, 1H), 3.40 (m, 1H), 3.62 (s, 6H, OCH3 ꢃ 2),
10.09 (br. s, D2O exch., 1H, NH), 10.36 (br. s, D2O exch., 1H, NH); 13
C
3.64 (s, 3H, OCH3), 4.29 (m, 1H), 4.39 (d, J ¼ 8.4 Hz, 1H), 6.43 (s, 2H),
7.53 (d, J ¼ 9.0 Hz, 1H), 7.60 (d, J ¼ 2.2 Hz, 1H), 7.93 (dd, J ¼ 9.0 Hz,
J ¼ 2.2 Hz, 1H), 11.36 (s br. s, D2O exch., 1H, NH), 12.08 (br. s, D2O
NMR (75 MHz, acetone-d6):
d
¼ 34.3, 39.0, 40.4, 41.5, 55.4, 55.5 (x 2),
59.7, 100.3, 104.7, 105.9 (x 2), 113.2, 116.6 (q, 1JC-F ¼ 286.1 Hz, CF3),
120.4,123.3,129.3,131.0,137.2,139.1,144.2,153.4 (x 2),153.9 (q, 2JC-
exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 33.5, 38.3, 40.5,
F ¼ 36.3 Hz, COCF3),176.1,178.8; IR (NaCl):
n
0 3404, 2938, 2834,1713,
41.2, 56.2 (x 2), 60.5,105.9 (x 2),112.2,115.0,116.1,116.9,125.2,133.1,
136.8, 139.3, 140.4, 140.5, 153.2 (x 2), 177.1, 180.2; MS (ZQ2000/
ESIþ): m/z 474 [M þ Na]þ, 490 [M þ Na]þ; HRMS (ESIþ): calcd for
C23H21N3O7Na [M þ Na]þ 474.1277, found 474.1284.
1592, 1462 cmꢂ1; MS (ZQ2000/ESIþ): m/z 548 [M þ H]þ, 570
[M þ Na]þ, 586 [M þ K]þ; Anal. C26H24F3N3O7 (C, H, N).
6.2.9. 2,2,2-Trifluoro-N-(1,2,3,10-tetrahydro-7-methoxy-5-(30,40,50-
trimethoxyphenyl)-1,3-dioxopyrrolo[3,4-a]carbazol-8-yl)-
acetamide (32)
6.2.6. (þ/ꢂ)-(3aR,5R,10bR)-8-Bromo-7-methoxy-5-(30,40,50-
trimethoxyphenyl)-4,5,10,10b-tetrahydropyrrolo[3,4-a]carbazole-
1,3(2H,3aH)-dione (15)
Carbazole 32 is an orange amorphous solid (60 mg, 13%). Mp
(acetone/n-hexane): 331e333 ꢀC; 1H NMR (300 MHz, acetone-d6):
General procedure 2 was performed with 6-bromo-5-methox-
yindole (0.86 mmol) and 25 mL (0.33 eq) of 35% HCl. After 24 h, the
crude precipitate (181 mg) consisting of a mixture of carbazole 31
and tetrahydrocarbazole 15 in a 10:90 ratio (as estimated by 1H NMR
analysis; yields: 4 and 37% respectively) was purified with difficulty
d
¼ 3.57 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.81 (s, 6H, OCH3 ꢃ 2),
6.93 (s, 3H), 7.43 (s, 1H), 7.87 (s, 1H), 10.75 (br. s, D2O exch., 1H, NH),
11.22 (br. s, D2O exch., 1H, NH), 12.25 (br. s, D2O exch., 1H, NH); 13
NMR (75 MHz, acetone-d6):
C
d
¼ 55.9, 56.6 (x 2), 60.7, 104.2, 106.6 (x
2), 109.6, 113.2, 114.0, 119.6, 125.0, 126.4, 130.4, 134.5, 134.9, 137.1,
138.1, 142.9, 147.0, 153.6 (x 2), 169.9, 170.5, COCF3 and CF3 not
visible; IR (NaCl): n0 3469, 3397, 3298, 2949, 2834, 1768, 1726, 1581,
1470 cmꢂ1; MS (ZQ2000/ESIþ): m/z 544 [M þ H]þ, 566 [M þ Na]þ,
582 [M þ K]þ; Anal. C26H20F3N3O7 (C, H, N).
by OPLC (CH2Cl2/EtOAc 8/2, flash volume: 750
mL, elution flow rate:
750 L), in order to obtain 31 (5 mg) and 15 (34 mg), respectively.
m
Carbazole 31, could also be obtained by oxidation of 15 (see below).
Tetrahydrocarbazole 15 is a beige amorphous solid. Mp (acetone/
n-hexane): 263e265 ꢀC; 1H NMR (300 MHz, acetone-d6):
d
¼ 2.20
(m, 1H), 2.46 (ddd, J ¼ 13.2 Hz, J ¼ 6.0 Hz, J ¼ 4.4 Hz, 1H), 3.48 (ddd,
J ¼ 9.8 Hz, J ¼ 8.6 Hz, J ¼ 6.0 Hz, 1H), 3.54 (s, 3H, OCH3), 3.69 (s, 6H,
OCH3 ꢃ 2), 3.71 (s, 3H, OCH3), 4.20 (ddd, J ¼ 8.7 Hz, J ¼ 4.4 Hz,
J ¼ 1.4 Hz,1H), 4.34 (dd, J ¼ 8.6 Hz, J ¼ 1.4 Hz,1H), 6.24 (s,1H), 6.56 (s,
2H), 7.68 (s, 1H), 10.08 (br. s, D2O exch., 1H, NH), 10.25 (br. s, D2O
6.3. General procedure 3 for the oxidation of tetrahydrocarbazoles
into their corresponding carbazoles: preparation of compounds
19e22, 24, 26, 29e31, 34
To a solution of tetrahydrocarbazole in 1,4-dioxane (concen-
tration of about 0.02 M) was added 2,3-dichloro-5,6-dicyanoqui-
none (2 eq). The reaction mixture was heated under reflux with
stirring during generally 30 min, cooled at room temperature and
filtered on Büchner. The precipitate was washed with CH2Cl2 and
the combined organic phases were successively washed with
saturated aqueous NaHCO3 and water until neutral, dried over
MgSO4, filtered and concentrated under reduced pressure, in order
to obtain the generally pure corresponding carbazole. If necessary,
further purification involved flash chromatography on silica gel.
exch., 1H, NH); 13C NMR (75 MHz, acetone-d6):
d
¼ 34.4, 39.1, 40.3,
41.5, 55.5 (x 3), 59.6,101.7,105.9 (x 3),113.2,115.5,125.8,129.6,132,5,
137.2, 139.2, 149.3, 153.4 (x 2), 176.0, 178.7; IR (KBr): n0 3290, 3199,
3079, 2939, 2851, 1786, 1700, 1594, 1467 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 539 [M þ Na]þ, 555 [M þ K]þ; Anal. C24H23BrN2O6 (C, H, N).
6.2.7. 8-Bromo-7-methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo
[3,4-a]carbazole-1,3(2H-10H)-dione (31)
Carbazole 31 is a pale orange amorphous solid. Mp (acetone/
MeOH): 358e360 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.57 (s, 3H,
OCH3), 3.76 (s, 3H, OCH3), 3.85 (s, 6H, OCH3 ꢃ 2), 6.91 (s, 3H), 7.43 (s,
6.3.1. 2-Benzyl-5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-a]
carbazole-1,3(2H,10H)-dione (19)
According to general procedure 3, carbazole 19 was obtained
from 5 (34 mg, 0.069 mmol) as a yellow amorphous solid (34 mg,
quantitative). Mp (CH2Cl2/n-hexane): 303e305 ꢀC; 1H NMR
1H), 7.84 (s,1H),11.24 (br. s, D2O exch.,1H, NH),12.21 (br. s, D2O exch.,
1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 56.2, 56.6 (x 2), 60.7,
104.7, 106.5 (x 2), 112.4, 113.4, 114.1, 116.8, 121.1, 126.2, 130.8, 134.4,
134.7, 138.0 (x 2), 143.2, 149.6, 153.6 (x 2), 169.8, 170.4; IR (NaCl): n0
3293, 3177, 3068, 2992, 2938, 2823, 2736, 1763, 1709, 1585, 1467,
1429 cmꢂ1; MS (ZQ2000/ESI-): m/z 511 [MꢂH]ꢂ; HRMS (ESI-): calcd
for C24H19Br(79)N2O6 [MꢂH]ꢂ 509.0348, found 509.0351 and calcd
for C24H19Br(81)N2O6 [MꢂH]ꢂ 511.0328, found 511.0350.
(400 MHz, CDCl3):
d
¼ 3.87 (s, 6H, OCH3 ꢃ 2), 4.00 (s, 3H, OCH3),
4.92 (s, 2H, CH2), 6.82 (s, 2H), 7.11 (td, J ¼ 7,8 Hz, J ¼ 1,2 Hz, 1H), 7.28
(m, 1H), 7.34 (t, J ¼ 7,2 Hz, 2H), 7.49 (m, 3H), 7.53 (d, J ¼ 7,8 Hz, 1H),
7.60 (s, 1H), 7.63 (d, J ¼ 7,8 Hz, 1H), 9.27 (br. s, D2O exch., 1H, NH);
13C NMR (75 MHz, CDCl3):
d
¼ 41.5, 56.2 (OCH3 ꢃ 2), 61.1 (OCH3),
6.2.8. 2,2,2-Trifluoro-N-((þ/ꢂ)-(3aR,5R,10bR)-1,2,3,3a,4,5,10,10b-
octahydro-7-methoxy-5-(30,40,50-trimethoxyphenyl)-1,3-
dioxopyrrolo[3,4-a]carbazol-8-yl)acetamide (16)
105.8 (x 2), 111.4, 111.7, 115.8, 120.7, 121.7, 123.2, 127.1, 127.7, 128.1,
128.4 (x 2), 128.7 (x 2), 129.1, 134.3, 134.9, 136.6, 138.2, 141.7, 143.4,
153.5 (x 2), 168.8 (x 2); IR (NaCl): v0 3374, 2932, 2823, 1756, 1694,
General procedure 2 was performed with 2,2,2-trifluoro-N-(5-
1581, 1494, 1456, 1429 cmꢂ1
;
MS (ZQ2000/ESIþ): m/z 515
methoxyindol-6-yl) acetamide (0.86 mmol) and 25
mL (0.33 eq) of
[M þ Na]þ, 531 [M þ K]þ; Anal. C30H24N2O5 (C, H, N).

3736
N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
6.3.2. 2-Benzyl-7-methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo
[3,4-a]carbazole-1,3(2H-10H)-dione (20)
According to general procedure 3, carbazole 20 was obtained from
6 (36 mg, 0.069 mmol) as a yellow amorphous solid (34 mg, 95%). Mp
(acetone/n-hexane): 324e326 ꢀC; 1H NMR (300 MHz, DMSO-d6):
2840,1759,1704,1581,1535,1472,1400 cmꢂ1; MS (ZQ2000/ESIþ): m/z
656 [M þ Na]þ, 672 [M þ K]þ; Anal. C33H26F3N3O7 (C, H, N).
6.3.7. 7-Methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-a]
carbazole-1,3(2H-10H)-dione (29)
d
¼ 3.57(s, 3H, OCH3),3.78(s, 3H, OCH3), 3.81(s,6H, OCH3 ꢃ2), 4.85(s,
According to procedure 3, carbazole 29 was obtained from 13
(30 mg, 0.069 mmol) as a bright yellow amorphous solid (29 mg,
96%). Mp (acetone/n-hexane): 317e319 ꢀC; 1H NMR (300 MHz,
2H), 6.87 (d, J ¼ 2.5 Hz,1H), 6.92 (s, 2H), 7.15 (dd, J ¼ 9.0 Hz, J ¼ 2.5 Hz,
1H), 7.36e7.25 (m, 5H), 7.47 (s,1H), 7.58 (d, J ¼ 9.0 Hz,1H),12.17 (br. s,
D2O exch.,1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 41.2, 55.5, 56.5
DMSO-d6):
d
¼ 3,57 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.81 (s, 6H,
(x 2), 60.7,105.1,106.6 (x 2),111.8,113.7,114.2,117.7,121.6,126.9,127.7
(x 2),127.8,129.1 (x 3),134.6,134.9,137.5,138.1 (x 2),143.2,153.6 (x 2),
153.8, 168.3, 169.0; IR (NaCl): n0 3368, 2927, 2829, 1756, 1694, 1582,
1490, 1464, 1431 cmꢂ1; MS (ZQ2000/ESIþ): m/z 545 [M þ Na]þ, 561
[M þ K]þ; Anal. C31H26N2O6 (C, H, N).
OCH3 ꢃ 2), 6.85 (d, J ¼ 2.5 Hz, 1H), 6.91 (s, 2H), 7.14 (dd, J ¼ 8.9 Hz,
J ¼ 2.5 Hz, 1H), 7.39 (s, 1H), 7.57 (d, J ¼ 8.9 Hz, 1H), 11.17 (br. s, D2O
exch., 1H, NH), 12.12 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz,
DMSO-d6):
d
¼ 55.5, 56.5 (x 2), 60.7, 105.0, 106.6 (x 2), 113.0, 113.6,
113.7, 117.5, 121.6, 126.6, 130.3, 134.5, 135.1, 138.0, 138.1, 143.1, 153.6
(x 2), 153.7, 170.0, 170.5; IR (KBr): n0 3315, 3179, 3058, 2991, 2829,
1760, 1708, 1582, 1508, 1489, 1463, 1417 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 455 [M þ Na]þ, 471 [M þ K]þ; Anal. C24H20N2O6 (C, H, N).
6.3.3. 2-Benzyl-8-methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo
[3,4-a]carbazole-1,3(2H,10H)-dione (21)
Tetrahydrocarbazole 7 (35 mg, 0.066 mmol) was oxidized accord-
ing to general procedure 3. The crude product was purified by flash
chromatography (CH2Cl2/EtOAc 98/2) to isolate the desired carbazole
21 as a bright yellow amorphous solid (24 mg, 70%). Mp (acetone/n-
6.3.8. 8-Methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-a]
carbazole-1,3(2H-10H)-dione (30)
General procedure 3 was applied to tetrahydrocarbazole 14
(30 mg, 0.069 mmol). The crude product was purified by flash
chromatography (CH2Cl2/EtOAc 80/20) to isolate the desired
carbazole 30 as a bright yellow amorphous solid (25 mg, 85%). Mp
(acetone/n-hexane): 344e346 ꢀC; 1H NMR (300 MHz, DMSO-d6):
hexane): 275e277 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.79 (s, 3H,
OCH3), 3.80 (s, 6H, OCH3 ꢃ 2), 3.83 (s, 3H, OCH3), 4.84 (s, 2H), 6.75 (dd,
J ¼ 9.0 Hz, J ¼ 2.3 Hz,1H), 6.92 (s, 2H), 7.14 (d, J ¼ 2.3 Hz,1H), 7.36e7.26
(m, 5H), 7.39 (d, J ¼ 9.0 Hz, 1H), 7.46 (s, 1H), 12.20 (br. s, D2O exch., 1H,
NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 41.1, 55.8, 56.5 (x 2), 60.7, 95.9,
d
¼ 3.77 (s, 6H, OCH3 ꢃ 3), 3.80 (s, 3H, OCH3), 6.72 (dd, J ¼ 8.6 Hz,
106.5 (x 2), 110.0, 111.4, 114.9, 115.1, 123.7, 127.3, 127.7 (x 3), 127.8, 129.1
(x 2),134.4,135.1,137.5,138.0,141.6,145.1,153.6 (x 2),160.4,168.3,168.9;
IR (NaCl): n0 3368, 2938, 2840, 1695, 1624, 1583, 1456, 1412 cmꢂ1; MS
(ZQ2000/ESIþ): m/z 545 [M þ Na]þ, 561 [M þ K]þ; HRMS (ESIþ): calcd
for C31H26N2O6Na [M þ Na]þ 545.1689, found 545.1673.
J ¼ 1.8 Hz, 1H), 6.88 (s, 2H), 7.11 (d, J ¼ 1.8 Hz, 1H), 7.35 (d, J ¼ 8.6 Hz,
1H), 7.36 (s, 1H), 11.11 (br. s, D2O exch., 1H, NH), 12.12 (br. s, D2O
exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 55.8, 56.4 (x 2),
60.7, 95.9, 106.5 (x 2), 109.8, 112.6, 114.6, 114.9, 123.5, 126.9, 129.0,
134.2, 135.2, 137.9, 141.4, 144.9, 153.5 (x 2), 168.2, 169.9, 170.5; IR
(NaCl): n0 3396, 3229, 2918, 2849, 1758, 1701, 1626, 1580,
1468 cmꢂ1; MS (ZQ2000/ESI-): m/z 431 [MꢂH]ꢂ; HRMS (ESI-):
calcd for C24H19N2O6 [MꢂH]ꢂ 431.1243, found 431.1226.
6.3.4. 2-Benzyl-5-(30,40,50-trimethoxyphenyl)-7-nitropyrrolo[3,4-a]
carbazole-1,3(2H-10H)-dione (26)
According to general procedure 3, carbazole 26 was obtained
from 10 (40 mg, 0.074 mmol) as a bright yellow solid (40 mg,
quantitative).
6.3.9. 5-(30,40,50-Trimethoxyphenyl)-7-nitropyrrolo[3,4-a]
carbazole-1,3(2H-10H)-dione (34)
General procedure 3 was applied to tetrahydrocarbazole 17
(128 mg, 0.28 mmol) with a reaction time of 16 h. Carbazole 34
precipitated upon cooling as a yellow amorphous solid and was
collected by filtration, washed several times with saturated aqueous
NaHCO3 and water, dried under vacuum. A further crop was
obtained from mother liquors and was treated as above mentioned
(total: 125 mg, quantitative). Because of its poor solubility, NMR
analyses were performed at 50 ꢀC. 1H NMR (300 MHz, DMSO-d6):
6.3.5. 2-Benzyl-8-bromo-7-methoxy-5-(30,40,50-trimethoxyphenyl)
pyrrolo[3,4-a]carbazole-1,3(2H-10H)-dione (22)
According to procedure 3, carbazole 22 was obtained from 8
(42 mg, 0.069mmol)asabrightyellowamorphous solid (40 mg, 95%).
Mp (acetone/n-hexane): 250e252 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.58(s, 3H, OCH3),3.76(s, 3H, OCH3),3.81(s, 6H, OCH3 ꢃ 2), 4.85(s,
2H), 6.93 (s, 2H), 6.94 (s, 1H), 7.36e7.27 (m, 5H), 7.51 (s, 1H), 7.84 (s,
1H), 12.26 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 3.85 (s, 6H, OCH3 ꢃ 2), 3.86 (s, 3H, OCH3), 7.00 (s, 2H), 7.58 (s,1H),
d
¼ 41.2, 56.2, 56.6(x2), 60.7,104.8,106.5(x2),112.2,112.6,114.6,116.7,
7.82 (d, J ¼ 9.0 Hz, 1H), 8.35 (dd, J ¼ 9.0 Hz, J ¼ 2.0 Hz, 1H), 8.49 (d,
J ¼ 2.0 Hz,1H),11.20 (br. s, D2O exch.,1H, NH),12.72 (br. s, D2O exch.,
121.1,126.5,127.7 (x 2),127.9,129.1 (x 2),129.6,134.6 (x 2),137.4,138.2
(x 2), 143.4, 149.7, 153.7 (x 2), 168.2, 168.9; IR (NaCl): n0 3330, 2927,
2823,1756,1701,1582,1466,1427 cmꢂ1; MS (ZQ2000/ESIþ): m/z 625
[M þ Na]þ; Anal. C31H25BrN2O6 (C, H, N).
1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 56.9 (x 2), 60.8, 107.4 (x
2), 113.3, 114.4, 115.7, 119.2, 121.2, 123.2, 126.5, 132.2, 134.1, 135.6,
139.4, 141.3, 144.0, 146.6, 154.0 (x 2), 169.2, 169.9; IR (KBr): n0 3277,
2938, 2840, 1764, 1735, 1618, 1582, 1490, 1459 cmꢂ1; MS (ZQ2000/
ESIþ): m/z 470 [M þ Na]þ, 486 [M þ K]þ; HRMS (ESI-): calcd for
C23H16N3O7 [MꢂH]ꢂ 446.0988, found 446.1006.
6.3.6. N-(2-Benzyl-1,2,3,10-tetrahydro-7-methoxy-5-(30,40,50-
trimethoxyphenyl)-1,3-dioxopyrrolo[3,4-a]carbazol-8-yl)-2,2,2-
trifluoroacetamide (24)
According to procedure 3, carbazole 24 was obtained from 9
(62 mg, 0.097 mmol) as a bright yellow amorphous solid (61 mg,
quantitative). Mp (acetone/n-hexane): 236e238 ꢀC; 1H NMR
6.3.10. 8-Bromo-7-methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo
[3,4-a]carbazole-1,3(2H-10H)-dione (31)
According to procedure 3, carbazole 31 was obtained from 15
(66 mg, 0.13 mmol) as a pale orange amorphous solid (59 mg, 90%).
(300 MHz, DMSO-d6):
d
¼ 3.59(s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.82 (s,
6H, OCH3 ꢃ 2), 4.85 (s, 2H), 6.94 (s, 2H), 6.96 (s,1H), 7.36e7.27 (m, 5H),
7.51 (s,1H), 7.90 (s,1H),10.74 (br. s, D2O exch.,1H, NH),12.30 (br. s, D2O
6.3.11. 2,2,2-Trifluoro-N-(1,2,3,10-tetrahydro-7-methoxy-5-
(30,40,50-trimethoxyphenyl)-1,3-dioxopyrrolo[3,4-a]carbazol-8-yl)
acetamide (32)
General procedure was applied to tetrahydrocarbazole 16
(40 mg, 0.073 mmol). Purification of the resulting crude product by
exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 41.2, 55.9, 56.6 (x 2),
60.7,104.2,106.6 (x 2),109.6,112.0,114.4,119.6,125.2,126.7,127.7 (x 2),
127.8, 129.1 (x 2), 129.2, 134.6, 134.7, 137.2, 137.5, 138.1, 143.1, 147.1, 153.6
(x 2), 168.2, 168.9, COCF3 and CF3 not visible; IR (NaCl): v0 3401, 2932,

N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
3737
flash chromatography on silica gel (CH2Cl2/EtOAc 87/13) provided
carbazole 32 as a bright yellow solid (29 mg, 74%).
OCH3 ꢃ 2), 4.83 (br. s, D2O exch.*, 4H, CH2, NH*2 overlapped), 6.83
(m, 1H), 6.85 (dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, 1H), 6.90 (s, 2H), 7.35 (s, 1H),
7.36e7.26 (m, 5H), 7.37 (m, 1H), 11.87 (br. s, D2O exch., 1H, NH); 13
C
6.4. General procedure 4 for the reduction of nitro groups:
preparation of compounds 11, 18, 25, 27, 33 and 35
NMR (75 MHz, DMSO-d6):
d
¼ 41.1, 56.5 (x 2), 60.7, 106.0, 106.7 (x
2), 111.0, 113.1, 114.2, 118.2, 122.0, 126.6, 127.6 (x 2), 127.8, 128.4,
129.1 (x 2), 134.5, 135.1, 136.1, 137.6, 138.1, 142.8, 143.0, 153.5 (x 2),
168.4, 169.1; IR (NaCl): n0 3352, 2931, 2836, 1750, 1698, 1581, 1494,
1461, 1428, 1403 cmꢂ1; MS (ZQ2000/ESIþ): m/z 530 [M þ Na]þ, 546
[M þ K]þ; Anal. C30H25N3O5 (C, H, N).
To a solution of nitrotetrahydrocarbazole or nitrocarbazole (1
eq) in THF (C ¼ 0.2 M) were successively added activated zinc
powder (30 eq) and acetic acid (20 eq). The reaction mixture was
stirring 2 h at room temperature until disappearance of starting
material then, diluted with EtOAc and filtered on a celite pad. The
organic phase was washed with saturated aqueous NaHCO3 and
brine, dried over MgSO4, filtered and concentrated under vacuo.
Crude product was purified by flash chromatography on silica gel in
order to afford the desired amino compound.
6.4.4. 7-Amino-5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-a]
carbazole-1,3(2H-10H)-dione (35)
General procedure 4 was applied to nitrocarbazole 34 (79 mg,
0.176 mmol) with a reaction time of 3 h. The crude product was
purified by flash chromatography on silica gel (CH2Cl2/EtOAc 50/50)
to provide the corresponding aminocarbazole 35 as a red amor-
6.4.1. (þ/ꢂ)-(3aR,5R,10bR)-7-Amino-2-benzyl-5-(30,40,50-
trimethoxyphenyl) tetrahydropyrrolo[3,4-a]carbazole-1,3(2H-10H)-
dione (11)
General procedure 4 was applied to nitrotetrahydrocarbazole 10
(62 mg, 0.114 mmol) with a reaction time of 2 h. The crude product
was purified by flash chromatography on silica gel (CH2Cl2/EtOAc
70/30) to provide the corresponding aminotetrahydrocarbazole 11
as a pink lacquer (51 mg, 87%). Mp (THF/n-hexane): 271e273 ꢀC; 1H
phous solid (30 mg, 41%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.81 (s,
9H, OCH3 ꢃ 3), 4.77 (br. s, D2O exch., 2H, NH2), 6.81 (m, 1H), 6.84
(dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, 1H), 6.89 (s, 2H), 7.28 (s, 1H), 7.37 (d,
J ¼ 8.6 Hz,1H),11.06 (br. s, D2O exch., 1H, NH),11.80 (br. s, D2O exch.,
1H, NH); 13C NMR (75 MHz, DMSO-d6):
d
¼ 56.5 (x 2), 60.7, 106.0,
106.6 (x 2), 112.3, 113.0, 113.7, 118.0, 122.0, 126.2, 129.7, 134.4, 135.3,
136.0, 138.1, 142.7, 142.9, 153.4 (x 2), 170.1, 170.7; IR (NaCl): n0 3428,
3388, 3357, 3221, 2924, 2852, 1753, 1702, 1580, 1473 cmꢂ1; MS
(ZQ2000/ESIþ): m/z 418 [M þ H]þ; HRMS (ESIþ): calcd for
C23H20N3O5 [M þ H]þ 418.1403, found 418.1409.
NMR (300 MHz, acetone-d6):
d
¼ 2.07 (m, 1H), 2.48 (ddd,
J ¼ 13.1 Hz, J ¼ 5.9 Hz, J ¼ 4.4 Hz,1H), 2.97 (br. s, D2O exch., 2H, NH2),
3.55 (ddd, J ¼ 10.3 Hz, J ¼ 8.4 Hz, J ¼ 5.9 Hz, 1H), 3.66 (s, 6H,
OCH3 ꢃ 2), 3.72 (s, 3H, OCH3), 4.19 (ddd, J ¼ 8.9 Hz, J ¼ 4.4 Hz,
J ¼ 1.5 Hz, 1H), 4,40 (dd, J ¼ 8.4 Hz, J ¼ 1.5 Hz, 1H), 4,53 (d,
J ¼ 14.6 Hz,1H), 4,60 (d, J ¼ 14.6 Hz,1H), 5.94 (d, J ¼ 2.0 Hz,1H), 6.47
(dd, J ¼ 8.5 Hz, J ¼ 2.0 Hz, 1H), 6.49 (s, 2H), 7.31e7.22 (m, 5H), 7.41
6.4.5. 8-Amino-2-benzyl-7-methoxy-5-(30,40,50-trimethoxyphenyl)
pyrrolo[3,4-a]carbazole-1,3(2H-10H)-dione (25)
To a solution of 24 (40 mg, 0.063 mmol) in 1,4-dioxane (4 mL)
was added
a 0.05 M aqueous solution of K2CO3 (2.5 mL,
(dd, J ¼ 8.5 Hz, J ¼ 0.6 Hz, 1H), 10.23 (br. s, D2O exch., 1H, NH); 13
C
0.125 mmol). The mixture was stirring at room temperature during
17 h until the disappearance of the starting trifluoroacetamide. The
reaction was then quenched by addition of 250 of water and
extracted by EtOAc. The combined organic layers were washed with
water and brine, dried over MgSO4, filtered and concentrated
under vacuo. The crude product was purified by flash chromatog-
raphy on silica gel (CH2Cl2/EtOAc 88/12) in order to obtain carba-
zole 25 as a bright red lacquer (26 mg, 76%). Mp (acetone/n-
NMR (75 MHz, acetone-d6):
d
¼ 35.4, 39.1, 39.3, 40.4, 41.8, 55.4 (x
2), 59.6, 105.6 (x 2), 109.0, 111.2, 112.8, 115.1, 126.2, 127.5, 128.0 (x 2),
128.4 (x 3), 133.9, 136.4, 137.0, 139.6, 144.0, 153.3 (x 2), 175.3, 178.0;
IR (NaCl): n0 3372, 2916, 2845, 1783, 1701, 1589, 1460 cmꢂ1; MS
(ZQ2000/ESIþ): m/z 534 [M
þ
Na]þ, 550 [M
þ
K]þ; Anal.
C30H29N3O5 (C, H, N).
6.4.2. (þ/ꢂ)-(3aR,5R,10bR)-7-Amino-5-(30,40,50-trimethoxyphenyl)
tetrahydropyrrolo[3,4-a]carbazole-1,3(2H-10H)-dione (18)
General procedure 4 was applied to nitrotetrahydrocarbazole 17
(30 mg, 0.066 mmol) with a reaction time of 7 h. The crude product
waspurified byflash chromatographyonsilica gelusing agradientof
EtOAc in CH2Cl2/EtOAc (50/50 / 30/70) to provide the corre-
sponding aminotetrahydrocarbazole 18 as a pale orange amorphous
hexane): 225e227 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 3,62 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 3.90 (s, 6H, OCH3 ꢃ 2), 4.81 (s, 2H),
5.05 (br. s, D2O exch., 2H, NH2), 6.83 (s, 1H), 6.93 (s, 2H), 6,99 (s, 1H),
7.25 (t, J ¼ 7.5 Hz, 2H), 7.32 (t, J ¼ 7.5 Hz, 2H), 7.38 (s, 1H), 7.40 (d,
J ¼ 7.5 Hz, 1H), 10.73 (br. s, D2O exch., 1H, NH); 13C NMR (75 MHz,
DMSO-d6):
d
¼ 40.7, 54.9, 55.8 (x 2), 59.9, 95.1, 103.1, 106.4 (x 2),
111.0, 114.1, 125.9, 127.3, 127.7 (x 2), 128.5 (x 3), 133.7, 135.5, 137.7,
138.3, 139.4, 139.6, 139.8, 140.4, 143.0, 153.7 (x 2), 168.2, 168.7; IR
(NaCl): n0 3484, 3368, 3001, 2935, 2827, 1753, 1694, 1632, 1577,
1489 cmꢂ1; MS (ZQ2000/ESIþ): m/z 560 [M þ Na]þ, 576 [M þ K]þ;
Anal. C31H27N3O6 (C, H, N).
solid (23 mg, 82%). 1H NMR (300 MHz, CDCl3):
d
¼ 2.04 (m, 1H), 2.57
(m, 1H), 3.43 (m, 1H), 3.69 (s, 6H, OCH3 ꢃ 2), 3.82 (s, 3H, OCH3), 4.15
(m, 2H), 6.01 (s, 1H), 6.34 (s, 2H), 6.59 (d, J ¼ 8.3 Hz, 1H), 7.29 (d,
J ¼ 8.3 Hz, 1H), 8.46 (br. s, D2O exch., 1H, NH), 8.63 (br. s, D2O exch.,
1H, NH); 13C NMR (75 MHz, CDCl3):
d
¼ 34.7, 38.9, 40.3, 41.3, 56.0 (x
2), 60.9,104.9,105.0 (x 2),111.6,113.0,113.1,126.7,127.0,131.6,136.6,
138.7, 139.2, 153.1 (x 2), 176.1, 178.6; IR (NaCl): n0 3387, 3332, 3250,
2936, 2837, 1775, 1714, 1590, 1504, 1456 cmꢂ1; MS (ZQ2000/ESIþ):
m/z 422 [M þ H]þ, 444 [M þ Na]þ, 460 [M þ K]þ; HRMS (ESIþ): calcd
for C23H24N3O5 [M þ H]þ 422.1716, found 422.1728.
6.4.6. 8-Amino-7-methoxy-5-(30,40,50-trimethoxyphenyl)pyrrolo
[3,4-a]carbazole-1,3(2H-10H)-dione (33)
Carbazole 32 (40 mg, 0.063 mmol) was treated in the same
conditions than 24. The crude product was purified by flash
chromatography (CH2Cl2/EtOAc 50/50) in order to obtain the cor-
responding aminocarbazole 33 as a red lacquer (24 mg, quanti-
6.4.3. 7-Amino-2-benzyl-5-(30,40,50-trimethoxyphenyl)pyrrolo[3,4-
a]carbazole-1,3(2H-10H)-dione (27)
tative). 1H NMR (400 MHz, DMSO-d6):
d
¼ 3.52 (s, 3H, OCH3), 3.76
(s, 3H, OCH3), 3.81 (s, 6H, OCH3 ꢃ 2), 5.40 (br. s, D2O exch., 2H,
NH2), 6.66 (s, 1H), 6.85 (s, 1H), 6.88 (s, 2H), 7.29 (s, 1H), 10.96 (br. s,
D2O exch., 1H, NH), 11.72 (br. s, D2O exch., 1H, NH); 13C NMR
General procedure 4 was applied to nitrocarbazole 26 (45 mg,
0.084 mmol) with a reaction time of 2 h. The crude product was
purified by flash chromatography on silica gel (CH2Cl2/EtOAc 70/30)
to provide the corresponding aminocarbazole 27 as a red amor-
phous solid (47 mg, quantitative). Mp (acetone/n-hexane):
(75 MHz, DMSO-d6):
d
¼ 55.4, 56.4 (x 2), 60.6, 95.2, 103.0, 106.5 (x
2), 110.1, 112.0, 113.8, 126.8, 128.2, 133.5, 135.6, 137.7, 139.3, 140.1,
140.9, 142.7, 153.4 (x 2), 170.2, 170.8; IR (NaCl): n0 3482, 3364,
3002, 2940, 2831, 1756, 1692, 1635, 1580, 1490 cmꢂ1; MS (ZQ2000/
274e276 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 3.81 (s, 9H,

3738
N. Ty et al. / European Journal of Medicinal Chemistry 45 (2010) 3726e3739
ESI-): m/z 446 [MꢂH]ꢂ; HRMS (ESIþ): calcd for C24H21N3O6
[M þ H]þ 447.1430, found 418.1425.
PME buffer (100 mM PIPES (1,4-piperazinebis(ethanesulfonic
acid)); 1 mM MgSO4; 2 mM EGTA) with 10 M DAPI and varying
concentrations of the test compounds using colchicine as an
internal control. After a preincubation of 45 min at room temper-
m
7. Biological evaluation
ature, 5 ml of 1 mM GTP was added to each well to initiate tubulin
7.1. Cytotoxicity
polymerization, and the plate was then transferred to a thermo-
stated Victor plate reader at 37 ꢀC for an additional 2 h. Fluores-
cence was then read at the excitation wavelength of 360 nm and
emission of 450 nm. The percent inhibition was determined as
Evaluation of cytotoxicity in murine B16 melanoma cells.
Murine B16 melanoma cells were grown in DMEM medium con-
taining 2 mM
penicillin and 100
L
-glutamine, 10% foetal bovine serum, 100 U/mL
follows: 1 ꢂ (
D
F(sample)/
D
F(control) ꢃ 100), where
D
F control ¼ F
m
g/mL streptomycin (3 ꢀC, 5% CO2). All
(no inhibition) ꢂ F(complete inhibition), and
D
F sample ¼ F
compounds were initially dissolved in DMSO at a stock concen-
tration of 2.5 mg/mL and were further diluted in cell culture
medium. For comparative purposes, CA-4 and colchicine were
routinely included in the experiments as reference compounds.
Exponentially growing B16 cells were plated onto 96 well plates at
5000 cells per well in 100
after plating, 100 l of medium containing the compound of
interest at final concentrations ranging from 0.01 to 100 M were
(sample) ꢂ F(complete inhibition with colchicine). The IC50 for
compound-induced inhibition of tubulin polymerization is the
concentration of compound at which the extent of inhibition of
polymerization is 50% of the maximum value as determined from
the semi-logarithmic plot of percent inhibition as a function of the
drug concentration using the nonlinear regression software Sig-
maPlot (Jandel Scientific).
ml of culture medium. Twenty-four hour
m
m
added to the wells (in triplicate) containing the cells, and incubated
for 48 h at 37 ꢀC and 5% CO2. After the 48 h exposure period to the
test compounds, cell viability was assayed using the MTT test [17]
and absorbance was read at 56 m in a microplate reader (Bio-
Kinetics Reader, EL340). Appropriate controls with DMEM only and
MTT were run to substract background absorbance. Results are
presented as percent of controls containing 1% DMSO, which was
not cytotoxic at this concentration. The concentration of compound
that inhibited cell viability by 50% (inhibitory concentration for 50%
of cells, or IC50) was determined using the GraphPad Prism soft-
ware. Results are presented as the mean ꢁ SEM of 3e7 independent
experiments each run in triplicate.
Acknowledgements
The authors are grateful to the “Ministère de l’Enseignement
Supérieur et de la Recherche” and the “Association pour la
Recherche sur le Cancer” (ARC, Villejuif, France) for financial
support to N.T. and G.D. This research was supported by the “Centre
National de la Recherche Scientifique” (CNRS), by the “Institut
National de la santé et de la recherche médicale” (Inserm), and by
a grant from the “Institut National du Cancer” (INCa, Boulogne
Billancourt, France).
Appendix. Supplementary data
7.2. Effect on the morphology of EA hy 926 cells
Supplementary data associated with article can be found in
online version at doi:10.1016/j.ejmech.2010.05.022.
Effect on the morphology of transformed HUVEC cells (EA$hy
926 cells). To assess the effects of the compounds on the
morphology of endothelial cells, we used the EA$hy 926 endothelial
cell line which is derived from the fusion of human umbilical vein
endothelial cells (HUVEC) with the permanent human cell line
A549 [18]. The EA$hy 926 cell line is considered as one of the best
immortalized HUVEC cell lines because these cells express most of
the biochemical markers of parental HUVEC EA$hy 926 cells [19],
originally obtained from Dr Cora-Jean S. Edgell (Pathology
Department, University of North Carolina, Chapel Hill, NC 27599-
7525, USA) were used with her permission, and were grown in
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