Synthesis and biological evaluation of novel naphthocarbazoles as potential anticancer agents

Article abstract of DOI:10.1021/jm049213f

We report the efficient synthesis involving palladium-catalyzed reactions and biological evaluation of new naphthocarbazoles designed as potential anticancer agents. The use of 5- and 6-benzyloxyindoles generated three substitution sites which were succes

Full text of DOI:10.1021/jm049213f

J. Med. Chem. 2005, 48, 1401-1413
1401
Synthesis and Biological Evaluation of Novel Naphthocarbazoles as Potential
Anticancer Agents
Sylvain Routier,†,* Paul Peixoto,† Jean-Yves Me´rour,† Ge´rard Coudert,† Nathalie Dias,‡ Christian Bailly,‡
Alain Pierre´,§ Ste´phane Le´once,§ and Daniel-Henry Caignard§
Institut de Chimie Organique et Analytique, UMR CNRS 6005, Universite´ d’orle´ans, Rue de Chartres, BP 6759,
45067 Orle´ans Cedex 2, France, INSERM U-524, IRCL, Place de Verdun, 59045 Lille, France, and Institut de Recherches
Servier, Division Recherche Cance´rologie, 125 chemin de Ronde, 78290 Croissy sur Seine, France
Received September 29, 2004
We report the efficient synthesis involving palladium-catalyzed reactions and biological
evaluation of new naphthocarbazoles designed as potential anticancer agents. The use of 5-
and 6-benzyloxyindoles generated three substitution sites which were successively exploited
to introduce several hydrophilic side chains. The cytotoxicity of the newly designed compounds
was evaluated on three cell lines. Several compounds showed a marked cytotoxicity with IC50
values in the sub-micromolar range. This is the case for the 3-hydroxy-naphthopyrrolocarba-
zoledione 37, bearing a dimethylaminoethyl side chain, which is extremely cytotoxic to L1210
and DU145 cells (IC50: 36 nM, 108 nM) and induces an accumulation of L1210 cells in the
G2+M phases of the cell cycle. Some of the most cytotoxic compounds were tested for inhibition
of CDK-5, GSK-3 and topoisomerase I, and their interaction with DNA was also evaluated.
Interaction with DNA was detected, suggesting that nucleic acids represent a privileged target
for these molecules.
Introduction
J-107088) which increases the water solubility and
enhances the molecular interaction with the target.9-13
In the same vein, several modifications of the sugar
moiety have been proposed to improve the water solu-
bility and modulate the topoisomerase I inhibition
property. The SAR relationships in the indolocarbazole
series have been extensively studied in the context of
topoisomerase I inhibition.1
We have recently described the bioisosteric replace-
ment of an indole moiety by a 7-azaindole unit affording
the first symmetrical and dissymmetrical 7-azaindolo-
carbazoles I and II.14 The cytotoxic properties of the
N-glycosylated derivatives of I and II have been re-
ported too.15,16 Recently, macrocyclic polyoxygenated bis-
indoylmaleimide and 7-azaindolylmaleimide III were
described as potent and selective inhibitors of the
glycogen synthase kinase-3â (GSK-3), a multisubstrate
kinase directly involved in diverse vital cellular pro-
cesses and which might represent an interesting thera-
peutic target.17,18 GSK-3 inhibitors might be useful for
the treatment of diabete, Alzheimer’s disease and
protection against cell death in general. Cyclin depend-
ent kinases (CDK) have also raised a considerable
attention because of their central role in the regulation
of cell cycle progression, and these CDKs can be targeted
with indolocarbazoles.19
Indolo[2,3-a]pyrrolo[3,4-c]carbazole alkaloids form a
class of compounds endowed with potent antitumor,
antiviral and/or antimicrobial activities.1 This family
has raised considerable attention because of their
central role in the regulation of cell cycle progression
and specific enzymatic inhibitions.2,3 Structure activity
relationships (SAR) have been reported and can be used
as a guide to design more potent compounds. Closely
related compounds such as arcyriaflavins have been
described as inhibitors of different isoforms of protein
kinase C. This is the case for arcyriaflavin-A (Figure 1)
which prevents the replication of the human cytome-
galovirus via an inhibition of the protein kinase pUL97.4
In contrast, compounds bearing a pyrroloindolocarbazole
with one N-glycosidic bond, such as the antibiotic
rebeccamycin, generally function as DNA topoisomerase
I inhibitors whereas similar compounds with two N-
glucosidic bonds, such as staurosporine, are mainly
nonspecific protein kinase C (PKC) inhibitors.1 A few
rebeccamycin analogues such as NSC655649 (also known
as BMS 181176) and NB-506 have entered clinical trials
for cancer treatment.5 The later compound NB-506 has
been subsequently replaced with a more potent deriva-
tive J-107088 which is also an indolocarbazole with a
functionalized maleimide nitrogen.6,7 Very recently, a
promising series of fluorinated analogues targeting
topoisomerase I has also been described.8 One of the key
molecular element of these compounds is their hydro-
philic side chain (e.g. diethylaminoethyl chain for
NSC655649, bis-(hydroxymethyl)methylamino chain for
To develop selective kinase inhibitors acting in the
ATP binding site, modifications of the indolocarbazole
moiety appeared as a valid alternative to the restrictive
synthesis of glycosylated compounds. For example, some
derivatives containing an indeno moiety fused to the
carbazole skeleton equipped with a lactam staurosporin-
like structureIV have been described as potent vascular
endothelial growth factor R2 (VEGF-R2) tyrosine ki-
nase inhibitor. VGEF is a key mediator of angiogenesis
and evidence has accumulated that tumor VEGF ex-
* Corresponding author. Tel: 33 2 38494853. Fax: 33 2 38417281.
e-mail: sylvain.routier@univ-orleans.fr.
† Universite´ d’orle´ans.
‡ INSERM U-524.
§ Institut de Recherches Servier.
10.1021/jm049213f CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/12/2005

1402 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Routier et al.
Figure 1. Modifications of the indolocarbazole skeleton.
pression was clinically associated with solid malignan-
cies.20 In the maleimide-containing series of rebecca-
mycin aglycons, indole versus (hetero)arylcarbazoles
replacement represents an interesting strategy. Granu-
latimide and isogranulatimide (Figure 1) have been
described as G2 checkpoints inhibitors.21-24 For our own
part we reported a preliminary synthesis of some
naphthalenic compounds V.25 Replacing also one of the
two indoles by a phenyl, pyridine, naphthalene, quino-
line or isoquinoline led to the identification of novel
potent cyclin D1/CDK1 or cyclin D1/CDK4 inhibitors.26-29
These examples indicated also that a slight chemical
modification of the indolocarbazole framework can have
a profound impact on the mechanism of action and the
biological activity. All these considerations prompted us
to describe our extensive efforts to design novel anti-
cancer agents in the naphthalenic series.
Figure 2. Retrosynthetic route.
trated solution, which limits the amount of formed
product.38-40 Palladium-catalyzed reactions or Wacker
type reactions represent interesting alternatives to the
photochemical step. By contrast, cross-coupling reac-
tions were scarcely described.41-44 Recently such strat-
egy was also used by Cephalon or Lilly groups20,26-29
using a similar approach. All these studies indicated
that Suzuki, Stille and intramolecular Heck reactions
were quite efficient but never used in the same sequence
to complete the synthesis of indolocarbazoles and related
structures.
Chemistry
Various synthetic methods30 have been used to modify
the indolocarbazole skeleton and to diversify the rep-
resentative panel of symmetrical or unsymmetrical
indolocarbazoles. Direct formation of the maleimide
framework through reaction of indole-3-acetamidate31
or by an oxidative coupling of indolyl acetate dianion
have been reported.32 Generation of the second indoyl
moiety via nitrene insertion, use of 2,2′-bisindole as
diene for a (4 + 2) Diels Alder cycloaddition with
dimethyl acetylene dicarboxylate or N-phenylmaleimide
have also been described.33-37 An alternative and more
common synthesis consisted of the selective 3-alkylation
of indole with a dibromomaleimide using Grignard
reagents to produce bisindoylmaleimides. This proce-
dure often requires an oxidative photochemical benz-
annulation, carried out by irradiation in a low concen-
To avoid the inconstant irradiation step, which is
always a limiting synthetic factor, we designed an
original route to naphthocarbazoles using a multistep
catalyzed palladium synthesis. The ideal straightfor-
ward retrosynthetic scheme consisted of the introduction
of the naphthyl parts by Stille or Suzuki procedures
followed by an intramolecular Heck reaction (Figure 2).
Our first efforts25 consisted of the synthesis of com-
pound 1, classically obtained from indole and 2,3-
dibromo-N-methylmaleimide in the presence of LiH-
MDS as a base according to a reported procedure.41
Compound 1 was either N-Boc or N-benzenesulfonyl
protected. These protective groups can be easily re-
moved either in acidic or basic conditions. N-Boc pro-
tected compound 2 was prepared using Boc2O, DMAP,
and triethylamine as a base whereas the benzenesulfon-

Naphthocarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1403
Scheme 1
Scheme 2
tive to temperature seems to be the best-adapted
protection of indolic moiety compared to the conven-
tional Boc group which was lost by heating.
A similar study was realized using the boronic acid 5
via a Suzuki procedure. Standard conditions involved
Pd(OAc)2 (10%) as a catalyst in the presence of K2CO3
as a base in a mixture of dioxane and water. These
cross-coupling reactions appeared more efficient than
Stille reactions with a shorter reaction time (2 h). From
compound 1 this approach gave access to compound 8
in a moderate 55% yield but obtained after 2 h at 100
°C (or 5 h at 90 °C). A similar yield was obtained using
N-Boc protected compound 2 and, as described before,
only the deprotected compound 8 was obtained. The best
result was obtained with the benzenesulfonyl compound
3 which gave compound 10 in 80% yield. In addition,
the deprotected compound 8 could be easily converted
into compound 10 with a 96% yield using NaH as a base
in the presence of benzenesulfonyl chloride.
The O-demethylation was equally performed on com-
pounds 8 and 10 using BBr3 to afford 11 and 12 in 72
and 98% yields, respectively (Scheme 1). The hydroxy
compounds 11 and 12 were converted into triflates 13
and 14 in 88 and 98% yields by reaction with triflic
anhydride in the presence of triethylamine (no reaction
was observed on the indolic nitrogen atom with com-
pound 11). The central six-membered ring was then
obtained by an intramolecular Heck reaction (Table 1).
This reaction was carried out at 100 °C using Pd(OAc)2
as a catalyst, PPh3 as a ligand, NaOAc (2 equiv) as a
base and Bu4NCl (1 equiv) as a phase transfer agent.
The N-unprotected indolic compound 13 in DMA or
dioxane afforded the desired compound 15 in only a
modest 40% yield. Compound 14 gave access to a
mixture of 15 and 16 which ratio was under the
influence of the conditions used; surprisingly, the ben-
zenesulfonyl group was partially removed under these
conditions. Compounds 15 and 16 were obtained in 51%
and 10% yields, respectively (entry 2), using the condi-
tions described for 13.
yl protective group was introduced on compound 1 using
NaH, to yield 3 in 85% yield.
Direct introduction of a 2-methoxynaphthyl group on
compound 2 was performed using the very efficient
Suzuki or Stille coupling reaction with palladium de-
rivatives as catalysts (Scheme 1).
First, the (3-methoxynaphthalen-2-yl)-trimethylstan-
nate 4 or (3-methoxy-naphthalen-2-yl)-boronic acid 5
was prepared by selective lithiation of the 2-meth-
oxynaphthalene 6 at -10 °C with n-BuLi (1.85 equiv).45
To attribute the regioselectivity of proton abstraction,
quenching the reaction after 1 h with D2O showed a
complete lithiation with the exclusive formation of the
compound 7. Several assays were then performed with
other electrophiles. Addition of trimethylstannyl chlo-
ride was performed at -78 °C to afford the compound 4
in 95% yield. Similar conditions using an excess of
trimethylborate as electrophile did not afford the desired
boronic acid 5; however, 5 could be obtained in 78% yield
by allowing the reaction mixture to reach room tem-
perature. (Scheme 2).
Having obtained the partners for the Stille or Suzuki
reactions, we performed several assays to optimize these
cross-coupling reactions between indolic derivatives 1-3
and naphthalene organometallic agents 4 and 5. Orga-
nostannane 4 and compounds 1-3 using Pd(PPh3)4 as
catalyst and CuI in dioxane or DMF gave only limited
yield of coupled products 8-10. After some experiments,
compound 8 is obtained in 56% yield from 1 using the
catalytic system Pd2(dba)3, AsPh3 as a ligand and CuI
as an additive. In the case of the N-Boc derivative 2,
compound 8 was only obtained with 12% yield using Pd-
(PPh3)4 as a catalyst and CuI in dioxane or DMF with
no trace of compound 9, while in the presence of LiCl
(3 equiv) in place of CuI, the yield of 8 from 2 increased
to 54%. Finally 10 was obtained from 3 in 64% yield
using the same catalytic system. This Stille coupling
reaction was more efficient in the presence of a N-indolic
protected compound. A benzenesulfonyl group insensi-
Increasing the amount of catalyst to 30% (entry 3)
enhanced the reaction turnover but the deprotected
product 15 was formed and the reaction stopped again.
After 3 days no trace of the protected compound 16 could
be detected. To complete the reaction, we deliberately

1404 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Table 1. Heck Cyclization Procedure on Compounds 13 and 14
Routier et al.
solvent,
temp
time,
h
product
(yield)
entry
1
reactant
catalyst
conditions
13
Pd(OAc)2 10%
PPh3 0.2 equiv,
Bu4NCl 1 equiv,
NaOAc 2 equiv
PPh3 0.2 equiv,
Bu4NCl 1 equiv,
NaOAc 2 equiv
DMA,
100°C
12
15 (40)
2
14
Pd(OAc)2 10%
dioxane,
100°C
16
16 (10)
15 (51)
15 (63)
3
4
14
14
Pd(OAc)2 30%
PPh3 0.2 equiv,
Bu4NCl 1 equiv,
NaOAc 2 equiv
PPh3 0.2 equiv,
Bu4NCl 1 equiv,
NaOAc 2 equiv
dioxane,
100°C
72
3
Pd(OAc)2 1 equiv
dioxane,
100°C
16 (70),
15 (12);
15 (91)a
a Same assay realized with an additional Bu4NF treatment.
Scheme 3
used a stoichiometric amount of palladium acetate. The
reaction rate was considerably increased, the starting
material 14 was fully converted in only 3 h and the final
products 15, 16 were isolated in an overall yield of 82%.
Purification of the protected compound 16 was not so
easy, and a small amount was contaminated with 15
during the purification. The full deprotection of the
crude mixture, using Bu4NF in refluxing THF,46 simpli-
fied the purification procedure and afforded the desired
compound 15 in a 91% yield for the two steps.
To diversify the naphthocarbazole skeleton, we de-
cided to introduce on the indolic moiety hydroxy groups
and to replace the indole moiety by a 7-azaindole unit
to modulate the biological activity by creating potential
supplementary hydrogen bonds with the biological
target(s). Preparation of the 3- indolic or 3-azaindolic
bromomaleimide compounds was realized using de-
scribed procedures. N-protected compounds 17 and 18
were synthesized from the corresponding unprotected
indolic nitrogen derivatives using benzenesulfonyl chlo-
ride in the presence of NaH in 85 and 92% yields,
respectively. The 7-azaindolic derivative 19 was ob-
tained using similar conditions in a 74% yield (Scheme
3).14,41
in a 85% yield using BBr3 in dichloromethane; Suzuki
cross-coupling reactions of 17-19 with 20 afforded
compounds 21-23 in 82, 81 and 81% yields, respectively
(Scheme 3). These compounds were then reacted with
triflic anhydride to give compounds 24-26 in 88, 73 and
97% yields, respectively. Then the intramolecular Heck
reaction was directly followed by the benzenesulfonyl
deprotection using Bu4NF in boiling THF to afford
compounds 27 and 28 in 84 and 95% yields for the two
steps. Unfortunately, the cyclization reaction was in-
efficient using the azaindolic compound 26. The last step
to obtain the fully deprotected derivatives was per-
formed using BBr3 and led to compounds 29 and 30 in
93 and 98% yields, respectively.
The naphthocarbazoles 15, 28-30 could be easily
functionalized and several compounds were then syn-
thesized (Scheme 4) with the goal of reinforcing the
hydrophilic properties of the compounds and hopefully
the biological activity. On one hand, different side chains
were introduced on the N-free indolic nitrogen atom of
compound 15. On the other hand, substitutions were
performed on the maleimide group of 15, 29 and 30
maintaining in all cases the free indolic NH and/or OH
groups. The structures of compounds 31-47 were
reported in Table 2.
Generation of the hydroxy group from the methyl
ether on the naphthalene moiety was hampered by the
presence of the benzyloxy group on the indolic unit. For
this reason, we used a naphthalene boronic acid with a
2-hydroxy substituent instead of a 2-methyl ether
substituent. Transformation of compound 5 into (3-
hydroxy-naphthalen-2-yl)-boronic acid 20 was realized
In the presence of NaH as a base, the N-indolic atom
of 15 was alkylated in THF (entries 1-3). Using 4-(2-
chloroethyl)-morpholine or (2-chloroethyl)-dimeth-
ylamine, compounds 31 and 32 were synthesized in 49
and 35% yields, respectively. Similarly, reaction of
compound 15 with allyl bromide led to 33 in a 51% yield

Naphthocarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1405
Scheme 4
Table 2. Substitution of the Naphthocarbazoles 15, 28-30
a Quantitative on TLC; derivatives soluble in water.
which was converted to compound 34 in a 71% yield by
oxidation using aqueous KMnO4. The creation of this
2,3-propanediol side chain may be considered as a sugar
mimic.
The maleimide function was then modified (entries
5, 6). First, anhydride 35 was obtained by reaction of
compound 15 with KOH in ethanol in a 83% yield.
Further reaction of 35 with (2-aminoethyl)-dimeth-
ylamine generated compound 36 in a 83% yield. This
compound could be directly obtained from compound 15
by boiling in (2-aminoethyl)-dimethylamine in a 88%
yield.
The selective substitution of compounds 29 and 30
on the maleimide moiety while maintaining the combi-
nation of unprotected NH and 2- or 3-OH groups
simultaneously was performed with several primary
amines. Such structures maintain the presence of the
well-known hydroquinonimine system which is consid-
ered as an important element for the bioactivity of such
anticancer agents. These substitutions were realized
with four different chains in order to determine the best
added chain. All substitutions were performed by ex-
change of the N-methyl group in boiling aliphatic amine
to obtain compounds 37-44 in the range 27-100% yield
(entries 7-14).
The last goal concerned the preparation of (i) O-
alkylated compounds in the presence of the unprotected
indolic nitrogen atom and (ii) N-alkylated compounds
in the presence of the hydroxy group in order to
determine the influence of the substitution of each atom
(O, N) by a similar dimethylaminoethyl side chain. First
we realized the direct alkylation of benzyl compound 28
as described for compound 15 to afford compound 45 in
a 85 % yield. Unfortunately, the benzyl deprotection was
unsuccessful despite numerous efforts (acidic deprotec-
tion, hydrogenolysis in the presence of Pd(C) 10%, Pd-
(OH)2...). Therefore, we realized a selective O-silylation
of the deprotected compounds 29 and 30. Starting from
compound 30 the O-TBDMS compound 46 was quanti-
tatively obtained, using TBDMSCl, imidazole and ce-
sium carbonate as a base (Scheme 5); under identical
conditions, compound 29 gave a degradation of the
mixture.
The p-quinoneimine system was too sensitive in this
basic medium. The protected compound 46 treated with
(2-chloroethyl)-dimethylamine (Scheme 5) afforded the

1406 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Routier et al.
Scheme 5
O-alkylated compound 47 in a 69% yield with no
N-alkylation as a result of a prior desylilation reaction
under basic conditions. The same compound 47 was
directly synthesized by the alkylation of compound 30,
in a 41% yield.
hydroxylated compounds. The homologation of the di-
methylamino side chain (addition of one supplementary
methylene unit) decreased sensibly the activity of
compounds 39 and 40. An extension of the chain longer
than four carbon atoms may disfavor the cytotoxic
activity. The introduction the mitoxantrone-type basic
side chain gave compounds 41 and 42 with reduced
activity, thus corroborating the observation that the
extension of the basic chain is detrimental to cytotox-
icity.
The activity is partially restored with compound 44
bearing the ramified bis-hydroxylated side chain but not
with the related molecule 43. Similarly, the capping of
the hydroxy group by a TBDMS protective group or the
dimethylamino side chain (compounds 46 and 47,
respectively) decreased the cytotoxicity to the micromo-
lar range. The position of the free hydroxyl groups is
apparently crucial. It is however difficult to provide a
rational basis for the influence of the position of R2
substituent (compare 37/38 vs 43/44).
Results and Discussion
In Vitro Antiproliferative Activities. The anti-
proliferative activities of the compounds were tested
against three tumor cell lines: murine L1210 leukemia,
human prostate DU145 and human colon HT29 carci-
nomes. The cytotoxicity of the compounds was measured
using a conventional microculture tetrazolium assay.
The IC50 values are collected in Table 3. In the L1210
inhibition assay, the N-methyl maleimide substituted
compounds 15, 29 and 30 showed micromolar activities.
The presence of hydroxyl groups sensibly decreased the
cytotoxicity. The comparison of the IC50 values obtained
with the unsubstituted derivative 15 and the N-indolic
alkylated compounds 31-34 indicates that the intro-
duction of a hydrophilic substituent induces a slight
decrease of activity for compounds 31, 32 and 34
whereas the incorporation of an allyl group decreased
more significantly the activity, by 10 times as seen for
compound 33.
Therefore, a hydrophilic substitution might be prefer-
able to a hydrophobic group. Moreover, the presence at
this position of the 2,3-dihydroxypropyl side chain seems
to be slightly better than no chain or the addition of
basic centers. Whereas these modifications on the
indolic nitrogen center were unsuccessful, replacing the
N-methyl group of 15 by an N-2-dimethylaminoethyl
side chain on 36 increases 5 to 10-fold the cytotoxicity
to 210 nM.
The cytotoxicities of the N-methyl compounds 29 and
30 are comparable to that of 15 with micromolar IC50.
The introduction of the 2-dimethylamino ethyl side
chain on the maleimide moiety enhances markedly the
cytotoxic potential to reach out nanomolar activities as
it is the case for compounds 37 and 38 (IC50: 36 and
110 nM, respectively). This observation tends to confirm
the idea that the addition of a basic side chain on the
maleimide generally improves the efficacy of the com-
pound, whatever the series (indolocarbazole or naph-
thocarbazole).
It is worth mentioning here that similar naphthocar-
bazoles have been recently described by the Lilly’s
group.26,27 In their report, the complete suppression of
the side chain attached to the maleimide skeleton led
to poorly active compounds (micromolar IC50), in agree-
ment with our own observations.
The results obtained with the two human cell lines
DU145 and HT29 are also very informative. Here again,
compounds 36, 37 and 38 show potent activities. In
general, for the different compounds the sensitivity of
the cells follows the order L1210 > DU145 > HT29. This
is not really surprising as leukemia cells are almost
always much more chemosensitive than colon cancer
cells. If one focus on the data obtained with the prostate
DU145 cell line, it appears that the presence of the
indolyl group or the N-hydrophilic substitution in-
creased the cytotoxicity. The position of the indolic
hydroxyl group combined with the presence of the
glycerol-like side chain is more favorable in position-2
than in position-3 (compare activities for compounds 43
and 44). Interestingly, compound 44 is particularly
efficient against the prostate cells (IC50: 6.8 nM) and
may thus represent an interesting lead molecule.
In conclusion of the cytotoxicity measurements, it
appears that the combination of N-dimethylamino
maleimide with an hydroxy group in position 2 or 3
generated four potent drug candidates 37-39 and 44,
Substitutions on the northern part of the molecules
were performed to further modulate the activity of the

Naphthocarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1407
Table 3. In Vitro Antiproliferative Activities [IC50 (µM)] against Three Cell Lines: a Murine Leukemia (L1210), a Human Prostate
Cancer (DU145), a Colon Carcinoma (HT29)a
a IC50 values are presented as means of duplicate experiments. ND: Not determined.
which are all significantly more active than the refer-
ence compound rebeccamycin.16 In addition, the intro-
duction on the maleimide moiety of an alkylamino
pharmacophore group, frequently found in antitumor
agents (e.g. mitoxantrone), led to very cytotoxic com-
pounds 36-42. These new derivatives are the first
naphthocarbazoles with nanomolar cytotoxic activities
(depending on the cell type). They are also the first
isostere analogues of the rebeccamycin aglycone and
represent potential candidates for further in vivo evalu-
ations, currently ongoing.
Cell Cycle Effect and Mechanism of Action.
Three compounds with a dimethylaminoethyl side chain
on the maleimide moiety, 32, 37 and 38, were used to
investigate the cycle cell effects on L1210 leukemia cells.
In all three cases, a significant accumulation of the cells
in the G2+M phases of the cell cycle was observed,
reaching 56, 51 and 61% for compounds 32, 37 and 38,
respectively. The effect is comparable to that observed
with rebeccamycin (69% G2 + M) which exhibited lower
cytotoxicity (IC50 L1210: 0.14 µM) than compounds 37
and 38.16 The molecular target(s) of these compounds
are not known. By analogy with previous studies, we
postulated that the compounds may interfere with
topoisomerases and certain kinases. Indeed, it was
reported recently by the Lilly group that similar naph-
thocarbazoles can inhibit cyclin dependent kinases with
nanomolar IC50 values.27 This prompted us to investi-
gate the effect of our new compounds on CDK-5, but no
significant activity was observed (IC50 > 10 µM).47
Similarly, we tested the compounds for inhibition of the
glycogen synthase kinase-3â (GSK-3) which is a multi-
substrate kinase considered as a promising target for
drugs active against diabetes, Alzheimer‘s disease and
possibly cancer, but here again, no active compounds
was found (IC50 > 10 µM). Topoisomerases represent
another target category for this molecules, as it is the
cases for many indolocarbazoles derived from rebecca-
mycin and NB-506, both inhibiting topoisomerase I,1 or
derived from the synthetic molecule NSC655649 (BMS

1408 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Routier et al.
Figure 3. Effect of increasing concentrations of the compound 37 on the relaxation of plasmid DNA by human topoisomerases
I (a) and II (b). Native supercoiled pKMp27 DNA (0.5 µg) (lane DNA) was incubated with 4 units topoisomerase in the absence
(lane Topo) or presence of the drug at the indicated concentration (µM). The camptothecin (CPT) and etoposide (Etop.) were used
as positive controls (25 µM each). Reactions were stopped with sodium dodecyl sulfate and treatment with proteinase K. DNA
samples were separated by electrophoresis on agarose gels containing ethidium bromide (1 µg/mL). The gels were photographed
under UV light. Nck, nicked; Lin, linear; Rel, relaxed; Sc, supercoiled DNA.
The interesting point is that the enhanced interaction
with DNA coincides with a superior cytotoxicity.
With L1210 cells, compounds 15, 29 and 30 show IC50
values in the 3-5 µM range whereas their analogues
bearing the cationic side chain exhibit much lower IC50,
in the sub-micromolar range. The replacement of the
N-methyl group of 29 with a N-dimethylaminoethyl
chain strengthens the binding to DNA (∆Tm increases
complex
Figure 4. Variation in melting temperature (∆Tm ) Tm
- TmDNA) for the different drugs bound to calf thymus DNA
(grey bars) or poly(dAT)2 (black bars) at a drug-DNA-
(nucleotide) ratio 0.5. Tm measurements were performed in
BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA), at 260 nm with a heating rate of 1 °C/min.
from 1 to 13 °C with CT DNA) and increases the
cytotoxicity 100 fold (IC50 of 3.9 µM and 36 nM for 29
and 37, respectively). These observations further il-
lustrate the key role of such a cationic side chain which
promotes hydrophilicity and target interaction. Al-
though we have yet no direct evidence that the enhanced
cytotoxicity effectively derived from the increased bind-
ing to DNA, this hypothesis is however plausible
because similar effects have been previously observed
with other indolocarbazoles.1 DNA can be considered as
a target for the naphthocarbazoles, at least the cationic
molecules. However, the lack of interaction of the
N-methyl compounds which are also cytotoxic suggests
that targets other than nucleic acids must be involved
in their mechanism of action, as it is generally the case
with these type of molecules.
In conclusion, we have synthesized a large series of
naphthocarbazole derivatives. Some of these molecules
exhibit potent cytotoxic properties toward cancer cells.
DNA binding likely contributes to the antiproliferative
activity of the most cytotoxic compounds which warrant
further in vitro and in vivo evaluations. The efficient
synthetic routes reported here provides novel opportuni-
ties to design DNA-targeted antitumor agents.
181176) described as a catalytic inhibitor of topo-
isomerase II (without inducing enzyme-mediated DNA
double strand breaks).5
This additional screening was not more successful as
no potent inhibitor of either topoisomerase I or topo-
isomerase II was found when using a plasmid relaxation
assay (Figure 3). Only a very minor effect on topo-
isomerase II was noted with compound 37, but it is
considerably weaker than that observed with the refer-
ence drug etoposide (Figure 3b). Unlike the reference
molecule camptothecin, this compound 37 does not
promote DNA cleavage by topoisomerase I, but it
induces a dose-dependent reduction of the electro-
phoretic mobility of DNA in agarose gels (Figure 3a).
Interaction with DNA was evaluated by melting
temperature measurements with both calf thymus DNA
(CT DNA) and the synthetic polymer poly(dAT)2. The
compounds equipped with a dimethylaminoethyl side
chain strongly stabilize duplex DNA whereas the neu-
tral compounds (including 44) showed no significant
effect. Under our experimental conditions, poly(dAT)2
and CT DNA melt at 44 °C and 67 °C, and these melting
temperatures remain practically unchanged in the
presence of the N-methyl compounds such as 15, 29 and
30. In contrast, the equivalent molecules 36, 37 and 38
bearing a (CH2)2N(CH3)2 strongly protect DNA against
heat denaturation (Figure 4).
Experimental Section
1
Chemistry. H and 13C NMR spectra were recorded on a
Bruker 250 instrument using CDCl3 or DMSO-d6. The chemi-
cal shifts are reported in ppm (δ scale), and all J values are
in Hz. The following abbreviations are used: singlet (s),
doublet (d), doubled doublet (dd), triplet (t), multiplet (m),
quaternary carbon (Cq). Melting point are uncorrected. IR
absorptions were recorded on a Perkin-Elmer PARAGON 1000
PC and values were reported in cm-1. MS spectra (ion spray)
were recorded on a Perkin-Elmer Sciex PI 300. Monitoring of
the reactions was performed using silica gel TLC plates (silica
Merck 60 F254). Spots were visualized by UV light at 254 and
356 nm. Column chromatography were performed using silica
gel 60 (0.063-0.200 mm, Merck).
The presence of a hydroxyl group on the naphthocar-
bazole chromophore reinforces the interaction with
DNA, and a slightly superior effect was observed with
the 3-OH derivative 37 compared to the equivalent
2-OH analogue 38, both with poly(dAT)2 and CT DNA.
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-bromo-1-methyl-
pyrrole-2,5-dione (3). A solution of 3-bromo-4-(1H-indol-3-

Naphthocarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1409
yl)-1-methyl-pyrrole-2,5-dione41 (1.1 g, 3.62 mmol) in dry THF
(35 mL) was added under argon at 0 °C to a suspension of
NaH (217 mg, 5.42 mmol, 60% in oil) in dry THF (10 mL).
After 40 min, benzenesulfonyl chloride (1.60 g, 9.04 mmol) was
added dropwise. The solution was stirred at room temperature
for 12 h, and a saturated solution of NH4Cl (50 mL) was added.
After extraction with EtOAc (2 × 100 mL), the organic layers
were washed with a saturated solution of NaCl (20 mL), dried
over MgSO4, filtered and evaporated under reduced pressure.
Flash chromatography (petroleum ether/EtOAc 5/5) afforded
compound 3 as an orange solid in 89% yield (1.443 g). Anal.
(C19H13BrN2O4S): C, H, N.
(3-Methoxy-naphthalen-2-yl)-trimethyl-stannane (4). A
solution of 2-methoxynaphthalene (6) (500 mg, 3.16 mmol) in
dry THF (20 mL) was cooled to -10 °C. A solution of n-BuLi
(3.65 mL, 584 mmol, 1.6 M in hexanes) was slowly added and
the temperature allowed to reach 0 °C. After 2 h, a solution of
trimethylstannyl chloride (943 mg, 4.74 mmol) in dry THF (10
mL) was added at -78 °C. The reaction mixture was stirred
for 5 h at -78 °C, and water was added (20 mL) at room
temperature. The organic layers were extracted with EtOAc
(2 × 20 mL). The combined organic layers were washed with
water (5 × 20 mL), dried over MgSO4, filtered and removed
under reduced pressure to afford compound 4 as a white solid
(963 mg, 95%). Anal. (C14H18OSn): C, H.
(3-Methoxy-naphthalen-2-yl)-boronic Acid (5). Same
procedure as described for compound 4 starting from 2-meth-
oxynaphthalene 6 (2 g, 12.65 mmol) and using trimethylborane
instead of trimethylstannyl chloride. Trimethylborane (4.03
g, 39.21 mmol) was added at -10 °C, and the reaction was
stirred at room temperature for 1 h. Hydrolysis was performed
by addition of a hydrochloric solution (0.2 N, 50 mL). The
solution was concentrated to 20 mL and extracted with CHCl3
(5 × 20 mL), and the solvent was removed under reduced
pressure. The residue was washed with petroleum ether (10
mL) and dried over MgSO4 to afford compound 5 as a white
solid (1.99 g, 78%).
Standard Suzuki Procedure. A solution containing the
desired halide compound (1-3, 17-19, 1 equiv), the appropri-
ate boronic acid (5, 20, 1.5 equiv), and K2CO3 (1.8 equiv) in
dioxane/water (80/20, 0.8 M) was saturated by argon during
20 min. Palladium acetate (10%) was added, and the mixture
was immediately poured in a preheated oil bath at 100 °C.
After 2 h the reaction mixture was cooled to room temperature
then filtered. The precipitate was washed with EtOAc. The
combined organic layers were washed with water, and the
solvents after drying over magnesium sulfate were removed
under reduced pressure.
dure as described for compound 11 starting from compound
10 (200 mg, 0.393 mmol) and BBr3 (2.5 M in dichloromethane,
1.00 mL, 2.5 mmol). The crude residue was purified by flash
chromatography. Anal. (C29H20N2O5S): C, H, N.
Trifluoromethanesulfonic Acid 3-[4-(1H-Indol-3-yl)-1-
methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-naphthalen-
2-yl Ester (13). A solution containing compound 11 (203 mg,
0.551 mmol) in dichloromethane (6 mL) and NEt3 (0.231 mL,
1.67 mmol) was cooled to 0 °C. Triflic anhydride (0.31 mL, 1.84
mmol) was added, and the temperature was allowed to reach
room temperature. After 5 h water (20 mL) was added, the
aqueous layer was extracted with CH2Cl2 (2 × 20 mL), the
combined organic layers were dried over MgSO4 and filtered
and the solvent was removed under reduced pressure. The
crude residue was purified by flash chromatography (petro-
leum ether/ EtOAc 4/6, NEt3 10%) to afford compound 13 as a
yellow solid (243 mg, 88%). Anal. (C24H15F3N2O5S): C, H, N.
Trifluoro-methanesulfonic Acid 3-[4-(1-Benzenesul-
fonyl-1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-
pyrrol-3-yl]-naphthalen-2-yl Ester (14). Same procedure as
described for compound 13 starting from compound 12 (750
mg, 1.47 mmol), NEt3 (0.725 mL, 5.29 mmol), and Tf2O (1 mL,
5.29 mmol), time reaction: 5 h. Compound 14 was obtained
directly after extraction without further purification as a
yellow solid (1.004 g, 98 Anal. (C30H19F3N2O7S2): C, H, N.
Heck Cyclization Procedure. A solution containing the
desired triflate compound (13-14, 24-25; 1 equiv), Bu4NCl
(1 equiv), NaOAc (2 equiv), and PPh3 (0.2 equiv) in dry dioxane
(0.1 N) was saturated by argon during 20 min at room
temperature. Palladium acetate was added, and the mixture
was immediately immersed in a preheated oil bath at 100 °C.
After the required time, the reaction mixture was cooled to
room temperature and filtered and the precipitate was washed
with CH2Cl2. The combined organic layers were washed with
water and dried over Na2SO4, and the solvents were removed
under reduced pressure
6-Methylnaphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,-
14H)-dione (15). (i) Heck cyclization procedure from 13 using
Pd(OAc)2 10%, time reaction: 12 h, flash chromatography
(petroleum ether/EtOAc 2/6) afforded compound 15 as a red
solid (40%). (ii) A solution containing compound 16 (400 mg,
0.82 mmol) and Bu4NF (1.63 mL, 1.63 mmol, 1 M in THF)
was refluxed in dry THF (20 mL) for 2 h. After cooling, water
was added and the aqueous layer was extracted with EtOAc
(3 × 50 mL). The combined organic layers were dried over
MgSO4. The crude product was purified by flash chromatog-
raphy (petroleum ether/EtOAc 2/6) to afford compound 15 (287
mg, quant.). Anal. (C23H14N2O2): C, H, N
3-(1H-Indol-3-yl)-4-(3-methoxy-naphthalen-2-yl)-1-meth-
yl-pyrrole-2,5-dione (8). Suzuki procedure from compound
1 and boronic acid 5: compound 8 was purified by flash
chromatography (petroleum ether/EtOAc 6:4) to afford an
orange solid (55%). Anal. (C24H18N2O3): C, H, N.
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(3-methoxy-naph-
thalen-2-yl)-1-methyl-pyrrole-2,5-dione (10). Suzuki pro-
cedure from compound 3 and boronic acid 5: compound 10
was purified by flash chromatography (petroleum ether/EtOAc
8:2) to afford an orange solid (80%). Anal. (C30H22N2O5S): C,
H, N.
3-(3-Hydroxy-naphthalen-2-yl)-4-(1H-indol-3-yl)-1-meth-
yl-pyrrole-2,5-dione (11). A solution containing compound
8 (200 mg, 0.261 mmol) in distilled dichloromethane (5 mL)
was cooled under argon to 0 °C. A solution of BBr3 (1 M in
dichloromethane, 0.5 mL, 0.5 mmol) was slowly added to the
mixture. After 1 h, the reaction mixture was poured into ice
and the aqueous layer was extracted with CH2Cl2 (2 × 20 mL).
The combined organic layers were washed with water, dried
over MgSO4, and filtered, and the solvents were removed under
reduced pressure. The crude residue was purified by flash
chromatography (petroleum ether/EtOAc 5/5) to afford com-
pound 11 as an orange solid (207 mg, 72%). Anal. (C23-
H16N2O3): C, H, N.
6-Methyl-14-benzenesulfonyl-naphtho[2,3-a]pyrrolo-
[3,4-c]carbazole-5,7(6H,14H)-dione (16). Heck cyclization
procedure from 14 using Pd(OAc)2 1.0 equiv, 3 h, flash
chromatography (petroleum ether/EtOAc 7/3) afforded com-
pound 16 as a red solid (91%). Anal. (C29H18N2O4S): C, H, N
3-(1-Benzenesulfonyl-5-benzyloxy-1H-indol-3-yl)-4-bro-
mo-1-methyl-pyrrole-2,5-dione (17). Same procedure as
described for compound 3: 3-(5-Benzyloxy-1H-indol-3-yl)-4-
bromo-1-methyl-pyrrole-2,5-dione41 (1 g, 2.26 mmol), NaH (234
mg, 7.5 mmol, 60% in oil), benzenesulfonyl chloride (1.34 g,
2.44 mmol), time reaction 12 h, flash chromatography (petro-
leum ether/EtOAc 3:7). Compound 17 was obtained as an
orange solid in 85% yield (1.143 g). Anal. (C26H19BrN2O5S):
C, H, N.
3-(1-Benzenesulfonyl-6-benzyloxy-1H-indol-3-yl)-4-bro-
mo-1-methyl-pyrrole-2,5-dione (18). Same procedure as
described for compound 3: 3-(6-Benzyloxy-1H-indol-3-yl)-4-
bromo-1-methyl-pyrrole-2,5-dione41 (1 g, 2.26 mmol), NaH (234
mg, 7.5 mmol, 60% in oil), benzenesulfonyl chloride (1.34 g,
2.44 mmol), 4 h, flash chromatography (petroleum ether/EtOAc
3:7). Compound 18 was obtained as an orange solid in 92%
yield (2.467 g). Anal. (C26H19BrN2O5S): C, H, N.
(3-Hydroxy-naphthalen-2-yl)-boronic Acid (20). A stirred
solution of compound 5 (1.500 g, 7.42 mmol) in distilled
dichloromethane (30 mL) was cooled at 0 °C. A solution of BBr3
(2.11 mL, 22.27 mmol) was slowly added. After 2 h at room
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(3-hydroxy-naph-
thalen-2-yl)-1-methyl-pyrrole-2,5-dione (12). Same proce-

1410 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Routier et al.
temperature, the reaction mixture was poured into cold water
(10 mL) and extracted with EtOAc (4 × 20 mL). The combined
organic layers were evaporated under reduced pressure to
afford compound 20 as a white solid (1.185 g, 85%).
followed by the Bu4NF deprotection, time reaction 1 h. Crude
compound 28 was washed with EtOAc to afford 28 as a red
solid without further purification (95%). Anal. (C30H20N2O3):
C, H, N.
3-(1-Benzenesulfonyl-5-benzyloxy-1H-indol-3-yl)-4-(3-
hydroxy-naphthalen-2-yl)-1-methyl-pyrrole-2,5-dione (21).
Suzuki procedure starting from compound 17 and boronic acid
20: time reaction 2 h, flash chromatography (petroleum ether/
EtOAc 7/3). Compound 21 was obtained as an orange solid in
82% yield. Anal. (C36H26N2O6S): C, H, N.
3-(1-Benzenesulfonyl-6-benzyloxy-1H-indol-3-yl)-4-(3-
hydroxy-naphthalen-2-yl)-1-methyl-pyrrole-2,5-dione (22).
Suzuki procedure starting from compound 18 and boronic acid
20: time reaction 2 h, flash chromatography (petroleum ether/
EtOAc 7/3). Compound 22 was obtained as an orange solid in
81% yield. Anal. (C36H26N2O6S): C, H, N.
3-(Hydroxy)-6-methylnaphtho[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7(6H,14H)-dione (29). Same procedure as described
for compound 11: compound 27 (500 mg, 1.09 mmol), BBr3 (1
M in dichloromethane, (2.34 mL, 2.34 mmol), time reaction 6
h. A flash chromatography (petroleum ether/EtOAc 8/2 then
5/5) afforded compound 29 as a red solid (371 mg, 93%). Anal.
(C23H14N2O3): C, H, N.
2-(Hydroxy)-6-methylnaphtho[2,3-a]pyrrolo[3,4-c]car-
bazole-5,7(6H,14H)-dione (30). Same procedure as described
for compound 11: compound 28 (200 mg, 0.439 mmol), BBr3
(1 M in dichloromethane, 6.0 mL, 6 mmol), time reaction 2 h.
A flash chromatography (petroleum ether/EtOAc 6/4 then
EtOAc) afforded compound 30 as a red solid (157 mg, 98%).
Anal. (C23H14N2O3): C, H, N.
6-Methyl-14-[2-(4-Morpholinyl)ethyl]-naphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (31). A solution
of compound 15 (77 mg, 0.219 mmol) in freshly distillated DMF
(2 mL) was stirred under argon at 0 °C and NaH (17 mg, 0.70
mmol, 60% in oil) was added. After 30 min, a suspension of
N-(2-chloroethyl)-morpholine hydrochloride (158 mg, 1.0 mmol),
NaH (41 mg, 1.71 mmol, 60% in oil) in dry DMF (5 mL) was
added. The reaction mixture was warmed to 100 °C for 6 h.
After cooling, cold water (30 mL) was added and the aqueous
layer was extracted with dichloromethane (3 × 50 mL). The
combined organic layers were evaporated under reduced
pressure, and the crude residue was purified by flash chro-
matography (EtOAc) to afford compound 31 as a red solid (50
mg, 49%). Anal. (C29H25N3O3): C, H, N.
6-Methyl-14-[2-dimethylamino)-ethyl]-naphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (32). Same pro-
cedure as described for compound 31: compound 15 (100 mg,
0.285 mmol), NaH (17 mg, 0.7 mmol), and (2-chloroethyl)-
dimethylamine hydrochloride (122 mg, 0.847 mmol) with NaH
(41 mg, 1.61 mmol), 90 °C, time reaction, 12 h. After evapora-
tion, the crude residue was dissolved in EtOAc (10 mL) and
filtered off. The filtrate was concentrated under reduced
pressure, and the organic material was purified by flash
chromatography (EtOAc/MeOH 9/1) to afford compound 32 as
a red solid (45 mg, 35%). Anal. (C27H23N3O2): C, H, N
6-Methyl-14-allyl-naphtho[2,3-a]pyrrolo[3,4-c]carbazole-
5,7(6H,14H)-dione (33). A suspension containing compound
15 (100 mg, 0.285 mmol) and NaH (18 mg, 0.75 mmol) in
freshly distilled DMF (2 mL) was stirred for 30 min at 0 °C.
Allyl bromide (0.150 mL, 1.7 mmol) was added, and the
reaction mixture was stirred at room temperature for 9 h. After
evaporation, the crude residue was precipitated in MeOH (10
mL) and filtered off to afford compound 33 as an orange solid
(57 mg, 51%). Anal. (C26H18N2O2): C, H, N
6-Methyl-14-(2,3-dihydroxypropyl)-naphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (34). A solution
containing KMnO4 (34.5 mg, 0.218 mmol) dissolved in a
mixture acetone/water (3/1, 20 mL) was slowly added to a
solution of compound 33 (40 mg, 0.102 mmol) in acetone (4
mL) at 0 °C. The resulting mixture was stirred at room
temperature for 6 h and filtered off, and the precipitate was
washed with CHCl3 (30 mL). The filtrate was extracted with
CHCl3 (20 mL), and the combined organic layers were removed
under reduced pressure. The crude material was purified by
flash chromatography (petroleum ether/EtOAc 1/9) to afford
compound 34 as a red solid (31 mg, 71%). Anal. (C26H20N2O4):
C, H, N.
3-(1-Benzenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-
(3-hydroxy-naphthalen-2-yl)-1-methyl-pyrrole-2,5-di-
one (23). Suzuki procedure from 7-azaindole derivatives 1914
and boronic acid 20: time reaction 2 h, flash chromatography
(petroleum ether/EtOAc 7/3). Compound 23 was obtained as
1
an yellow solid in 81% yield. H NMR (CDCl3): 3.22 (s, 3H),
6.61 (m, 2H), 7.22-7.57 (m, 10H), 8.24 (m, 3H), 7.49 (s, 1H);
13C NMR (CDCl3): 25.0 (CH3), 107.2 (Cq), 112.3 (CH), 117.9
(Cq), 118.5 (CH), 123.3 (CH), 125.1 (CH), 126.9 (Cq), 127.1
(CH), 127.3 (2CH), 128.1 (2CH), 129.1 (Cq), 129.5 (CH + Cq),
130.2 (Cq), 131.3 (CH), 131.9 (Cq), 133.4 (CH), 134.4 (Cq),
136.6 (Cq), 141.8 (CH), 144.3 (CH), 150.3 (CH + Cq), 168.9
(CO), 171.7 (CO); MS (IS) 510 (M + 1)+. Anal. (C28H19N3O5S):
C, H, N.
Trifluoromethanesulfonic Acid 3-[4-(1-Benzenesul-
fonyl-5-benzyloxy-1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-
dihydro-1H-pyrrol-3-yl]-naphthalen-2-yl Ester (24). Same
procedure as for compound 13: Compound 21 (1.00 g, 1.62
mmol), NEt3 (0.687 mL, 4.86 mmol), triflic anhydride (0.912
mL, 5.42 mmol), 3 h. A flash chromatography (petroleum ether/
EtOAc 7/3 + NEt3 1%) afforded compound 24 as an orange
1
solid (1.066 g, 88%). H NMR (CDCl3,): 3.27 (s, 3H), 5.89 (s,
2H), 6.76-6.81 (m, 3H), 7.22-7.27 (m, 4H), 7.49-7.65 (m, 6H),
7.82-7.97 (m, 5H), 8.23 (s, 1H), 8.44 (s, 1H); 13C NMR
(CDCl3,): 24.6 (CH3), 69.1 (Cq), 103.4 (CH), 111.0 (Cq), 114.4
(CH), 115.6 (CH), 120.1 (Cq), 120.8 (CH), 122.3 (Cq), 127.0 (Cq
+ 3CH), 127.5 (Cq), 127.7 (2CH), 128.1 (2CH + Cq), 128.4
(2CH), 128.8 (CH), 129.2 (CH), 129.4 (2CH), 131.5 (CH), 131.8
(CH), 132.8 (CH), 133.2 (Cq), 133.4 (Cq), 134.1 (CH), 135.8
(Cq), 137.5 (Cq), 144.9 (Cq), 155.3 (Cq), 169.6 (CO), 169.9 (CO);
MS (IS) 747 (M + 1)+. Anal. (C37H25F3N2O8S2): C, H, N.
Trifluoromethanesulfonic Acid 3-[4-(1-Benzenesul-
fonyl-6-benzyloxy-1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-
dihydro-1H-pyrrol-3-yl]-naphthalen-2-yl Ester (25). Same
procedure as for compound 13: Compound 22 (1.00 g, 1.62
mmol), NEt3 (0.687 mL, 4.86 mmol), triflic anhydride (0.912
mL, 5.42 mmol), time reaction 3 h. A flash chromatography
(petroleum ether/EtOAc 7/3 + NEt3 1%) afforded compound
25 as an orange solid (887 mg, 73%). Anal. (C37H25F3N2O8S2):
C, H, N.
Trifluoromethanesulfonic Acid 3-[4-(1-Benzenesul-
fonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1-methyl-2,5-dioxo-
2,5-dihydro-1H-pyrrol-3-yl]-naphthalen-2-yl Ester (26).
Same procedure as described for compound 13: Compound 23
(0.888 g, 1.75 mmol), NEt3 (0.730 mL, 5.24 mmol), and triflic
anhydride (0.881 mL, 5.24 mmol), 2 h. A flash chromatography
(petroleum ether/EtOAc 6/4 + NEt3 1%) afforded compound
26 as a yellow solid (1.118 g, 97%). Anal. (C29H18F3N3O7S2):
C, H, N.
3-(Benzyloxy)-6-methylnaphtho[2,3-a]pyrrolo[3,4-c]-
carbazole-5,7(6H,14H)-dione (27). Heck cyclization proce-
dure from 24 using Pd(OAc)2 (1 equiv), time reaction 4 h,
followed by the Bu4NF deprotection, time reaction 1 h. Flash
chromatography (petroleum ether/EtOAc 7/3) afforded com-
pound 27 as a red solid (84%). Anal. (C30H20N2O3): C, H, N.
2-(Benzyloxy)-6-methylnaphtho[2,3-a]pyrrolo[3,4-c]-
carbazole-5,7(6H,14H)-dione (28). Heck cyclization proce-
dure from 25 using Pd(OAc)2 (1 equiv), time reaction 6 h,
5H-Furo[3,4-c]naphtho[2,3-a]pyrrolo[3,4-c]carbazole-
5,7(14H)-dione (35). To a solution of compound 15 (100 mg,
0.285 mmol) in ethanol (40 mL) was added a solution of
aqueous KOH (5 N, 10 mL) at room temperature. The reaction
mixture was refluxed for 5 h. After coolingbeing cooled to room
temperature, the solution was concentrated then neutralized
by addition of a 10% HCl aqueous solution. After filtration,
the precipitate was washed with water (10 mL) then with

Naphthocarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1411
EtOAc (2 × 10 mL), dried under vacuum to afford compound
35 as a red solid (80 mg, 83%). Anal. (C22H11NO3): C, H, N.
2-(Benzyloxy)-6-methyl-14-[2-(dimethylamino)ethyl]-
naphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-di-
one (45). Same procedure as described for compound 31:
compound 28 (100 mg, 0.219 mmol), NaH (17 mg, 0.7 mmol),
(2-chloroethyl)-dimethylamine hydrochloride (122 mg, 0.847
mmol) with NaH (41 mg, 1.61 mmol), time reaction 12 h, 90
°C. After evaporation, the crude residue was dissolved in
EtOAc (10 mL) and filtered off. The filtrate was concentrated
under reduced pressure, and the organic material was purified
by flash chromatography (EtOAc) to afford compound 45 as a
red solid (98 mg, 85%). Anal. (C34H29N3O3): C, H, N.
2-{[(tert-Butyldimethyl)silyl]oxy}-6-methylnaphtho-
[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (46). A
solution containing imidazole (330 mg, 4.97 mmol) and TB-
DMSCl (620 mg, 4.13 mmol) dissolved in DMF (15 mL) was
stirred under argon at 0 °C. After 30 min, cesium carbonate
(267 mg, 0.82 mmol) and compound 30 (150 mg, 0.41 mmol)
were added. After stirring for 4 h at room temperature, water
(20 mL) was added and extractions were performed with
EtOAc (3 × 20 mL). The combined organic layers were
removed under reduced pressure, and the crude residue was
purified by flash chromatography (petroleum ether/EtOAc 7/3,
NEt3 1%) to afford compound 46 as an orange solid (196 mg,
quant.). Anal. (C29H28N2O3Si): C, H, N.
2-[2-(Dimethylamino)ethyl]-6-methylnaphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (47). (i) Same
procedure as described for compound 31: compound 46 (50
mg, 0.10 mmol), NaH (7 mg, 0.29 mmol, 60% in oil), DMF,
(2-chloroethyl)-dimethylamine hydrochloride (77 mg, 0.534
mmol), de NaH (35 mg 1.45 mmol, 60% in oil), time reaction
3 h, 100 °C. After treatment, the product was dissolved in
EtOAc, and petroleum ether was added until a precipitate
appeared. The suspension was filtered, the precipitate was
dried under vacuum to afford compound 47 as a red solid (31
mg, 69%). (ii) Idem. starting from compound 30 (130 mg, 0.355
mmol), (2-chloroethyl)-dimethylamine hydrochloride (111 mg,
0.77 mmol), NaH (90 mg, 3.75 mmol, 60% in oil) in one portion,
DMF, 100 °C, 3 h. Precipitation in MeOH afforded compound
47 (65 mg, 41%). Anal. (C27H23N3O3): C, H, N
Growth Inhibition Assays. Tumor cells were provided by
American Type Culture Collection (Frederick, MD). Three cell
lines were used: one murine leukemia (L1210), one human
prostate cancer (DU145), and one colon carcinomia (HT29)
were cultivated in RPMI 1640 medium (Life cience Technolo-
gies, Cergy-Pontoise, France) supplemented with 10% fetal calf
serum, 2 mM l-glutamine, 100 units/mL penicillin, 100 µg/mL
streptomycin and 10 mM HEPES buffer (pH ) 7.4). Cyto-
toxicity was measured by the microculture tetrazolium assay
described.48 Cells were continuously exposed to graded con-
centrations of the compounds for four doubling times, 15 µL
of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide was added to each well, and the plates were
incubated for 4 h at 37 °C. The medium was then aspirated
and the formazan solubilized by 100 µL of DMSO. Results are
expressed as IC50, the concentration which was reduced by 50%
the optical density of treated cells with respect to untreated
controls. IC50 were determined by nine serial dilutions per-
formed in duplicate.
6-[2-(Dimethylamino)ethyl]-naphtho[2,3-a]pyrrolo[3,4-
c]carbazole-5,7-(6H,14H)-dione (36). (i) A solution of com-
pound 15 (50 mg, 0.148 mmol) was heated at 80 °C in the
presence of a large excess of N1,N1-dimethyl-ethane-1,2-
diamine (1 mL). After 8 h, water (50 mL) and EtOAc (50 mL)
were added and the aqueous layers were extracted with EtOAc
(2 × 30 mL). The solvents were removed under reduced
pressure, and the crude residue was purified by flash chro-
matography (acetone then THF) to afford compound 36 as a
red solid (50 mg, 83%). (ii) Same procedure starting from
compound 35 (50 mg, 0.142 mmol) affording compound 36 (51
mg, 88%). Anal. (C26H21N3O2): C, H, N.
3-(Hydroxy)-6-[2-(dimethylamino)ethyl]naphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (37). Same pro-
cedure as described for compound 36: compound 29 (200 mg,
0.546 mmol), N1,N1-dimethyl-ethane-1,2-diamine (4 mL), time
reaction 8 h, rflx. A flash chromatography (acetone) afforded
compound 37 as a red solid (201 mg, 87%). Anal. (C26-
H21N3O3): C, H, N.
2-(Hydroxy)-6-[2-(dimethylamino)ethyl]naphtho[2,3-a]-
pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (38). Same pro-
cedure as described for compound 36 : compound 30 (50 mg,
0.136 mmol), N1,N1-dimethyl-ethane-1,2-diamine (1 mL), 24
h, rflx. A flash chromatography (acetone) afforded compound
38 as a red solid (32 mg, 55%). Anal. (C26H21N3O3): C, H, N.
3-(Hydroxy)-6-[3-(dimethylamino)propyl]naphtho[2,3-
a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (39). Same
procedure as described for compound 36: compound 29 (100
mg, 0.273 mmol), N1,N1-dimethyl-propyl-1,3-diamine (2 mL),
5 h, rflx. A flash chromatography (acetone, NEt3 5%) afforded
compound 39 as a red solid (201 mg, 87%). Anal. (C27-
H23N3O3): C, H, N.
2-(Hydroxy)-6-[3-(dimethylamino)propyl]naphtho[2,3-
a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-dione (40). Same
procedure as described for compound 36: compound 30 (50
mg, 0.136 mmol), N1,N1-dimethyl-propyl-1,3-diamine (0.5 mL),
time reaction 3 h, rflx. A flash chromatography (acetone, NEt3
5%) afforded compound 40 as a red solid (47 mg, 80%). Anal.
(C27H23N3O3): C, H, N.
3-(Hydroxy)-6-{2-[(2-hydroxyethyl)amino]ethyl}-
naphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-di-
one (41). Same procedure as described for compound 36:
compound 29 (85 mg, 0.232 mmol), 2-(2-aminoethylamino)-
ethanol (2 mL), time reaction 3 h, 120 °C. After evaporation
of the solvents, the residue was precipitared with MeOH (20
mL), filtered, washed three time with MeOH (10 mL), and
dried under vacuum to afford compound 41 as a red solid (100
mg, quant.). Anal. (C26H21N3O4): C, H, N.
2-(Hydroxy)-6-{2-[(2-hydroxyethyl)amino]ethyl}-
naphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-di-
one (42). Same procedure as described for compound 36:
compound 30 (50 mg, 0.136 mmol), 2-(2-aminoethylamino)-
ethanol (1 mL), time reaction 5 h, 120 °C. After evaporation
of the solvents, the residue was washed with water (4 × 20
mL) then with EtOAc (4 × 20 mL), dried under vacuum to
afford compound 42 as a red solid (16 mg, 27%). Anal.
(C26H21N3O4): C, H, N.
3-(Hydroxy)-6-[2-hydroxy-1-(hydroxymethyl)ethyl]-
naphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-di-
one (43). Same procedure as described for compound 36:
compound 29 (50 mg, 0.136 mmol), 2-amino-1,3-propandiol (0.2
mL), time reaction 3 h, 120 °C. A flash chromatography
(EtOAc) afforded compound 43 as a red solid (38 mg, 65%).
Anal. (C25H18N2O5): C, H, N.
Cell Cycle Analysis. For the cell cycle analysis, L1210 cells
(2.5 × 105 cells/mL) were incubated for 21 h with various
concentrations of the compounds and fixed by 70% ethanol (v/
v), washed, and incubated in PBS containing 100 µg/mL
RNAase and 25 µg/mL propidium iodide for 30 min at 20 °C
for each sample, 104 cells were analyzed on a XL/MCL flow
cytometer (Beckman Coulter). The fluorescence of propidium
iodide was collected through a 615 nm long-pass filter. These
experiments were performed at drug concentrations between
IC50 and IC90 values. Dose-dependent effects were observed
in all cases.
DNA Relaxation Experiments. Supercoiled pKMp27
DNA (0.5 µg) was incubated with 4 units human topoisomerase
I or II (TopoGen Inc.) at 37 °C for 1 h in relaxation buffer (50
mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl2, 1 mM dithio-
threitol, 1 mM EDTA) in the presence of varying concentra-
2-(Hydroxy)-6-[2-hydroxy-1-(hydroxymethyl)ethyl]-
naphtho[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H,14H)-di-
one (44). Same procedure as described for compound 36:
compound 30 (50 mg, 0.136 mmol), 2-amino-1,3-propandiol (0.5
mL), time reaction 3 h, 120 °C. A flash chromatography
(petroleum ether/EtOAc 2/8) afforded compound 44 as a red
solid (18 mg, 31%). Anal. (C25H18N2O5): C, H, N.

1412 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Routier et al.
(11) Ohe, Y.; Tanigawara, Y.; Jujii, H.; Ohtsu, T.; Wakita, H.;
Igarashi, T.; Minami, H.; Eguchi, K.; Shinkai, T.; Tamura, T.;
Kunotoh, H.; Saijo, N.; Okada, K.; Ogino, H.; Sasaki, Y. Phase
I and Pharmacology Study of 5-days infusion of NB-506. Proc.
Am. Soc. Clin. Oncol. 1997, 16, 199a.
(12) Dowlati, A.; Remick, S. C.; Majka, S.; Ingalis, S.; Hoppel, C.;
Spiro, T.; Gerson, S.; Willson, J. K. V. A phase I Clinical and
Pharmacocinetic Study of Rebeccamycin Analogue (NCS655649)
given Daily for 5 Consecutive Days. Proc. Am. Soc. Clin. Oncol.
1999, 18, 181a.
(13) Long, B. H.; Balasubramanian, B. N. Non Camptothecin Topo-
isomerase I Active Compounds as Potential Anticancer Agents.
Expert Opin. Ther. Pat. 2000, 10, 635-666.
(14) Routier, S.; Ayerbe, N.; Me´rour, J. Y.; Coudert, G.; Bailly, C.;
Pierre´, A.; Pfeiffer, B.; Caignard, D. H.; Renard, P. Synthesis
and Biological Activity of 7-Azaindolocarbazoles. Tetrahedron
2002, 58, 6621-6630.
tions of the drug under study. Reactions were terminated by
adding SDS to 0.25% and proteinase K to 250 mg/mL. DNA
samples were then added to the electrophoresis dye mixture
(3 mL) and electrophoresed in a 1% agarose gel containing
ethidium bromide (1 mg/mL), at room temperature for 2 h at
120 V. Gels were washed and photographed under UV light.49
DNA Binding Measurements. Melting curves were mea-
sured using an Uvikon 943 spectrophotometer coupled to a
Neslab RTE111 cryostat. For each series of measurements, 12
samples were placed in a thermostatically controlled cell-
holder, and the quartz cuvettes (10 mm path length) were
heated by circulating water. The Tm measurements were
performed in BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM NaH2-
PO4, 1 mM EDTA). The temperature inside the cuvette was
measured with a platinum probe; it was increased over the
range 20-100 °C with a heating rate of 1 °C/min. The
“melting” temperature Tm was taken as the midpoint of the
hyperchromic transition.
(15) Marminon, C.; Pierre´, A.; Pfeiffer, B.; Pe´rez, V.; Le´once, S.;
Renard P.; Prudhomme, M.; Syntheses and Antiproliferative
Activities of Rebeccamycin Analogues Bearing two 7-Aazaindole
Moieties. Bioorg. Med. Chem. 2003, 11, 679-687.
(16) Marminon, C.; Pierre´, A.; Pfeiffer, B.; Pe´rez, V.; Le´once, S.;
Joubert, A.; Bailly, C.; Renard, P.; Hickman, J.; Prudhomme M.
Syntheses and Antiproliferative Activities of 7-Azarebeccamycin
Analogues Bearing One 7-Azaindole Moiety. J. Med. Chem.
2003, 46, 609-622.
Acknowledgment. This research was supported by
grants from the Ligue Nationale Contre le Cancer
(Comite´ du Nord) and the Institut de Recherches sur le
Cancer de Lille (IRCL), (C.B.) and from the Association
pour la Recherche sur le Cancer (ARC), the Cance´ropoˆle
Grand Ouest and the Re´gion Centre (S.R.). We thank
Dr L. Meijer (Roscoff, France) for performing the kinases
studies. The expert technical assistance of B. Baldeyrou
is also acknowledged.
Supporting Information Available: 1H NMR, 13C NMR,
IR, and MS data and results of elemental analysis for
compounds 3-5, 8, 10-18, 20-47 are available free of charge
via the Internet at http://pubs.acs.org.
(17) Zhang, H.-C.; White, K. B.; Ye, H.; McComsey, D. F.; Derian, C.
K.; Addo, M. F.; Andrade-Gordon, P.; Eckardt, A. J.; Conway,
B. R.; Westover, L.; Xu, J. Z.; Look, R.; Demarest, K. T.;
Emanueland, S.; Maryanoff, B. E. Macrocyclic Bisindolylmale-
imides as Inhibitors of Protein Kinase C and Glycogen Synthase
Kinase-3. Bioorg. Med. Chem. Lett. 2003, 13, 3049-3053.
(18) Kuo, G.-H.; Prouty, C.; DeAngelis, A.; Shen, L.; O’Neill, D. J.;
Shah, C.; Connolly, P. J.; Murray, W. V.; Conway, B. R.; Cheung,
P.; Westover, L.; Xu, J. Z.; Look, R. A.; Demarest, K. T.; Emanuel,
S.; Middleton, S. A.; Jolliffe, L.; Pat Beavers, M.; Chen X.
Synthesis and Discovery of Macrocyclic Polyoxygenated Bis-7-
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