Synthesis and biological evaluation of novel phenylcarbazoles as potential anticancer agents

Article abstract of DOI:10.1021/jm050945x

We here report the synthesis and biological evaluation of new phenylcarbazole derivatives designed as potential anticancer agents. Indole and hydroxyindole were used to generate three scaffolds that were successively exploited to introduce various substit

Full text of DOI:10.1021/jm050945x

J. Med. Chem. 2006, 49, 789-799
789
Synthesis and Biological Evaluation of Novel Phenylcarbazoles as Potential Anticancer Agents
Sylvain Routier,*,† Jean-Yves Me´rour,† Nathalie Dias,‡ Ame´lie Lansiaux,‡ Christian Bailly,‡,| Olivier Lozach,§ and
Laurent Meijer§
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 Station Biologique, place G. Teissier, B.P. 74,
29682 ROSCOFF Cedex, France
ReceiVed September 22, 2005
We here report the synthesis and biological evaluation of new phenylcarbazole derivatives designed as
potential anticancer agents. Indole and hydroxyindole were used to generate three scaffolds that were
successively exploited to introduce various substituents on the maleimide moiety. The synthesis includes a
final intramolecular key Heck-type reaction, which was carried out with a triflate derivative or with a
bromophenyl derivative. Each step was optimized and the complete chemical strategy is detailed. Several
compounds showed a marked cytotoxicity against CEM human leukemia cells with IC50 values in the 10-
100 nM range. Precise structure-activity relationships were delineated. Cell cycle analysis, topoisomerase
I inhibition, and interaction with DNA were evaluated, and inhibition of CDK activity was also investigated.
Although binding of the drugs to DNA likely contributes to the cytotoxic action, the exact molecular targets
of these molecules remain undiscovered. The efficient chemical routes reported here for the design of highly
cytotoxic compounds provide novel opportunities to identify antitumor agents in the phenylcarbazole series.
Introduction
carbazoles designed so far for kinase inhibition are equipped
with an unsubstituted maleimide, whereas in general the most
cytotoxic compounds bear hydrophilic side chains. It is impor-
tant to mention also that closely related (het)arylcarbazoles (type
III) have been described as inhibitors of cyclin D1/CDK4.13,14
This strategy was previously adopted for granulatimide and iso-
granulatimide (Figure 1) acting as G2 checkpoint inhibitors.15-17
Cyclin-dependent kinases (CDKs) were also targeted with
indolocarbazoles such as bis N-indolyl alkylated arcyriaflavins
recently described as CDK1, CDK2, and CDK4 inhibitors.19,20
In the NH maleimide series, indole versus (hetero)arylcarbazoles
replacement represents also an interesting strategy.
With this in mind, we envisaged the synthesis of different
naphthalenic compounds (V).21 The replacement of one of the
two indoles by a naphthalene ring produced a highly cytotoxic
naphtho[2,3-a]pyrrolo[3,4-c]carbazole derivative (V), suggesting
that the naphthalene is effectively a suitable bioisostere for
indole. However, no kinase inhibition was detected in that series.
At the same time, we developed a phenylcarbazole series (IV)
in order to broaden our SAR knowledge. Recently, this approach
has been followed by a group at Eli Lilly for similar aryl or
heteroaryl carbazoles. The activity of only one phenylcarbazole
and the synthesis of two other analogues were reported, and
most of this work consisted of the variation of one aromatic
moiety.13 This interesting data prompted us to present here our
phenylcarbazole series IV. A new efficient synthesis of such
compounds, including indolic substitution and maleimide varia-
tions, is presented together with preliminary pharmacological
studies.
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 the central role of these molecules in the
regulation of cell cycle progression and specific enzyme
inhibition.2,3 Structure-activity relationships (SAR) in the
indolocarbazole series have been extensively studied in the
context of topoisomerase I inhibition and tumor cell killing.
Compounds bearing a pyrroloindolocarbazole and equipped with
one N-glycosidic bond, such as the antibiotic rebeccamycin,
generally function as DNA topoisomerase I inhibitors. A few
analogues, such as NB-506 and J-107088 (also known as
edotecarin), have entered clinical trials for cancer treatment.4-6
More recently, fluorinated derivatives of such molecules have
been reported and their topoisomerase I-dependent anticancer
activity looks promising.7-9
For our part, we have recently described the bioisosteric
replacement of an indole moiety by a 7-azaindole unit, affording
the first symmetrical and dissymmetrical 7-azaindolocarbazoles
I and II.10 In the same vein, the cytotoxic properties of the
N-glycosylated derivatives of I and II have been reported by
others.11,12 For these different molecules, the role of topo-
isomerase I inhibition in the cytotoxic action seems relatively
minor compared to the NB-506-type series, and evidence
suggests that other signaling proteins, particularly kinases, may
play a role.
To develop selective kinase inhibitors, fitting in the ATP
binding site, modifications of the aromatic heterocyclic indolo-
carbazole moiety appeared as a valid alternative to the synthesis
of glycosylated compounds. In addition, most of the aryl
Chemistry
Various synthetic methods have been used to synthesize a
representative panel of symmetrical or unsymmetrical indolo-
carbazoles.22 Lilly’s group choose to build, on one hand, the
indolic glyoxalates and, on the other hand, a 2-bromophenyl
acetamidate, leading to the indoylarylmaleimide.23 Final benz-
annulation was performed by palladium intramolecular Heck
reaction (70% yield). One disadvantage of this approach is the
* Corresponding author. Tel: +33 228 41 49 53. Fax: +33 238 41 72
81. E-mail: sylvain.routier@univ-orleans.fr.
† Universite´ d’Orle´ans.
‡ INSERM U-524.
§ Station Biologique.
| Present address: Centre de Recherche en Oncologie Expe´rimentale,
Institut de Recherche Pierre Fabre, 3 rue des satellites, 31140 Toulouse,
France.
10.1021/jm050945x CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/23/2005

790 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Routier et al.
Figure 1. Modifications of the indolocarbazole skeleton.
Scheme 1. Clockwise Retrosynthesis of Naphthocarbazoles
need for various acetamidate and glyoxalate skeletons not
directly available and another one was the decrease of yield
using substituted indoles. An alternative and more convenient
synthesis consisted of the selective 3-alkylation of indole with
2,3-dibromomaleimide, using Grignard reagents to produce
3-indoylmaleimides.24 The second (hetero)aromatic group could
be introduced via palladium(0) catalyzed Stille or Suzuki
reactions, whereas the final benzannulation required an intra-
molecular Heck reaction.
absence of insertion of the palladium entity in the C-OTf bond.
Despite a reported successful condensation of 4-indolyl triflate
at the position-2 of an indole derivative in the presence of
Pd(OAc)2 with dppe as a ligand and an excess of triethylamine
at 110 °C,26 we were unable to apply these conditions to 5 to
obtain the desired cyclized product. As such, we tried to cleave
solely the benzenesulfonyl group to modify the electronic and
steric behavior of the 2,3-ethylenic bond of the pyrrole moiety
implicated in the formation of the six-membered ring. Com-
pound 5 was reacted with 2.2 equiv of Bu4NF in refluxing
THF.27 Unfortunately, the triflate group was also cleaved and
the fully deprotected compound 6 was generated in a 77% yield.
All our efforts to obtain the triflate derivative 7 from 6 led to
a degradation of the mixture. Therefore, we decided to perform
the first Suzuki reaction using 2-bromophenyl boronic acid 8
on either the unprotected N-indolic compound 9 or the protected
compound 1 (Scheme 3). Two procedures differing by the nature
of the palladium catalyst (Pd (OAc)2 or Pd(PPh3)4 (Table 1,
entries 1 and 2) were carried out with compound 9. The expected
derivative 12 was only obtained by using Pd(OAc)2 in a
moderate yield (50%). In this case, the presence of an unsub-
stituted indolic nitrogen atom was detrimental to the synthesis.
In contrast, the benzenesulfonyl protected compound 1, treated
with 8, afforded the desired compound 11 in an excellent yield
(98%) with a trace amount (1%) of the cyclized compound 13
(entry 3), indicating the possibility of a one-pot Suzuki-Heck
tandem reaction (entries 4-6).
In our previous paper,21 we described the synthesis of new
naphthocarbazoles V (Scheme 1) in only four steps using a
clockwise synthetic sequence starting from indole and involving
(i) the introduction of the maleimide, (ii) a Suzuki cross-coupling
reaction on the indolic bromomaleimide for the introduction of
the functionalized naphthalene moiety, (iii) the activation of the
naphthyl as a naphthyl triflate, and (iv) an intramolecular Heck
reaction directly followed by a cleavage of the protecting group.
This strategy also represents a major simplification in the
synthesis of parent indolocarbazole derivatives.
Following this strategy, we began the synthesis of phenyl-
carbazole by treating compound 1 with 2-methoxyphenylboronic
acid 2 in the presence of Pd(OAc)2, dioxane, water, and K2CO3
as a base at 80 °C according to a Suzuki cross-coupling reaction;
compound 3 was quantitatively obtained (Scheme 2). Compound
4 was obtained in a 94% yield by demethylation of 3 by using
BBr3 in dichloromethane. The hydroxy group of 4 was then
converted to the triflate 5 with an 85% yield (Scheme 2).
Several attempts of cyclization of 5, using our previously
described conditions, were unproductive.19,21 Only the starting
material and detriflated compound 4 were isolated whatever the
reaction time and temperature, thus indicating a complete
One of the differences between the Suzuki (entry 3) and the
Heck-type cross-coupling reactions was the presence or the
absence of an additional ligand for the catalyst, whereas both
procedures involved the use of palladium acetate. So triphenyl-

Phenylcarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 791
Scheme 2a
a (i) 2-Methoxyphenylboronic acid 2 (1.1 equiv), dioxane, water, K2CO3 (2 equiv), Pd(OAc)2 10%, 80 °C, 4 h, quant.; (ii) BBr3 (7.5 equiv), CH2Cl2, rt.,
1 h, 94%; (iii) Tf2O (1 equiv), NEt3 (3 equiv), CH2Cl2, rt, 2 h, 85%; (iv) Bu4NF (2 equiv), THF, reflux, 2 h, 77%.
Scheme 3a
a (i) From 1 Pd(OAc)2 (0.1 equiv), 2-BrPhB(OH)2 8 (3 equiv), K2CO3 (4 equiv, dioxane/water 4/1, 100 °C, 8 h, 98%; from 9, idem, 4 h, 9 50%; from
10, 8 (1.5 equiv), 15 58%, and 16 31%; (ii) from 12, Pd(PPh3)4 5%, AcOK (1.1 equiv), DMA, 130 °C, 4 h, quant.; from 16, idem, quant.; (iii) from 11,
Bu4NF (1.2 equiv), THF, reflux, 2 h, 93%; from 13, idem, quant.; from 15, idem, 86%; (iv) BBr3 (1.0 equiv), CH2Cl2, rt, 1 h, quant.
Table 1. Suzuki and Heck Procedures
entry
compds
catalyst (equiv)
additives (equiv)
solvent; temp, °C
time, h
product (yield %)
12 (50)
1
2
3
4
5
9
9
1
1
1
Pd(OAc)2 (0.1)
Pd(PPh3)4 (0.2)
Pd(OAc)2 (0.1)
Pd(OAc)2 (0.2)
(i) Pd(OAc)2 (0.1)
(ii) Pd(OAc)2 (0.1)
(i) Pd(OAc)2 (0.1)
(ii) Pd(OAc)2 (1.0)
Pd(OAc)2 (1.0)
Pd(PPh3)4 (0.2)
Pd(PPh3)4 (0.2)
Pd(PPh3)4 (0.05)
Pd(OAc)2 (0.1)
Pd(PPh3)4 (0.05)
K2CO3 (4)
NaHCO3 satd
K2CO3 (4)
dioxane/water; 100
toluene, EtOH; reflux
dioxane/water; 100
dioxane/water; 100
dioxane/water; 100
4
12
8
24
(i) 6
(ii) 18
(i) 6
(ii) 12
6
6
0.5
4
degradation
11 (98), 13 (1)
11 (51), 13 (10)
11 (70), 13 (20)
K2CO3 (4), PPh3 (0.4)
(i) K2CO3 (4),
(ii) PPh3 (0.2)
(i) K2CO3 (4)
(ii) PPh3 (2)
PPh3 (2)
AcOK (1.1)
AcOK (1.1)
AcOK (1.1)
K2CO3 (4)
6
1
dioxane/water; 100
11 (34)
13 (20)
13 (83)
13 (15), 12 + 11 (50)
14 (quant.)
14 (quant.)
15 (58), 16 (31)
17 (quant.)
7
8
9
10
11
12
11
11
12
12
10
16
dioxane; 90
DMA; 130
DMA; 130
DMA; 130
dioxane/water; 100
DMA; 130
8
4
AcOK (1.1)
phosphine was introduced at the beginning of the reaction in
our first experiments (entry 4). Increasing the quantity of
palladium acetate (0.2 equiv) and PPh3 (0.4 equiv) slightly
increased the yield of the tandem reaction (compound 13 was
obtained in a 10% yield) after 24 h, but the Suzuki product 11
was obtained in only 51% yield with a complete disappearance
of the starting material 1. Therefore, we decided to introduce
the second amount of palladium catalyst and triphenylphosphine
after 6 h of reaction, which is the time necessary for the full
Suzuki conversion of 1 into 11. Thus, upon sequentially adding
10% catalyst and 20% of PPh3, the reaction afforded, after 24
h, 70% of the Suzuki derivative 11 and 20% of the annulated
product 13, increasing the yields of 11 and 13, but the Heck
reaction was never completed (entry 5). Not really satisfied by
this, we performed a further attempt based on our knowledge
of the palladium intramolecular reaction in the naphthalene
series.21 In this case, the reaction time was reduced, in the
presence of a stoichiometric amount of catalyst, and fortunately,
the yields were enhanced. So adding 1 equiv of Pd (OAc)2 and
2 equiv of phosphine, after 6 h of reaction, led to the same
amount of cyclized compound 13 (20%) but to only 34% of
compound 11 (entry 6).

792 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Routier et al.
Scheme 4a
a (i) KOH (5 N), EtOH, (10 equiv), reflux, then HCl, 0 °C, pH 5; from 14, 5 h, 99%; from 17, idem, 83%; (ii) BBr3 (1.0 equiv), CH2Cl2, rt, 1 h, quant.;
(iii) (a) from 19, NH4OH 35%, DMF, reflux, 8 h, 22 93%; from 21, idem, 120 °C, 24 h, 23 96%; (b) from 19, NH2(CH2)2N(CH3)2, DMF, 120 °C, 12 h, 24
80%; from 21, idem, 25 71%; (c) from 19, HOCH2CH(NH2)CH2OH, DMF, reflux, 12 h, 26 75%; from 21, idem, 27 68%; (d) from 19, histamine (1.5 equiv),
DMF, 130 °C, 3 days, sealed tube, 28 97%; from 21, idem, 29 91%.
Confronted with difficulties in enhancing the yield of the
tandem cross-coupling reactions, we then decided to realize the
synthesis step-by-step (entry 7). Starting from 11, we found that
a stoichiometric amount of palladium acetate led to the cyclized
compound 13 in a 83% yield with a reaction time of 6 h. Several
assays were also performed to optimize this step. As an example,
we have changed the nature of the catalyst according to a
published procedure.23 Thus, using 0.2 equiv of Pd(PPh3)4 as a
catalyst, AcOK as a base in DMA at 130 °C led to 13 in only
15% accompanied by a inseparable mixture of deprotected
compound 12 and 11 (2/1, 50%, entry 8), suggesting also a
thermal sensitivity of the benzenesulfonyl group in the presence
of the base.
We have also cleaved the protective group of 11 by Bu4NF
in THF, which afforded 12 in a 93% yield, and applied to this
compound the Heck cyclization conditions Pd(PPh3)4 (0.2
equiv), AcOK, (1.1 equiv), DMA at 130 °C. In this case, we
obtained the cyclized compound 14 in a quantitative yield after
a remarkably short reaction time (30 min, entry 9). Decreasing
also the amount of Pd(PPh3)4 to 0.05 equiv increased the reaction
time to 4 h, but the yield of 14 was not affected (entry 10).
Alternatively, compound 14 was prepared from 13 using Bu4NF
in THF in only 2 h in the same yield (93%). These results
showed also the difference of reactivity between deprotected
and protected indoles during intermolecular Heck reaction.28-30
Thus, compound 14 could be obtained by a Suzuki reaction on
1, leading to 11, followed by a deprotection reaction affording
12, and then a quantitative Heck cyclization led to 14 in an
overall yield of 91%. On the other hand, the deprotection step
could be carried out after the Heck reaction, leading to 13. By
this second route the overall yield decreased slightly to 82%.
Both of these methods, starting from 1, led to the desired
unprotected compound 14 in three steps.
On the basis of our previous results in the naphthalene series,
we decided to introduce a hydroxyl group which is generally
the most biologically effective substituent on the indolic moiety.
Commercially available 5-benzyloxyindole led to compound 10
in three easy steps. The Suzuki procedure carried out with the
boronic acid 8 afforded compound 15 in a 58% yield. In
addition, the N-indolic deprotected compound 16 was isolated
in a 31% yield (entry 11). This reaction was nevertheless not
optimized. The synthetic sequence was directly carried on with
deprotection of compound 15 into 16 in the presence of Bu4NF
in 86% yield. Using the same conditions as for 12, the final
Heck cross-coupling of 16 gave compound 17 in a quantitative
yield (entry 12). Deprotection of the benzyl group was
performed with BBr3 at room temperature to afford 18 in a
quantitative yield. In the 5-benzyloxyindole series, compound
17 was obtained from 9 in a 89% overall yield in three steps.
The phenylcarbazoles 14 and 17 could be easily substituted
on the maleimide moiety. It should be noted that the direct
displacement of the NCH3 group by a primary amine failed. So
the intermediate anhydrides 19 and 20 were prepared by treating
14 or 17 with a boiling KOH hydroalcoholic solution during a
few hours and then an acidification to pH 4 (Scheme 4). These
two compounds were isolated in 99 and 83% yields, respec-
tively. Debenzylation of 20 with BBr3 afforded the anhydride
21 in a quantitative yield. Anhydrides 19 and 21 were treated
successively with ammonia or 2-dimethylaminoethylamine or
2-amino-1,3-propandiol and histamine to afford compounds 22-
29 in moderate (51%) to excellent (97%) yields. In the case of
histamine, only a slight excess of this reagent was used; 3 days
of reaction in a sealed tube were necessary to complete the
reaction, giving compounds 28 and 29 in excellet yields.
Results and Discussion
In Vitro Antiproliferative Activities. The antiproliferative
activities of the compounds were tested against CEM and
camptothecin-resistant CEM/C2 human leukemia cell lines.
Their cytotoxicity was measured using a conventional micro-
culture tetrazolium-based assay. IC50 values are collated in Table
CEM
2. The relative resistance index (i.e. the ratio IC50CEM/C2/IC50
)
never exceeds 3 (versus >2000 for camptothecin).31 Therefore,
in all cases the use of the topoisomerase I-deficient CEM/C2
cell line (carrying a mutation on the top1 gene) has only a
marginal effect on the cytotoxicity of the molecules. Topo-
isomerase I is surely not the target of these compounds (unless
they are not sensitive to this specific enzyme mutation, but this
is very unlikely; see below).
The phenylcarbazole 22 was totally inactive (IC50 > 100 µM)
in both cell lines, whereas the corresponding anhydride 19
showed a slight increase in the cytotoxic activity, ranging over
25 µM. The presence of an additional hydroxyl group on the
carbazole moiety sensibly increased the cytotoxicity, and the
introduction of a hydrophilic substituent induces an increase of
activity by at least 2-fold (compare 22 and 19 with 23 and 21).
The introduction of various modifications on the indolic nitrogen
center enhanced markedly the cytotoxic potential of the different
molecules tested. Substitution on the maleimide moiety of 22
by a methyl group has no effect on its cytotoxicity, as observed
with compound 14 (IC50 > 100 µM). In sharp contrast, the
replacement the N-methyl group by an N-2-dimethylaminoethyl
side chain on 24 considerably improves the efficacy of this
compound to reach submicromolar activities (IC50 ) 110 nM).
However, the substitution of 22 by a ramified bis-hydroxylated
side chain or by histamine was less successful. The cytotoxicities
of compounds 26 and 28 were increased (IC50 ) 3.71 and 1.58
µM, respectively) when compared to those of the unsubstituted

Phenylcarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 793
Table 2. In Vitro Cytotoxicity and Kinase Activity Assays IC50 (µM)a
a ND ) not determined; RRI ) relative resistance index.
or N-methylated compounds 22 and 14 but did not reach the
high nanomolar range observed with the N-2-dimethylamino-
ethyl compound 24. Interestingly, the introduction of a hydroxyl
group on the carbazole moiety of the substituted compounds
24 and 28 increased their cytotoxic activities 2-10-fold (IC50
) 12.2 and 715 nM, respectively, for compounds 25 and 29).
A remarkable level of cytotoxicity has been reached with
compound 25.
To sum up the cytotoxicity data, it appears that substitution
of the maleimide moiety by a N-dimethylamino side chain leads
to a highly cytotoxic molecule (24, IC50 ) 110 nM) and this
potent activity is further increased when this modification is
combined with the presence of a hydroxyl group on the
carbazole moiety (25, IC50 ) 12.2 nM). These data confirm
the idea that the presence of a basic chain on the maleimide
generally improves the efficacy of the compound.21,32 This is
not entirely surprising, as the protonation of the terminal nitrogen
at physiological pH must facilitate the interaction with the DNA
polyanion (as well as enhancing its solubility).
Figure 2. Cell cycle distribution in CEM cells treated for 72 h with
graded concentrations (0, 0.1, 0.5, 1, 2.5, 5, and 10 µM) of compounds
24, 25, 28, and 29. Cells were analyzed with a FACScan flow cytometer.
Data are the result of two independent experiments.
Cell Cycle Effect and Mechanism of Action. The effects
of the different compounds on cell cycle progression of leukemia
CEM cells were investigated using propidium iodide staining
and FACScan flow cytometry. No changes in the cell cycle
profiles were observed after treatment of CEM cells with 1 µM
of phenylcarbazole 22 or the corresponding anhydride 19. The
presence of an additional hydroxyl group on the carbazole
moiety, as for compounds 23 and 21, does not affect the
accumulation of CEM cells in the different cell cycle phases
(data not shown). The introduction of a methyl group or a
ramified bis-hydroxylated side chain on the indolic nitrogen of
22 has also no effect on the cell cycle, as seen for compounds
14 and 26 (29% G2/M). However, substitution on the maleimide
moiety of 22 by a histamine residue induces a significant
accumulation of cells in the G2/M phase of the cell cycle,
reaching about 80% after treatment with 5 µM of compound
28 (Figure 2). The addition of a hydroxyl group on the carbazole
moiety of those three compounds induces first an accumulation
of cells in the G2/M phases, reaching 34%, 33%, and 45% for
compounds 18, 27, and 29, respectively, and then followed by
an S phase accumulation when cells were treated with a higher
concentration (5-10 µM) (Figure 2). The addition to 22 of an
N-2-dimethylaminoethyl side chain significantly changes the
profile of the CEM cell cycle when compared with the other

794 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Routier et al.
DNA/molecule complex
Figure 3. Variation of melting temperatures (∆Tm ) Tm
- TmDNA) for the different derivatives bound to calf thymus DNA (open
bars) or poly(dAT)2 (plain bars) at a drug/DNA ratio of 0.5. Tm
measurements were performed in pH 7.1 BPE buffer (6 mM Na2HPO4,
2 mM NaH2PO4, 1 mM EDTA) at 260 nm with a heating rate of 1
°C/min.
molecules studied. Treatment with 1 µM 24 provokes an
accumulation of cells in the S phase (26%) and the appearance
of a small population of apoptotic sub-G1 cells (5%). The
introduction of a hydroxyl group to this molecule triggers a
similar but more potent effect, as 15% of the cells are apoptotic
after treatment with 0.5 µM 25. This data set might be correlated
with the gain of cytotoxic activity measured when the hydroxyl
group was added (IC50 ) 110 and 12.2 nM for 24 and 25,
respectively).
To understand how those molecules induce their cellular
effects, we have investigated their effect on purified human
topoisomerase I. None of these compounds were able to induce
topoisomerase I-dependent DNA strand breaks, as seen with
the reference drug camptothecin (data not shown). The molec-
ular assay confirms the cell data to conclude that topoisomerase
I is not the target of these molecules, despite the capacity of
some compounds to bind to DNA. Topoisomerase II inhibition
was also investigated but no effect was detected (data not
shown).
DNA binding was studied by melting temperature experi-
ments using both calf thymus DNA (CT-DNA) and the polymer
poly(dAT)2 (Figure 3). In our experimental conditions, CT-DNA
and poly(dAT)2, respectively, melt at 66 and 43 °C. The melting
temperatures (Tm) remained unchanged when nucleic acids were
incubated with the phenylcarbazole 22 or its corresponding
anhydride 19.
The introduction of various chemical modifications at the
indolic nitrogen center stabilizes the duplex DNA against heat
denaturation. Whereas the N-methyl derivative 14 shows no
significant effect on DNA stability, the replacement of the Me
group by a longer side chain, such as a N-dimethylaminoethyl
(24), a ramified bis-hydroxylated (26), or a histamine (28) group,
leads to a significant increase of the Tm values with both CT-
DNA and poly(dAT)2. These compounds do exhibit significant
interaction with nucleic acids. In all cases, the addition of a
hydroxyl group on the carbazole chromophore promotes DNA
binding (Figure 3). This enhanced affinity for DNA correlates
with the gain of cytotoxicity and the drug capacity to block
CEM cells in G2/M and S phases of the cell cycle. Therefore,
it is tempting to conclude that DNA interaction plays a role in
the cytotoxic action of these compounds, but this is certainly
not sufficient to explain the remarkable antiproliferative activity
of compounds such as 24 and 25.
Figure 4. DNase I footprinting for compounds 22-25 on the 3′-end
labeled (a) 117-bp and (b) 265-bp EcoR I-PVu II DNA restriction
fragments from the pBS plasmid. The products of nuclease digestion
were resolved on 8% polyacrylamide gels containing 7 M urea. Control
tracks (cont) contain no ligand. The concentrations (µM) of the tested
products are shown at the top of the appropriate gels. Tracks labeled
“G” represent dimethyl sulfate-piperidine markers specific for guanines.
Numbers on the left side of the gels refer to the standard numbering
scheme for the nucleotide sequence of the DNA fragment.
were subjected to DNase I digestion in the presence of increasing
drug concentrations. In most cases, the molecules do not affect
the DNase I cleavage pattern and show no sequence preference.
However, clear modulation of the enzyme cutting was observed
with 24 and 25, both possessing the dimethylaminoethyl side
chain. The footprinting gels in Figure 4 show the concentration
dependence for the modulation of DNase I cleavage by these
two molecules and the densitometric analysis of the gels attests
to the sequence preference (Figure 5).
The differential cleavage plots (Figure 5) for the 117- and
265-bp fragments in the presence of 22-25 show marked
differences between the molecules. The sequences protected
from the enzyme cleavage (i.e. the presumptive drug binding
sites) by 24 and 25 contain mixed AT and CG bp but are
essentially GC-rich, such as 5′-CGCC and 5′-CGTTGT in the
117-bp fragment and the sequence 5′-CTCACT in the 265-bp
fragment. Strongly enhanced DNase I-cleaved regions were also
characterized in the vicinity of the protected sites (such as around
sites 45 and 65 on the 117-mer). These sites reflect structural
perturbations of the DNA at sequences flanking the binding sites,
and this is generally a signature of intercalative drug binding.
We next tested the compounds for potential inhibition of
cyclin-dependent kinases (CDK1 and CDK5) and glycogen
synthase kinase-3 (GSK-3). Only compounds 22 and 23 showed
a significant inhibitory activity. None showed a selectivity for
CDKs or GSK-3. Despite these kinase inhibitory activities
(particularly compound 23), the compounds were inactive in
the cell proliferation assay. The most active compounds on
proliferation (24 and 25) were inactive on the kinases tested. It
is thus unlikely that the antiproliferative effects of the synthe-
sized compounds can be accounted for by a direct effect on
A DNase I footprinting approach was also performed to
evaluate the sequence selectivity of the different compounds.
Two DNA restriction fragments of 117 and 265 base pairs (bp)

Phenylcarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 795
points are uncorrected. IR absorptions were recorded on a Perkin-
Elmer PARAGON 1000 PC, and values are reported in cm-1. MS
spectra (ion spray) were performed on a Perkin-Elmer Sciex PI
300 or on Avatar 320 using an ATR (Ge) technique. 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. Flash chromatography columns were performed using silica
gel 60 (0.063-0.200 mm, Merck).
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(2-methoxyphenyl)-1-
methylpyrrole-2,5-dione (3). A solution containing 2-methoxy-
phenylboronic acid 2 (150 mg, 0.99 mmol), compound 1 (400 mg,
0.9 mmol), and K2CO3 (224 mg, 1.8 mmol) in a mixture of dioxane
(9 mL) and water (1.7 mL) was degassed by argon bubbling for
20 min. Pd(OAc)2 (20 mg, 0.9 mmol) was added in one portion
and the reaction mixture immersed into a preheated oil bath at 80
°C for 4 h. After cooling, water (50 mL) and EtOAc (50 mL) were
added, and the solution was filtered. The precipitate was washed
with EtOAc (2 × 25 mL), and the aqueous layers were separated.
The organic layer was evaporated under reduced pressure and the
crude material was purified by flash chromatography (petroleum
ether/EtOAc 8/2) to afford compound 3 as a yellow solid (423 mg,
quant.). Mp: 142 °C. 1H NMR (DMSO, 250 MHz): δ 2.66 (s, 3H,
NCH3), 3.05 (s, 3H, OCH3), 6.50 (d, 1H, J ) 7.8 Hz), 6.78 (d, 1H,
J ) 8.8 Hz), 6.89 (t, 1H, J ) 8.8 Hz), 7.03 (t, 1H, J ) 7.3 Hz),
7.24 (t, 1H, J ) 7.8 Hz), 7. 37 (m, 2H), 7.62 (m, 2H), 7.70 (d, 1H,
J ) 7.1 Hz), 7.96 (d, 1H, J ) 8.3 Hz), 8.05 (m, 3H). MS (IS): 473
(M + 1)+. Anal. (C26H20N2O5S): C, H, N.
Figure 5. Differential cleavage plots indicating the susceptibility of
(a) 117-bp and (b) 265-bp fragments to DNase I cutting in the presence
of indicated compounds (10 µM each). Negative values correspond to
a ligand-protected site and positive values represent enhanced cleavage.
Vertical scales are in units of ln(fa) - ln(fc), where fa is the fractional
cleavage at any bond in the presence of the drug and fc is the fractional
cleavage of the same bond in the control, given closely similar extents
of overall digestion. Only the region of the restriction fragment analyzed
by densitometry is shown.
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(2-hydroxyphenyl)-1-
methylpyrrole-2,5-dione (4). To a solution containing compound
3 (500 mg, 1.06 mmol) in distillated CH2Cl2 (8 mL) at 0 °C was
slowly added a solution of BBr3 (1M in CH2Cl2, 8.0 mL, 8.0 mmol).
After 1 h at room temperature, the reaction mixture was poured
into ice (20 g). The aqueous layer was extracted with CH2Cl2 (2 ×
20 mL), and the combined organic layers were evaporated under
reduced pressure. Flash chromatography (petroleum ether/EtOAc
3/7) afforded compound 4 as an orange solid (455 mg, 94%). Mp:
CDKs or GSK-3. However, the reason for the lack of cellular
effects of compounds 22 and 23 remains to be identified. These
compounds constitute interesting scaffolds from which more
potent and more selective inhibitors could be designed. A
structural analogy can be found between known highly cytotoxic
antitumor agents and the most potent compounds reported here,
such as 24, which bears a dimethylaminoethyl side chain
appended to a flat hydroxylated chromophore as for the
triazoloacridine drug C-1305.33 This is also the case for
compound 27, which is structurally comparable to the indolo-
carbazole derivative J-107088 (edotecarin), mentioned in the
Introduction.34 In conclusion, we have devised a novel efficient
synthetic strategy to prepare a family of phenylcarbazole
derivatives and established structure-activity relationships. The
synthesis of phenylcarbazoles was realized in only four very
efficient steps starting from indoles. The strategy involving
palladium-catalyzed step represents a general method that could
be easily scale-up and applied to other (hetero)aromatic systems.
Some of the molecules, in particular those containing an
N-dimethylaminoethyl side chain on the maleimide nitrogen,
show potent cytotoxic properties, and interaction with DNA (but
not topoisomerase I) may contribute to this antiproliferative
activity. DNA binding, presumably by intercalation at defined
AT/GC-containing sites, likely accounts for their propensity to
block CEM cell cycle progression in the G2/M or S phases.
The phenylcarbazole series certainly warrants further investiga-
tions, and the chemical routes reported here offer interesting
perspectives to introduce a variety of substituents.
1
172-174 °C. H NMR (DMSO, 250 MHz): δ 3.05 (s, 3H), 6.76
(m, 2H), 6.97 (t, 1H, J ) 7.0 Hz), 6.89 (m, 3H), 7.25 (t, 1H, J )
7.3 Hz), 7.61 (m, 2H), 7.70 (d, 1H, J ) 7.3 Hz), 7.89 (m, 3H),
8.70 (s, 1H), 9.61 (s, 1H, exchangeable D2O). MS (IS): 459 (M +
1)+. Anal. (C25H18N2O5S): C, H, N.
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(2-trifluoromethyl-
sulfonatephenyl)-1-methylpyrrole-2,5-dione (5). To a solution of
compound 4 (800 mg, 1.75 mmol) and NEt3 (730 µL, 5.26 mmol)
in CH2Cl2 (36 mL) cooled at 0 °C was added dropwise a solution
of triflic anhydride (980 µL, 1.75 mmol) in CH2Cl2 (5 mL). After
2 h, water (20 mL) was added, and the aqueous layers were
extracted with CH2Cl2 (2 × 20 mL). The combined organic layers
were evaporated under reduced pressure, and the crude residue was
purified by flash chromatography (petroleum ether/EtOAc 7/3 NEt3
1%) to afford compound 5 as a yellow solid (875 mg, 85%). Mp:
1
94 °C. H NMR (DMSO, 250 MHz): δ 3.12 (s, 3H), 6.48 (d, 1H,
J ) 7.8 Hz), 6.93 (t, 1H, J ) 7.8 Hz), 7.27 (t, 1H, J ) 7.8 Hz),
7.40 (d, 1H, J ) 7.8 Hz), 7.59-7.78 (m, 6H), 7.96 (m, 3H), 8.15
(s, 1H). MS (IS): 591 (M + 1)+. Anal. (C26H17F3N2O7S2): C, H,
N.
3-(2-Hydroxyphenyl)-4-(1H-indol-3-yl)-1-methylpyrrole-2,5-
dione (6). A solution of compound 5 (150 mg, 0.255 mmol) and
Bu4NF (1M in THF, 510 µL, 0.510 mmol) in dry THF (15 mL)
under argon was refluxed for 2 h. After cooling, water (15 mL)
was added and the aqueous layer was extracted with EtOAc (2 ×
20 mL). The combined organic layers were evaporated under
reduced pressure and the crude material was purified by flash
chromatography (petroleum ether/EtOAc 7/3) to afford compound
6 as an orange solid (70 mg, 77%). Mp: 108 °C. 1H NMR (DMSO,
250 MHz): δ 3.02 (s, 3H), 6.54 (d, 1H, J ) 7.9 Hz), 6.65 (t, 1H,
J ) 7.9 Hz), 6.81 (m, 2H), 7.02 (t, 1H, J ) 7.8 Hz), 7.13 (d, 1H,
J ) 6.7 Hz), 7.22 (t, 1H, J ) 7.5 Hz), 7.36 (d, 1H, J ) 8.1 Hz),
7.97 (d, 1H, J ) 3.3 Hz), 9.41 (s, 1H, exchangeable D2O), 11.80
(s, 1H, exchangeable D2O). MS (IS): 319 (M + 1)+. Anal.
(C19H14N2O3): C, H, N.
Experimental Section
Chemistry. 1H and 13C NMR spectra were recorded on a Bruker
250 instrument using CDCl3 or DMSO-d6. Chemical shifts are
reported in ppm (δ scale) and all J values are in hertz. The following
abbreviations are used: singlet (s), doublet (d), doubled doublet
(dd), triplet (t), multiplet (m), quaternary carbon (Cq). Melting

796 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Routier et al.
3-(1-Benzenesulfonyl-1H-indol-3-yl)-4-(2-bromophenyl)-1-
methylpyrrole-2,5-dione (11). A solution containing 2-bromo-
phenylboronic acid 8 (712 mg, 3.26 mmol), compound 1 (410 mg,
0.92 mmol), and K2CO3 (509 mg, 3.68 mmol) in a mixture of
dioxane (5 mL) and water (1.2 mL) was degassed by argon bubbling
for 20 min. Pd(OAc)2 (26 mg, 0.09 mmol) was then added in one
portion and the reaction mixture poured into a preheated oil bath
at 100 °C for 9 h. After cooling, water (50 mL) and EtOAc (50
mL) were added, and the solution was filtered. The precipitate was
washed with EtOAc (2 × 25 mL), and the aqueous layers were
separated. The organic layer was evaporated under reduced pressure
and the crude material was purified by flash chromatography
(petroleum ether/EtOAc 9/1) to afford compound 11 as a yellow
3-(1-Benzenesulfonyl-1H-5-benzyloxyindol-3-yl)-4-(2-bromo-
phenyl)-1-methylpyrrole-2,5-dione (15). The same procedure as
described for compound 11, starting from 10, was used. The reaction
time was 8 h and flash chromatography (petroleum ether/EtOAc
8/2) afforded compound 15 (58%) as a yellow solid. Mp: 139-
141 °C. 1H NMR (DMSO, 250 MHz): δ 3.08 (s, 3H), 4.48 (s, 2H),
6.19 (d, 1H, J ) 3.2 Hz), 6.89 (dd, 1H, J ) 7.8, J ) 3.2 Hz),
7.32-7.47 (m, 8H), 7.60-7.70 (m, 4H), 7.80 (d, 1H, J ) 10.1
Hz), 7.97 (d, 2H, J ) 8.3), 8.16 (s, 1H). MS (IS): 627-629 (M +
1)+. Anal. (C32H23BrN2O5S): C, H, N.
3-(1H-5-benzyloxyindol-3-yl)-4-(2-bromophenyl)-1-methyl-
pyrrole-2,5-dione (16). The same procedure as described for
compound 12 was used. Flash chromatography afforded compound
16 (86%) as a orange solid. Mp: >250 °C. 1H NMR (DMSO, 250
MHz): δ 3.04 (s, 3H), 4.44 (s, 2H), 6.03 (s, 1H), 6.73 (d, 1H, J )
8.8 Hz), 7.35-7.46 (m, 9H), 7.81 (d, 1H, J ) 7.5 Hz), 8.11 (s,
1H), 11.94 (s, 1H, exchangeable D2O). MS (IS): 487-489 (M +
1)+. Anal. (C26H19BrN2O3): C, H, N.
1
solid (469 mg, 98%). Mp: 160 °C. H NMR (DMSO, 250 MHz):
δ 3.08 (s, 3H), 6.77 (d, 1H, J ) 8.1 Hz), 7.00 (t, 1H, J ) 8.1 Hz),
7.33 (t, 1H, J ) 8.1 Hz), 7.44 (m, 3H), 7.63 (m, 3H), 7.73 (t, 1H,
J ) 7.3 Hz), 7.92 (m, 3H), 8.08 (s, 1H). MS (IS): 521-523 (M +
1)+. Anal. (C25H17BrN2O4S): C, H, N.
3-(2-Bromophenyl)-4-(1H-indol-3-yl)-1-methylpyrrole-2,5-
dione (12). To a solution of compound 11 (1.100 g, 2.11
mmol) in THF (20 mL) was added at room temperature a solution
of Bu4NF (1 M in THF, 2.53 mL, 2.53 mmol). After 2 h at reflux,
the mixture was cooled, water (20 mL) was added, and the aqueous
layer was extracted with EtOAc (2 × 20 mL). The combined
organic layers were evaporated under reduced pressure. The crude
residue was purified by flash chromatography (petroleum ether/
EtOAc 7/3) to afford compound 12 as a orange solid (747 mg,
93%). Mp: 196-198 °C. 1H NMR (DMSO, 250 MHz): δ 3.11 (s,
3H), 6.42 (d, 1H, J ) 8.1 Hz), 6.73 (t, 1H, J ) 8.1 Hz), 7.13 (t,
1H, J ) 7.6 Hz), 7.41 (m, 4H), 7.81 (dd, 1H, J ) 1.6 Hz, J ) 3.1
Hz), 8.13 (d, 1H, J ) 1.6 Hz), 12.60 (s, 1H, exchangeable D2O).
MS (IS): 381-383 (M + 1)+. Anal. (C19H13BrN2O2): C, H, N.
2-Methyl-8-(benzenesulfonyl)benzo[a]pyrrolo[3,4-c]carbazole-
1,3(2H,8H)-dione (13). A solution containing compound 11 (550
mg, 1.05 mmol), Bu4NCl (292 mg, 1.05 mmol), AcONa (172 mg,
2.10 mmol), and PPh3 (422.1 mg, 2.10 mmol) in dry dioxane (19
mL) was degassed by argon bubbling for 20 min. Pd(OAc)2 (235
mg, 2.10 mmol) was then added in one portion and the reaction
mixture immersed into a preheated oil bath at 90 °C for 6 h. After
cooling, water (50 mL) and EtOAc (50 mL) were added, and the
solution was filtered. The precipitate was washed with EtOAc (2
× 25 mL), and the aqueous layers were separated. The organic
layer was evaporated under reduced pressure and the crude material
was purified by flash chromatography (petroleum ether/EtOAc 8/2)
to afford compound 13 as a yellow solid (344 mg, 83%). Mp: 244
2-Methyl-5-(benzyloxy)benzo[a]pyrrolo[3,4-c]carbazole-1,3-
(2H,8H)-dione (17). The same procedure as described in procedure
A for compound 14, starting from 16, was used. Flash chromatog-
raphy afforded compound 17 (quant.) as a orange solid. Mp: 188-
190 °C. 1H NMR (DMSO, 250 MHz): δ 3.04 (s, 3H), 5.17 (s, 2H),
7.20 (d, 1H, J ) 8.1 Hz), 7.35-7.46 (m, 3H), 7.56-7.58 (m, 3H),
7.70-7.73 (m, 2H), 8.40 (s, 1H), 8.49 (d, 1H, J ) 8.1 Hz), 8.81
(d, 1H, J ) 8.2 Hz), 12.64 (s, 1H, exchangeable D2O). MS (IS):
407 (M + 1)+. Anal. (C26H18N2O3): C, H, N.
2-Methyl-5-hydroxybenzo[a]pyrrolo[3,4-c]carbazole-1,3-
(2H,8H)-dione (18). To a solution containing compound 17 (263
mg, 0.65 mmol) in dry CH2Cl2 (10 mL) was added dropwise a
solution of BBr3 (1M in CH2Cl2, 0.65 mL, 0.65 mmol). After 1 h
at room temperature, the reaction mixture was poured into ice (30
g), and the aqueous layers were extracted with EtOAc (2 × 30
mL). The organic layer was evaporated under reduced pressure to
dryness. The crude material was washed with in MeOH (3 × 10
mL) to afford compound 18 as an orange solid. Mp: >250 °C. 1H
NMR (DMSO, 250 MHz): δ 3.00 (s, 3H), 7.02 (dd, 1H, J ) 9.1
Hz, J ) 2.3 Hz), 7.47 (d, 1H, J ) 9.1 Hz), 7.64-7.68 (m, 2H),
8.26 (d, 1H, J ) 2.3 Hz), 8.44 (dd, 1H, J ) 9.1 Hz, J ) 4.3 Hz),
8.76 (dd, 1H, J ) 9.1 Hz, J ) 4.3 Hz), 9.24 (s, 1H), 12.50 (s, 1H,
exchangeable D2O). MS (IS): 317 (M + 1)+. Anal. (C19H12N2O3):
C, H, N.
5H-Furo[3,4-c]benzo[a]pyrrolo[3,4-c]carbazole-1,3(8H)-di-
one (19). To a solution containing compound 14 (500 mg, 1.66
mmol) in EtOH (10 mL) was added at room temperature an aqueous
solution of KOH (5 N) under vigorous stirring. The reaction mixture
was refluxed during 5 h. After cooling, the reaction mixture was
acidified using a hydrochloric solution (5 N) until the pH raised to
5 and then concentrated to 6 mL under reduced pressure. The
precipitate was filtered, washed with cold MeOH (3 × 10 mL),
and dried under vacuum to afford compound 19 as a red solid (473
mg, 99%). Mp: >250 °C. 1H NMR (DMSO, 250 MHz): δ 7.40 (t,
1H, J ) 7.5 Hz), 7.59 (d, 1H, J ) 7.5 Hz), 7.75 (d, 1H, J ) 7.5
Hz), 7.78 (m, 2H), 8.66 (d, 1H, J ) 7.0 Hz), 8.79 (m, 2H), 12.35
(s, 1H, exchangeable D2O). MS (IS): 288 (M + 1)+. Anal.
(C18H9NO3): C, H, N.
5-Benzyloxy-5H-furo[3,4-c]benzo[a]pyrrolo[3,4-c]carbazole-
1,3(8H)-dione (20). The same procedure as described for compound
19, starting from compound 17, yielded compound 20 as a orange
solid (83%). Mp: 243 °C dec. 1H NMR (DMSO, 250 MHz): δ 5.13
(s, 2H), 7.20 (d, 1H, J ) 8.2), 7.35-7.46 (m, 6H), 7.76-7.81 (m,
2H), 8.06 (s, 1H), 8.52 (d, 1H, J ) 8.2 Hz), 8.63 (d, 1H, J ) 8.0
Hz), 12.90 (s, 1H, exchangeable D2O). MS (IS): 394 (M + 1)+.
Anal. (C25H15NO4): C, H, N.
1
°C dec. H NMR (DMSO, 250 MHz): δ 3.08 (s, 3H), 6.85 (m,
2H), 7.15 (m, 2H), 7.42 (m, 2H), 7.63 (t, 1H, J ) 7.5 Hz),
7.79 (m, 2H), 8.18 (d, 1H, J ) 8.3 Hz), 8.60 (d, 1H, J ) 7.8 Hz),
8.71 (m, 1H), 8.94 (m, 1H). MS (IS): 441 (M + 1)+. Anal.
(C25H16N2O4S): C, H, N.
2-Methylbenzo[a]pyrrolo[3,4-c]carbazole-1,3(2H,8H)-dione (14).
Procedure A from 12. A solution containing compound 12 (623
mg, 2.00 mmol) and AcOK (215 mg, 2.2 mmol) in dry DMA (27
mL) was degassed by argon bubbling for 20 min. Pd(PPh3)4 (119
mg, 0.1 mmol) was then added in one portion and the reaction
mixture was immersed into a preheated oil bath at 130 °C for 4 h.
After cooling, water (50 mL) and EtOAc (50 mL) were added, and
the solution was filtered. The precipitate was washed with EtOAc
(2 × 25 mL), and the aqueous layers were separated. The organic
layer was evaporated under reduced pressure and the crude material
was purified by flash chromatography (petroleum ether/EtOAc 5/5
then MeOH) to afford compound 14 as an orange solid (599 mg,
quant.).
Procedure B from 13. The same procedure as described for
compound 12 was used. Flash chromatography (petroleum ether/
1
EtOAc 7/3) afforded compound 14 (quant.). Mp: 244 °C dec. H
5-Hydroxy-5H-furo[3,4-c]benzo[a]pyrrolo[3,4-c]carbazole-
1,3(8H)-dione (21). The same procedure as described for compound
18 yielded compound 21 as a red solid (quant.). Mp: >250 °C. 1H
NMR (DMSO, 250 MHz): δ 7.07 (dd, 1H, J ) 9.1 Hz, J ) 2.2
Hz), 7.55 (d, 1H, J ) 9.1 Hz), 7.83-7.89 (m, 2H), 8.12 (d, 1H, J
) 2.2 Hz), 8.62 (d, 1H, J ) 8.4 Hz), 8.73 (d, 1H, J ) 9.1 Hz),
NMR (DMSO, 250 MHz): δ 3.11 (s, 3H), 7.34 (t, 1H, J ) 8.1
Hz), 7.56 (t, 1H, J ) 7.2 Hz), 7.71 (d, 1H, J ) 8.1 Hz), 7.81
(m, 2H), 8.65 (m, 1H), 8.87 (d, 1H, J ) 8.1), 8.96 (m, 1H), 12.92
(s, 1H, exchangeable D2O). MS (IS): 301 (M + 1)+. Anal.
(C19H12N2O2): C, H, N.

Phenylcarbazoles as Potential Anticancer Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 797
9.40 (s, 1H, exchangeable D2O), 12.92 (s, 1H, exchangeable D2O).
MS (IS): 304 (M + 1)+. Anal. (C18H9NO4): C, H, N.
After cooling, water (10 mL) was added. The precipitate was filtered
and washed with cold MeOH (5 mL) to afford compound 28 as an
1
Benzo[a]pyrrolo[3,4-c]carbazole-1,3(2H,8H)-dione (22).19 To
a solution of compound 19 (150 mg, 0.52 mmol) in DMF (2 mL)
was added a solution of ammonia (37%, 2 mL). The reaction
mixture was refluxed during 8 h. After cooling to room temperature,
water (20 mL) was added and pH was adjusted to 7 using
hydrochloric acid (1 N). The precipitate was filtered and washed
with EtOAc (3 × 10 mL) to afford compound 22 (138 mg, 93%).
5-Hydroxybenzo[a]pyrrolo[3,4-c]carbazole-1,3(2H,8H)-di-
one (23). The same procedure as described for compound 22 was
used. The solution was heated at 120 °C for 24 h. After filtration,
the precipitate was dissolved in acetone and purified by flash
chromatography (petroleum ether/EtOAc 7/3) to afford compound
23 as a red solid (95 mg, 96%). Mp: >250 °C. 1H NMR (DMSO,
250 MHz): δ 7.03 (d, 1H, J ) 9.0 Hz), 7.56 (d, 1H, J ) 9.0 Hz),
7.77 (m, 3H), 8.33 (d, 1H, J ) 2.0 Hz), 8.44 (d, 1H, J ) 9.0 Hz),
9.26 (s, 1H, exchangeable D2O), 11.07 (s, 1H, exchangeable D2O),
12.50 (s, 1H, exchangeable D2O). MS (IS): 303 (M + 1)+. Anal.
(C18H10N2O3): C, H, N.
orange solid (63 mg, 97%). Mp: 158-160 °C. H NMR (DMSO,
250 MHz): δ 2.95 (t, 2H, J ) 8.0 Hz), 3.88 (t, 2H, J ) 8.0 Hz),
6.85 (s, 1H), 7.34 (t, 1H, J ) 8.0 Hz), 7.51 (s, 1H), 7.58 (t, 1H, J
) 8.0 Hz), 7.74 (d, 1H, J ) 8.0 Hz), 7.80-7.83 (m, 2H), 8.63 (d,
1H, J ) 5.0 Hz), 8.86 (d, 1H, J ) 8.0 Hz), 8.69 (d, 1H, J ) 8.0
Hz), 11.85 (s, 1H, exchangeable D2O), 12.92 (s, 1H, exchangeable
D2O). MS (IS): 381 (M + 1)+. Anal. (C23H16N4O2): C, H, N.
2-(2-(1H-Imidazol-4-yl)ethyl)-5-hydroxybenzo[a]pyrrolo[3,4-
c]carbazole-1,3(2H,8H)-dione (29). The same procedure as de-
scribed for compound 28 was used. After treatment, compound 29
1
was obtained as a red solid (91%). Mp: 183-185 °C. H NMR
(acetone-d6, 250 MHz): δ 2.45 (t, 2H, J ) 7.0 Hz), 3.40 (t, 2H, J
) 6.8 Hz), 6.39 (s, 1H), 6.58 (dd, 1H, J ) 9.0 Hz, J ) 2 Hz), 7.07
(s, 1H), 7.10 (d, 1H, J ) 9 Hz), 7.30-7.33 (m, 2H), 7.45 (s, 1H),
7.88 (d, 1H, J ) 2.0 Hz), 8.15 (dd, 1H, J ) 8.0 Hz, J ) 4.0 Hz),
8.83 (s, 1H, exchangeable D2O), 11.53 (s, 1H, exchangeable D2O),
12.21 (s, 1H, exchangeable D2O). MS (IS): 397 (M + 1)+. Anal.
(C23H16N4O3): C, H, N.
DNA Binding Measurements. Melting curves were measured
using an Uvikon 943 spectrophotometer coupled to a Neslab
RTE111 cryostat. Titrations of the drug with DNA, covering a large
range of drug/DNA-phosphate ratios (D/P), were performed by
adding aliquots of a concentrated drug solution to a constant DNA
solution (20 µM). Tm measurements were performed in pH 7.1 BPE
buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA). The
temperature inside the cuvette (10 mm path length) 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.
2-(2-Dimethylaminoethyl)benzo[a]pyrrolo[3,4-c]carbazole-
1,3(2H,8H)-dione (24). A solution of compound 19 (100 mg, 0.35
mmol) in 2-dimethylaminoethylamine (0.5 mL) was heated at 120
°C for 12 h. After cooling to room temperature, water (20 mL)
was added. The precipitate was filtered and washed with cold
MeOH (3 × 10 mL) to afford compound 24 as a yellow solid (100
1
mg, 80%). Mp: >250 °C. H NMR (DMSO, 250 MHz): δ 2.21
(s, 6H), 2.57 (t, 2H, J ) 5.6 Hz), 3.79 (t, 2H, J ) 5.6 Hz), 7.35 (t,
1H, J ) 7.0 Hz), 7.54 (t, 1H, J ) 8.0 Hz), 7.72-7.83 (m, 2H),
8.33 (d, 1H, J ) 2.0 Hz), 8.66 (d, 1H, J ) 5.0 Hz), 8.89 (d, 1H,
J ) 8.0 Hz), 8.96 (d, 1H, J ) 5.0 Hz), 12.95 (s, 1H, exchangeable
D2O). MS (IS): 358 (M + 1)+. Anal. (C22H19N3O2): C, H, N.
2-(2-Dimethylaminoethyl)benzo[a]pyrrolo[3,4-c]carbazole-
1,3(2H,8H)-dione (25). The same procedure as described for
compound 24 was used. After treatment, compound 25 was isolated
DNase I Footprinting. The complete procedure has been
recently detailed.35
Cell Culture and Survival Assay. Human CEM and CEMC2
leukemia cells were obtained from the American Tissue Culture
Collection. Cells were grown at 37 °C in a humidified atmosphere
containing 5% CO2 in RPMI 1640 medium, supplemented with
10% fetal bovine serum, 4.5 g/L glucose, 10 mM HEPES, 1 mM
sodium pyruvate, penicillin (100 IU/mL), and streptomycin (100
µg/mL). The cytotoxicity of the tested compounds was assessed
using a cell proliferation assay developed by Promega (CellTiter
96 aqueous one-solution cell proliferation assay). Briefly, 2 × 104
exponentially growing cells were seeded in 96-well microculture
plates with graded drug concentrations in a volume of 100 µL. After
72 h incubation at 37 °C, 20 µL of the tetrazolium dye was added
to each well, and the samples were incubated for a further 2 h at
37 °C. Plates were analyzed on a Labsystems Multiskan MS (type
352) reader at 492 nm.
Cell Cycle Analysis. For flow cytometric analysis of DNA
content, 0.7 × 106 cells in exponential growth were treated with
graded concentrations of the tested drug for 24 h and then washed
with 1 mL of PBS. After centrifugation, the cell pellet was
resuspended in 1 mL of cold ethanol for 24 h at -20 °C. The
ethanol was removed, and the pellet was washed with 1 mL of
PBS and then incubated for 30 min in a solution containing 50
µg/mL PI and 100 µg/mL RNase. Samples were analyzed on a
Becton Dickinson FACScan flow cytometer using CellQuest
software, which was also used to determine the percent of cells in
the different phases of the cell cycle. PI was excited at 488 nm
and the fluorescence analyzed at 620 nm on channel Fl-2.
Protein Kinase Assays. Kinase activities were assayed in buffer
A or C, at 30 °C, at a final ATP concentration of 15 µM. Blank
values were subtracted and activities calculated as picomoles of
phosphate incorporated for a 10-min incubation. The activities are
usually expressed in percent of the maximal activity, i.e., in the
absence of inhibitors. Controls were performed with appropriate
dilutions of dimethyl sulfoxide.
1
as an orange solid (71%). Mp: >250 °C. H NMR (DMSO, 250
MHz): δ 2.25 (s, 6H), 2.52 (m, 2H), 3.74 (m, 2H), 7.04 (d, 1H, J
) 8.0 Hz), 7.52 (d, 1H, J ) 8.0 Hz), 7.76 (m, 2H), 8.34 (s, 1H),
8.59 (d, 1H, J ) 5.0 Hz), 8.92 (d, 1H, J ) 5.0 Hz), 9.27 (s, 1H,
exchangeable D2O), 12.62 (s, 1H, exchangeable D2O). MS (IS):
374 (M + 1)+. Anal. (C22H19N3O3): C, H, N.
2-[2-Hydroxy-1-(hydroxymethyl)ethyl]benzo[a]pyrrolo[3,4-c]-
carbazole-1,3(2H,8H)-dione (26). To a solution of compound 19
(150 mg, 0.38 mmol) in DMF (0.5 mL) was added 2-aminopropan-
1,3-diol (377 mg, 4.14 mmol). The reaction mixture was refluxed
for 12 h. After cooling, water (5 mL) was added and the pH was
adjusted to 5 using hydrochloric acid (1 N). The aqueous layer
was extracted with EtOAc (3 × 10 mL), and the combined organic
layers were evaporated under reduced pressure. The crude material
was washed with cold MeOH (2 mL) to afford compound 26 as a
orange solid (103 mg, 75%). Mp: >232 °C dec. 1H NMR (DMSO,
250 MHz): δ 3.76 (m, 2H), 3.93 (m, 2H), 4.37 (m, 1H), 4.94 (s,
2H, exchangeable D2O), 7.39 (t, 1H, J ) 7.0 Hz), 7.54 (d, 1H, J )
7.0 Hz), 7.74 (d, 1H, J ) 8.2 Hz), 7.85-7.88 (m, 2H), 8.69 (d,
1H, J ) 4.7 Hz), 8.92 (d, 1H, J ) 7.7 Hz), 9.02 (d, 1H, J ) 4.7
Hz), 12.92 (s, 1H, exchangeable D2O). MS (IS): 361 (M + 1)+.
Anal. (C21H16N2O4): C, H, N.
2-]2-Hydroxy-1-(hydroxymethyl)ethyl]-5-hydroxybenzo[a]-
pyrrolo[3,4-c]carbazole-1,3(2H,8H)-dione (27). The same pro-
cedure as described for compound 26 was used. After treatment,
compound 27 was isolated as a red solid (68%). Mp: 248 °C dec.
1H NMR (DMSO, 250 MHz): δ 3.74 (m, 2H), 3.89 (m, 2H), 4.33
(m, 1H), 4.94 (s, 2H, exchangeable D2O), 7.08 (dd, 1H, J ) 8.9
Hz, J ) 3.5 Hz), 7.55 (d, 1H, J ) 8.2 Hz), 7.77-7.81 (m, 2H),
8.36 (d, 1H, J ) 3.5 Hz), 8.71-8.74 (m, 1H), 8.98-9.01 (m, 1H),
9.29 (s, 1H, exchangeable D2O), 12.94 (s, 1H, exchangeable D2O).
MS (IS): 377 (M + 1)+. Anal. (C21H16N2O5): C, H, N.
2-(2-(1H-Imidazol-4-yl)ethyl)benzo[a]pyrrolo[3,4-c]carbazole-
1,3(2H,8H)-dione (28). In a sealed tube, histamine (28 mg, 1.5
equiv) was added to a solution of compound 19 (50 mg, 0.17 mmol)
in DMF (2 mL). The solution was heated to 130 °C for 3 days.
GSK-3R/â was purified from porcine brain by affinity chroma-
tography on immobilized axin.36 It was assayed, following a 1/100
dilution in 1 mg of BSA/mL of 10 mM DTT, with 5 µL of 40 µM
GS-1 peptide as a substrate, in buffer A [10 mM MgCl2, 1 mM

798 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Routier et al.
syl]-5H,13H-benzo[b]-thienyl[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-
dione (BMS-251873) with Curative Antitumor Activity against
Prostate Carcinoma Xenograft Tumor Model. J. Med. Chem. 2004,
47, 1609-1612.
EGTA, 1 mM DTT, 25 mM Tris-HCl (pH 7.5), 50 µg heparin/
mL], in the presence of 15 µM [γ-33P]ATP (3000 Ci/mmol; 1 mCi/
mL) in a final volume of 30 µL. After 30 min incubation at 30 °C,
25-µL aliquots of supernatant were spotted onto 2.5 × 3 cm pieces
of Whatman P81 phosphocellulose paper, and 20 s later, the filters
were washed five times (for at least 5 min each time) in a solution
of 10 mL of phosphoric acid/L of water. The wet filters were
counted in the presence of 1 mL of ACS (Amersham) scintillation
fluid.
CDK1/cyclin B was extracted from M phase starfish oocytes
and purified by affinity chromatography on p9CKShs1-sepharose beads
as previously described.37 The kinase activity was assayed in buffer
C [60 mM â-glycerophosphate, 15 mM p-nitrophenyl phosphate,
25 mM Mops (pH 7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT,
1 mM sodium vanadate, 1 mM phenyl phosphate], with 1 mg of
histone H1/mL, in the presence of 15 µM [γ-33P]ATP (3000 Ci/
mmol; 1 mCi/mL) in a final volume of 30 µL. After 10 min
incubation at 30 °C, 25-µL aliquots of supernatant were spotted
onto P81 phosphocellulose papers and treated as described above.
(8) Saulnier, M. G.; Balasubramanian, B. N.; Long, B. H.; Frennesson,
D. B.; Ruediger, E.; Zimmermann, K.; Eummer, J. T.; St. Laurent,
D. R.; Stoffan, K. M.; Naidu, B. N.; Mahler, M.; Beaulieu, F.;
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C.; Solomon, C.; Wong, H.; Martel, A.; Wright, J. J.; Kramer, R.;
Langley, D. R.; Vyas, D. M. Discovery of a Fluoroindolo[2,3-a]-
carbazole Clinical Candidate with Broad Spectrum Antitumor Activity
in Preclinical Tumor Models Superior to the Marketed Oncology
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Bioorg. Med. Chem. 2003, 11, 679-687.
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A.; Bailly, C.; Renard, P.; Hickman, J.; Prudhomme, M. Syntheses
and Antiproliferative Activities of 7-Azarebeccamycin Analogues
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(13) Sanchez-Martinez, C.; Shih, C.; Faul, M. M.; Zhu, G.; Paal, M.;
Somoza, C.; Li, T.; Kumrich, C. A.; Winneroski, L. L.; Xun, Z.;
Brooks, H. B.; Patel, B. K. R.; Schultz, R. M.; DeHahn, T. B.;
Spencer, C. D.; Watkins, S. A.; Considine, E.; Dempsey, J. A.; Ogg,
C. A.; Campbell, R. M.; Anderson, B. A.; Wagner, J. Aryl[a]pyrrolo-
[3,4-c]carbazoles as Selective Cyclin D1-CDK4 Inhibitors. Bioorg.
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T. B.; Kirmani, K.; Spencer, C. D.; Watkins, S. A.; Considine, E.
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granulatimide, a G2 Checkpoint Inhibitor. Syntheses of Didemnimide
C, Isodidemnimide A, Neodidemnimide A, 17-Methylgranulatimide,
and Isogranulatimides A-C. J. Org. Chem. 2000, 65, 530-535.
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(19) Faul, M. M.; Engler, T. A.; Sullivan, K. A.; Grutsch, J. L.; Clayton,
M. T.; Martinelli, M. J.; Pawlak, J. M.; LeTourneau, M.; Coffey, D.
S.; Pedersen, S. W.; Kolis, S. P.; Furness, K.; Malhotra, S.; Al-awar,
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CDK5/p25 was reconstituted by mixing equal amounts of
recombinant mammalian CDK5 and p25 expressed in Escherichia
coli as GST (glutathione-S-transferase) fusion proteins and purified
by affinity chromatography on glutathione-agarose (p25 is a
truncated version of p35, the 35 kDa CDK5 activator). Its activity
was assayed in buffer C as described for CDK1/cyclin B.
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, (A.L.),
from the Canceropoˆle Grand Ouest, the Re´gion Centre, and the
Association pour la Recherche sur le Cancer, (S.R.) and grants
from the EEC (FP6-2002-Life Sciences & Health, PRO-
KINASE Research Project) (L.M.) and the Canceropole Grand-
Ouest (L.M.). N.D. was the recipient of a fellowship from the
Ligue Nationale Contre le Cancer and the Institut de Recherches
sur le Cancer de Lille.
Supporting Information Available: 13C NMR, IR, and MS
data and results of elemental analysis for compounds 3-6, 11-
21, 23-29. This material is available free of charge via the Internet
at http://pubs.acs.org.
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