Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of pyrrolo[3,4-a]carbazole-1,3-diones, pyrrolo[3,4-c]carbazole-1,3-diones, and 2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tetraone

Article abstract of DOI:10.1016/j.ejmech.2007.03.026

The synthesis of substituted pyrrolo[3,4-a]carbazole-1,3-diones, pyrrolo[3,4-c]carbazole-1,3-diones, and 2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tetraone is reported. Their inhibitory properties toward Checkpoint 1 kinase (Chk1) have been

Full text of DOI:10.1016/j.ejmech.2007.03.026

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 282e292
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties
of pyrrolo[3,4-a]carbazole-1,3-diones, pyrrolo[3,4-c]carbazole-1,3-diones,
and 2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tetraone
a
Elisabeth Conchon , Fabrice Anizon , Bettina Aboab , Roy M. Golsteyn , Stephane Leonce ,
a
a
b
b
´
´
Bruno Pfeiffer b, Michelle Prudhomme a,
*
a Laboratoire SEESIB, Universite´ Blaise Pascal, UMR 6504 du CNRS, 24, avenue des Landais, 63177 Aubie`re, France
b Institut de Recherches SERVIER, Division de Recherche Cance´rologie, 125 Chemin de ronde, 78290 Croissy sur Seine, France
Received 26 October 2006; received in revised form 13 March 2007; accepted 15 March 2007
Available online 7 April 2007
Abstract
The synthesis of substituted pyrrolo[3,4-a]carbazole-1,3-diones, pyrrolo[3,4-c]carbazole-1,3-diones, and 2-aminopyridazino[3,4-a]pyr-
rolo[3,4-c]carbazole-1,3,4,7-tetraone is reported. Their inhibitory properties toward Checkpoint 1 kinase (Chk1) have been evaluated and their
in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, human colon carcinoma HT29 and HCT116 have been
determined. From the biological results, it appears that, in contrast with the upper E heterocycle, the lower D heterocycle is not absolutely re-
quired for Chk1 inhibition. The ATP binding pocket of Chk1 seems to be adaptable to substitution of the nitrogen of the imide E heterocycle with
a hydroxymethyl group, allowing the fundamental hydrogen bond with the Glu85 residue of the enzyme.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrrolo[3,4-a]carbazole-1,3-diones; 2-Amino-pyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tetraone; Antitumor agents; Chk1 inhibitors
1. Introduction
inhibit Chk1 with IC50 values of 2 and 3 mM respectively [8].
Several isomers and derivatives, in which the imidazole het-
erocycle is conserved, have been synthesized [7e9]. Like
staurosporine and UCN-01, isogranulatimide (Fig. 1) is an
ATP-competitive Chk1 inhibitor [9,10]. In the crystal struc-
tures of Chk1 in complex with staurosporine or UCN-01 or
isogranulatimide, two hydrogen bonds are observed between
the E lactam or imide heterocycle and the ATP binding site
of the enzyme, the first one between NH and the carbonyl ox-
ygen of Glu85, and the second one between the oxygen of the
carbonyl group on the left and the amide nitrogen of Cys87.
In the course of structureeactivity relationship studies on
granulatimide, we synthesized granulatimide analogues in
which the imidazole moiety was replaced by an imide or pyr-
role heterocycle [11e17]. Other granulatimide analogues in
which the imide E ring was replaced by a lactam ring and
the imidazole D heterocycle was replaced by an imide ring
were also synthesized [18]. The IC50 values toward Chk1 of
Chk1 kinase represents a relevant target for the conception
of new antitumor agents [1,2]. Chk1 plays a major role in the
G2 checkpoint that is activated in response to DNA damage. In
more than 50% of tumors cells, the p53 protein is mutated
leading to an inactive G1 checkpoint. In the p53-mutated cells,
only the G2 checkpoint is activated in response to DNA dam-
age and provokes a cell cycle arrest allowing time for DNA re-
pair [3]. Therefore, the combination of a DNA damaging agent
and a Chk1 inhibitor should drive, selectively cancer cells, to-
ward a lethal mitosis. Inhibitors of Chk1 kinase have triggered
considerable interest over the past ten years [2,4]. Granulati-
mide and isogranulatimide are natural products isolated from
the ascidian Didemnum granulatum [5e7]. These compounds
* Corresponding author. Tel.: þ33 4 73 40 71 24; fax: þ33 4 73 40 77 17.
E-mail address: michelle.prudhomme@univ-bpclermont.fr (M. Prudhomme).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.026

E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
283
R2
N
H
N
H
N
H
N
O
O
Y
R
X
O
O
O
E
E
E
O
C
C
R1
NH
A
A
D
B
B
N
D
N
N
NR3
N
N
N
O
N
H
N
H
R4
bis-imide granulatimide analogues X = Y =O
lactam-imides X = H2, Y = O or X = O, Y = H2
CH3
O
H
isogranulatimide
granulatimide
OCH3
NHCH3
staurosporine R = H
UCN-01 R = OH
H
H
H
N
N
O
N
O
O
O
O
O
O
NH
NH
NH
N
H
N
H
N
H
O
O
O
lactam-imide C
NH
2
lactam-imide B
bis-imide A
R
N
O
O
N
O
R1
R2
O
O
O
NH
NH
NR3
O
OEt
N
H
N
H
N
O
H
O
pyrrolo[3,4-a]carbazole-
1,3-diones
pyrrolo[3,4-c]carbazole
-1,3-diones
pyrrolo[3,4-c]pyridazino[3,4-a]
carbazole-1,3,4,7-dione
Fig. 1. Structures of granulatimide, isogranulatimide, bis-imides and lactam-imide analogues, staurosporine and UCN-01, pyrrolo[3,4-a]carbazole-1,3-diones,
pyrrolo[3,4-c]carbazole-1,3-diones, and 2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tetraone.
some bis-imides and lactam-imides are in the nanomolar range
[15e18]. To investigate the role of the C, D, and E rings, in-
dolylpyrazolones and an indolylpyridazinedione have been
synthesized [19]. The indolylpyridazinedione was found to
be inactive toward Chk1 and the indolylpyrazolones were
poor Chk1 inhibitors, suggesting that a more rigid system is
required for Chk1 inhibition. Accordingly, we have investi-
gated the Chk1 inhibitory capacities of compounds bearing
the A, B and C rings but in which the D or E heterocycles
are missing. In this connection, substituted pyrrolo[3,4-a]car-
bazole-1,3-diones, in which the E upper imide heterocycle is
missing and pyrrolo[3,4-c]carbazole-1,3-diones, in which the
D imide heterocycle is missing, were synthesized. Moreover,
2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7-tet-
raone, in which the D heterocycle is a 6-membered pyridazine
dione ring was also synthesized (Fig. 1). The inhibitory activ-
ities of the new compounds toward Chk1 were determined and
their cytotoxicities toward three tumor cell lines: murine leu-
kemia L1210, human colon carcinoma HT29 and HCT116
were evaluated.
was prepared from indole and ethyl 1-chloromalonate in the
presence of AlClEt2 as previously described [19]. Reaction of
ethyl 3-oxo-3-(1H-indol-3-yl)propionate with NaH then with
N-BOM-dibromomaleimide gave compound 1. The nucleo-
philic substitution leading to the coupling of the maleimide
was followed by HBr elimination yielding pyrrolocarbazole
1. By reaction of 1 with PCl5 in DMF, chlorinated compound
2 was obtained in 94% yield, whereas compound 3 without the
ethoxycarbonyl group was prepared from compound 1 in 96%
yield via a Krapcho decarboxylation [20] by reaction with
LiCl in DMF. Hydrogenolysis of 3 followed by aminolysis
led to compound 4 in 33% yield. Hydrogenolysis of 1 using
Pd(OH)2 as a catalyst led to compound 5 in 80% yield. Ami-
nolysis of 5 afforded imide 6 in 30% yield.
The sequence of reactions for the synthesis of a pyr-
rolo[3,4-a]carbazole-1,3-dione with the ethoxycarbonyl group
in the 5-position is outlined in Scheme 2. Reaction of ethyl
1-(phenylsulfonyl)indole-3-glyoxylate [21] 7 with methyl
magnesium bromide provided intermediate 8 in 77% yield.
Compound 8 was dehydrated in the presence of p-toluenesul-
fonic acid to give compound 9 in 95% yield. Compound 9 was
isolated as two conformers. The conformers’ ratio (6:1 in
2. Results and discussion
1
CDCl3) was determined from the H NMR spectrum on the
2.1. Chemistry
signals of the ethylenic CH2 at 6.02 and 6.45 ppm for the
major conformer and at 6.14 and 6.26 ppm for the minor con-
former. In DMSO, the conformers’ ratio (4:1) was determined
on the signals of the ethylenic CH2 at 6.27 and 6.49 ppm for
the major conformer and at 6.42 and 6.56 ppm for the minor
To obtain pyrrolo[3,4-a]carbazole-1,3-diones diversely
substituted in 4 and 5 positions, compound 1 was firstly syn-
thesized (Scheme 1). Ethyl 3-oxo-3-(1H-indol-3-yl)propionate

284
E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
O
O
O
O
HO
O
O
NaH, THF
N
N
H
BOM
N
BOM
N
H
O
O
O
1
Br
Br
O
PCl5, DMF
Cl
O
H2
Pd(OH)2
O
O
HO
O
O
N
BOM
N
N
H
CH2OH
N
O
2
H
5
LiCl, DMF
HO
O
NH4OH
HO
O
HO
O
O
O
O
1) H2, Pd(OH)2
2) NH4OH
N
NH
BOM
NH
N
N
H
3
H
N
O
O
H
4
O
6
Scheme 1. Synthetic scheme for compounds 1e6.
conformer. A DielseAlder cycloaddition between compound
9 and maleimide carried out in xylene led to the indoline iso-
mer 10 of the cycloadduct in 44% yield. The structure of the
deprotection of the indole nitrogen was carried out using tetra-
butylammonium fluoride (15 equiv.) in refluxing THF. Con-
comitant with the removal of the phenylsulfonyl protective
group, oxidation into carbazole was observed leading directly
to compound 12 in 83% yield.
To get an insight into the influence of the D imide hetero-
cycle, compound 13 (Scheme 3), in which this heterocycle is
missing, was prepared from indolylmaleimide and ethyl acry-
late, followed by oxidation of the DielseAlder cycloadduct in-
termediate. The position of ethyloxycarbonyl substituent was
1
indoline isomer 10 was assigned from NMR He1H COSY
correlations (Fig. 2). The two protons of the methylene group
at 2.11 (m) and 3.57 ppm (dd) are coupled with the proton at
3.19 ppm (ddd) which is coupled with the proton at 4.07 ppm
(dd) which is coupled with the proton at 4.42 ppm (dd). Oxi-
dation using DDQ led to N-phenylsulfonyl carbazole 11 in
a poor yield (20%). To improve the yield of the oxidation,
OEt
O
HO
O
O
H
O
O
O
N
O
O
MeMgBr
O
O
O
NH
toluene
N
N
N
N
PTSA, toluene
O
10
SO Ph
SO Ph
9
SO Ph
2
SO Ph
2
2
2
8
7
DDQ, dioxane
TBAF, THF
OEt
OEt
O
O
O
O
NH
NH
N
H
N
O
O
12
11
SO Ph
2
Scheme 2. Synthetic scheme for compounds 8e12.

E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
OEt
285
obtained as the hydrochloride, probably due to the electron with-
drawing effect of the maleimide. It was not surprising to isolate
compound 19 from the reaction with hydrazine hydrate, because
in rebeccamycin and indolocarbazole series, the amino group on
the maleimide nitrogen could never be converted into a hydro-
chloride [22].
3.57
H
2.11
3.19
O
H
H
O
NH
H
4.07
H
In rebeccamycin and indolocarbazole series, we previously
observed the conversion of a maleic anhydride or a maleimide
into a N-amino-maleimide or pyridazine dione (Fig. 4)
[23,24]. It seems that the pyridazine dione 6-membered het-
erocycle is favoured when the overall structure is more rigid.
In compound 19, the heterocycles D and E could be inversed.
Indeed, in the presence of hydrazine hydrate, the maleic anhy-
dride of compound 17 could lead to a pyridazine dione
whereas the D maleimide cycle could lead to a N-amino mal-
eimide [23,25], but the isolation of intermediate 18 is in the
favour of the formation of pyridazine dione as the D heterocy-
N
4.42
SO Ph
O
2
10
Fig. 2. 1He1H COSY correlations in compound 10.
assigned from NMR experiments (COSY He1H, HSQC, and
1
HMBC). The singlet at 8.23 ppm was coupled with the tertiary
carbon at 120.2 ppm. The HMBC spectrum showed that the
proton at 8.23 ppm was coupled with two C]O at 164.4,
and 169.3 ppm (Fig. 3).
With the aim of converting the ester function into a hydra-
zide function, the imide nitrogen of compound 13 was pro-
tected with a benzyloxymethyl substituent. Reaction of 13
with benzyloxymethylchloride in the presence of diisopropyle-
thylamine as a base led to compounds 14 and 15 bearing a ben-
zyloxymethyl and a hydroxymethyl substituent on the imide
nitrogen, in 76% and 17% yields, respectively.
Finally, to obtain a compound with a pyridazinedione upper
E heterocycle, with which the fundamental hydrogen bonds with
the Glu85 and Cys87 in the ATP binding site of the enzyme should
be conserved but with a six-membered ring heterocycle,
a DielseAlder reaction between 4-(indol-3-yl)pyridazin-3,6-di-
one [19] and maleimide was tried. Since the cycloaddition did
not occur, 1,3,4,6-tetrahydrofuro[3,4-a]pyrrolo-[3,4-c]carba-
zole-1,3,4,6-tetraone 17 (Scheme 4) was prepared from 3-(3-in-
dolyl)furane-2,5-dione [11]. A DielseAlder cycloaddition with
maleimide afforded indole 16 in 90% yield. The indole structure
of compound 16 was assigned from its two exchangeable pro-
tons shifted at about 11.5 ppm (in indolines, the NH proton is
shifted at about 8 ppm). Oxidation of 16 with DDQ led to carba-
zole 17 in 86% yield. Reaction of compound 17 with hydrazine
hydrate followed by acidification led to compounds 18 and 19 in
3% and 37% yields, respectively. Compound 19 was not
1
cle. In the H NMR spectrum of compound 18, the signals of
the exchangeable protons are shifted at 12.29 ppm as a broad
signal (3H) which is in agreement with the presence of a pyr-
1
idazine ring. In the H NMR spectrum of compound 19, two
exchangeable protons appear as a singlet shifted at 5.30 ppm
which is in agreement with NeNH2. Three other exchangeable
protons, shifted at chemical shifts >12 ppm, correspond to the
indolic NH and NHeNH of the hydrazide. The isolation of
compound 18 shows that the maleimide ring reacts more
quickly than the maleic anhydride ring with hydrazine
hydrate. Compound 18 should be an intermediate which in
the presence of a second molecule of hydrazine hydrate would
lead to compound 19 bearing a N-amino-maleimide as the E
heterocycle.
2.2. Chk1 inhibitory activities
Unfortunately, compound 19 was highly insoluble, there-
fore its biological activities could not be evaluated. In the
other newly synthesized structures, the D or E ring is missing.
In compounds 5 and 12, the position of the ethyl carboxylate is
inverted on the C ring, which should have an influence on the
Chk1 inhibitory activities. The Chk1 inhibitory properties
have been evaluated and compared with those of granulati-
mide, isogranulatimide, bis-imide A, lactam-imides B and C
H
N
H
R
N
O
O
N
O
O
O
O
DIPEA, BOMCl
1)
O
EtO
2) DDQ
N
H
N
OEt
N
OEt
H
H
O
O
13
14 R = BOM
15 R = CH2OH
Scheme 3. Synthetic scheme for compounds 13e15.

286
E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
Glu85 is necessary to stabilize the molecules inside the ATP
binding site. Compound 13, possessing the E imide heterocy-
cle but lacking the D heterocycle inhibited Chk1 although the
IC50 value was superior than 5 mM. Compound 15, in which
the D heterocycle is missing but bearing a hydroxymethyl sub-
stituent on the imide nitrogen of the E heterocycle, was
a strong Chk1 inhibitor with an IC50 value of 0.21 mM. This
result was somewhat surprising because in the previous series,
a free nitrogen at the imide E heterocycle was required for
Chk1 inhibitory activity. Molecular modelling studies have
been carried out to investigate the stabilization of compounds
13 and 15 in the ATP binding pocket of Chk1.
H
N
O
O
102.2 ppm
H
8.23 ppm
N
OEt
H
O
13
Fig. 3. 13Ce1H correlations in compound 13.
shown in Fig. 1 (Table 1). The percentage of Chk1 inhibition
were determined at a compound concentration of 10 mM. For
the potent Chk1 inhibitors, the IC50 values were determined.
Compound 4 in which the E ring is missing and which does
not bear a carbonyl group attached to the C ring was a poor
Chk1 inhibitor. Compounds 5 and 6 in which the E ring is
missing and in which a carbonyl group is attached to the C
ring at the 4-position were inactive. Compound 12, in which
the E ring is missing and in which a carbonyl group is attached
to the C ring at the 5-position, was a modest Chk1 inhibitor
whereas compounds 5 and 6 do not inhibit this kinase. These
results are in agreement with what observed in the lactam-
imide series [18]. When the E ring was a lactam, the Chk1
inhibitory activities of lactam-imides in which the carbonyl
of the lactam is oriented on the left (toward the indole) were
always stronger than those of lactam-imides in which the car-
bonyl of the lactam is oriented on the right (toward the imide)
[18]. These results might be explained by the possibility of the
first ones to establish the fundamental hydrogen bonds with
Glu85 and Cys87 residues in the ATP binding pocket of Chk1.
Anhydride 16 was a poor Chk1 inhibitor and anhydride 17
did not inhibit Chk1 suggesting that the hydrogen bond with
2.3. Molecular modelling studies
Molecular modelling with compounds 13 and 15 were
carried out using as model the complex structure of Chk1/
staurosporine [10] downloaded from the Protein Data Bank.
Interestingly, in both cases, the two fundamental hydrogen
bonds were conserved between Glu85 and Cys87 residues of
the enzyme and the D heterocycle. In both cases, there is a hy-
drogen bond between NH of Cys87 and the carbonyl group on
the left of the D imide heterocycle. The hydrogen bond with
the carbonyl of Glu85 was formed either with the imide NH
of compound 13 (Fig. 5B) or with the hydroxy group of com-
pound 15 (Fig. 5A). In both models, a hydrogen bond between
the carbonyl of the ethyl carboxylate and the indole NH stabi-
lized the conformation of the ethyl carboxylate. The two
models were superimposed to visualize the modification of
the orientation in the ATP binding site of Chk1 (Fig. 5C). It
can be observed that when compound 15 lies in the ATP bind-
ing pocket, a slight displacement of Glu85 allows a hydrogen
bond with the hydroxy group.
O
O
O
O
O
O
O
O
O
O
O
maleimide
xylene
DDQ
dioxane
NH
NH
N
N
N
H
H
H
O
O
17
16
1) H2N-NH2, H2O
60 °C
2) HCl
NH
2
N
E
O
O
E
O
O
O
O
O
NH
NH
NH
D
D
+
NH
N
N
H
H
O
O
19
18
Scheme 4. Synthetic scheme for compounds 16e19.

E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
287
NH2
N
NH2
N
HN NH
O
O
O
O
O
O
N
N
N
N
N
N
H
H
H
OH
OH
O
O
Ref 24a
Ref 23
HO
OCH3
HO
Ref 24b
OH
OCH3
Fig. 4. N-Amino maleimides and pyridazine heterocycles in indolocarbazole series.
2.4. In vitro antiproliferative activities
3. Conclusion
The in vitro antiproliferative activities against three tumor
cell lines: murine leukemia L1210, human HT29 and
HCT116 colon carcinoma were evaluated. The IC50 values
in mM are reported in Table 1. Compounds 4 and 6 were
poorly cytotoxic toward the tumor cell lines tested. The IC50
values for compounds 12, 13, 15 and 16 were in the micromo-
lar range and these compounds were not selective toward the
tumor cell lines tested. Checkpoint inhibitors are not expected
to be cytotoxic by themselves. They should be cytotoxic in as-
sociation with DNA damaging agents. However, the cytotoxic-
ity observed with compounds 12, 13, 15 and 16 could be due
to the inhibition of other kinases than Chk1. To get a first in-
sight into the kinase selectivity, the inhibitory activity of com-
pound 15 toward the tyrosine kinase Src has been evaluated.
Compound 15 did not behave as a Src inhibitor (percentage
of Src inhibition at 10 mM: 11.5%).
In conclusion, this work reports the synthesis granulatimide
analogues in which the D and E rings are modified or absent.
The biological activities of compound 19 in which the heterocy-
cles D þ E are modified could not be evaluated due to its insol-
ubility. Compared with bis-imide A and lactam-imide B the
newly synthesized compounds 4, 5, 6 and 12 in which the E
ring is missing were poor Chk1 inhibitors suggesting that this
ring is required for Chk1 inhibition. Compound 15 in which
the D ring is missing and in which the nitrogen of the E hetero-
cycle bears a hydroxymethyl group was found to be a potent
Chk1 inhibitor. At first sight, the D ring is not absolutely re-
quired. Moreover, a substitution of the imide nitrogen with a hy-
droxylmethyl group is compatible with Chk1 inhibitory activity
and allows hydrogen bonding with Glu85 in the ATP binding
pocket of Chk1. This result is especially interesting and pro-
vides a new avenue to the design of Chk1 inhibitors.
Table 1
Percentages of Chk1 inhibition at a drug concentration of 10 mM. IC50 values (mM) toward Chk1
Compound
% Chk1 inhibition at 10 mM
IC50 Chk1 (mM)
L1210
HCT116
HT29
Granulatimide
Isogranulatimide
93.9
89.7
0.08
0.44
0.02
0.05
0.37
2.8
10
6.1
13
5.7
13.7
9.7
Bis-imide A
Lactam-imide B
94.4
85.4
32.7
45.8
47.0
54.4
10.6
57.5
4.9
nd
10.2
58.9
34.4
11.5
33.6
3.8
8.2
Lactam-imide C
4
71.7
11
>100
46.7
31.7
84.9
6.4
5
Inactive
Inactive
45.3
6
12
13
15
16
17
71.7
72.4
>5
5.8
5.5
2.3
2.9
4.0
3.9
0.21
10.6
Inactive
5.7
11.9
nd
10.7
6.2
33.0
In vitro antiproliferative activities against three tumor cell lines: murine leukemia L1210, human HT29 and HCT116 colon carcinoma (IC50 mM). nd: not
determined.

288
E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
A
B
GLU 85
GLU 85
CYS 87
CYS 87
C
GLU 85
CYS 87
Fig. 5. Molecular modelling of compounds 13 and 15 in the ATP binding site of Chk1. 5A: compound 15, 5B: compound 13. 5C: superimposition of the two
models: compounds 13 (in pink) and 15 (in blue). (For interpretation of the references to colours in figure legends, the reader is referred to the web version of
this article.)
4. Experimental section
N-benzyloxymethyl-dibromomaleimide (490 mg, 1.31 mmol)
in THF (10 mL). The mixture was stirred at 0 ꢁC for 2 h before
addition of MeOH to remove the excess of NaH. After extraction
with EtOAc, the organic phase was dried over MgSO4 and the
solvent was removed. The solid residue was purified by flash
chromatography (eluent: AcOEt/cyclohexane from 1:9 to 3:7)
to give 1 (144 mg, 0.32 mmol, 30% yield) as a yellow solid.
Mp 193 ꢁC. IR (KBr) nC]O 1697, 1725, 1760 cmꢀ1, nNH,OH
3100e3600 cmꢀ1. HRMS (ESIþ) [M þ Na]þ calcd for
C25H20N2O6Na 467.1219, found 467.1243. 1H NMR
(400 MHz, DMSO-d6): 1.40 (3H, t, J ¼ 7.0 Hz), 4.45 (2H, q,
J ¼ 7.0 Hz), 4.65 (2H, s), 5.10 (2H, s), 7.26e7.41 (6H, m),
7.55 (1H, t, J ¼ 7.0 Hz), 7.70 (1H, d, J ¼ 8.0 Hz), 8.35 (1H,
d, J ¼ 8.0 Hz), 11.55 (1H, s, OH), 12.36 (1H, s, NH). 13C
NMR (100 MHz, DMSO-d6): 13.7 (CH3), 61.5, 66.6, 70.3
(CH2), 105.5, 108.6, 116.7, 121.0, 127.9, 135.2, 137.7,
141.3, 155.9 (C quat), 112.1, 120.8, 122.8, 127.0, 127.5
(3C), 128.2 (2C) (C tert), 165.5, 166.6, 166.7 (C]O).
4.1. Chemistry
IR spectra were recorded on a PerkineElmer 881 spectrom-
eter (n in cmꢀ1). NMR spectra were performed on a Bruker
AVANCE 400 (chemical shifts d in parts per million, the fol-
lowing abbreviations are used: singlet (s), broad singlet (br s),
doublet (d), doubled doublet (dd), triplet (t), doubled triplet
(dt), multiplet (m), quadruplet (q), tertiary carbons (C tert),
quaternary carbons (C quat)). Low resolution mass spectra
(ESIþ) and HRMS were determined on a MS Hewlett Packard
engine, mass spectra (FABþ) were determined at CESAMO
(Talence, France) on a high resolution Fisons Autospec-Q
spectrometer. Chromatographic purifications were performed
by flash silicagel Geduran SI 60 (Merck) 0.040e0.063 mm
column chromatography.
4.1.1. 10H-2-Benzyloxymethyl-4-ethoxycarbonyl-5-hydroxy-
1,3-dihydropyrrolo[3,4-a]carbazole-1,3-dione 1
To a solution of ethyl 3-oxo-3-(1H-indol-3-yl)propionate
(250 mg, 1.08 mmol) in THF (15 mL) was added NaH (60%
in oil, 85 mg, 2.16 mmol) at 0 ꢁC. The mixture was stirred
at 0 ꢁC for 30 min, before dropwise addition to a solution of
4.1.2. 10H-2-Benzyloxymethyl-5-chloro-4-ethoxycarbonyl-
1,3-dihydropyrrolo[3,4-a]carbazole-1,3-dione 2
To a solution of 1 (40 mg, 0.090 mmol) in DMF (3 mL)
was added PCl5 (19 mg, 0.090 mmol). The mixture was re-
fluxed for 18 h. Water was added, the precipitate was filtered

E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
289
off and washed with water to give 2 (39 mg, 0.084 mmol, 94%
yield) as an orange solid.
filtration over Celite, the solvents were removed and the resi-
due was purified by flash chromatography (eluent: EtOAc/cy-
clohexane 5:5) to give 5 (39 mg, 0.11 mmol, 80% yield) as
a yellow solid.
Mp > 280 ꢁC. IR (KBr) nC]C 1612, 1642 cmꢀ1, nC]O
1710, 1774 cmꢀ1, nNH 3380 cmꢀ1. HRMS (ESIþ) [M þ Na]þ
calcd for C25H19N2O5ClNa 485.0880, found 485.0898. 1H
NMR (400 MHz, DMSO-d6): 1.43 (3H, t, J ¼ 7.0 Hz), 4.52
(2H, q, J ¼ 7.0 Hz), 4.67 (2H, s), 5.15 (2H, s), 7.26e7.38
(5H, m), 7.46 (1H, t, J ¼ 8.0 Hz), 7.72 (1H, t, J ¼ 7.0 Hz),
7.81 (1H, d, J ¼ 8.0 Hz), 8.61 (1H, d, J ¼ 8.0 Hz), 12.82
(1H, s, NH). Due to its insolubility, the 13C NMR spectrum
could not be recorded.
Mp > 280 ꢁC. IR (KBr) nC]C 1619, 1644 cmꢀ1, nC]O
1706, 1720, 1750 cmꢀ1, nNH,OH 3300e3650 cmꢀ1. HRMS
(ESIþ) [M þ H]þ calcd for C18H15N2O6 355.0930, found
355.0939. 1H NMR (400 MHz, DMSO-d6): 1.40 (3H, t,
J ¼ 7.0 Hz), 4.44 (2H, q, J ¼ 7.0 Hz), 5.00 (2H, s), 6.36 (1H,
br s, OH), 7.34 (1H, t, J ¼ 7.5 Hz), 7.54 (1H, t, J ¼ 8.0 Hz),
7.70 (1H, d, J ¼ 8.0 Hz), 8.35 (1H, d, J ¼ 8.0 Hz), 11.58
(1H, br s), 12.30 (1H, s). 13C NMR (100 MHz, DMSO-d6):
13.8 (CH3), 59.9, 61.4 (CH2eO), 105.4, 108.7, 116.6, 121.1,
128.2, 135.2, 141.3, 156.1 (C quat), 112.1, 120.7, 122.8,
126.9 (C tert), 165.6, 166.5, 166.6 (CO).
4.1.3. 10H-2-Benzyloxymethyl-5-hydroxy-1,3-
dihydropyrrolo[3,4-a]carbazole-1,3-dione 3
To a solution of 1 (20 mg, 0.045 mmol) in DMF (1.5 mL)
was added LiCl (10 mg, 0.22 mmol). The mixture was re-
fluxed for 18 h. Water was added. The precipitate was filtered
off and then washed with water to give 3 (16 mg, 0.043 mmol,
96% yield) as a yellow solid.
4.1.6. 2H,10H-4-Ethoxycarbonyl-5-hydroxy-1,
3-dihydropyrrolo[3,4-a]carbazole-1,3-dione 6
To a solution of 5 (28 mg, 0.079 mmol) in THF (8 mL) was
added 28% aqueous NH4OH (17 mL). The mixture was stirred
for 24 h at room temperature. After evaporation, water was
added to the residue. After filtration, the solid residue was
washed with water to give 6 (10 mg, 0.031 mmol, 39% yield)
as a yellow solid.
Mp > 280 ꢁC. IR (KBr) nC]C 1619, 1645 cmꢀ1, nC]O
1662, 1700, 1725 cmꢀ1, nNH,OH 3300e3600 cmꢀ1. HRMS
(ESIþ) [M þ Na]þ calcd for C17H12N2O5Na 347.0644, found
347.0661. 1H NMR (400 MHz, DMSO-d6): 1.38 (3H, t,
J ¼ 7.0 Hz), 4.42 (2H, q, J ¼ 7.0 Hz), 7.33 (1H, dt,
J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.53 (1H, dt, J1 ¼ 8.0 Hz,
J2 ¼ 1.0 Hz), 7.68 (1H, d, J ¼ 8.0 Hz), 8.32 (1H, d,
J ¼ 8.0 Hz), 11.05 (1H, s), 11.39 (1H, s), 12.24 (1H, s). 13C
NMR (100 MHz, DMSO-d6): 13.2 (CH3), 60.8 (CH2), 111.5,
120.0, 122.2, 126.2 (C tert arom), 106.2, 107.6, 115.8,
120.5, 128.7, 134.5, 140.7, 155.2 (C quat arom), 165.2,
167.8, 167.9 (CO).
Mp > 250 ꢁC (decomposition). IR (KBr) nC]C 1610 cmꢀ1
,
nC]O 1689, 1756 cmꢀ1, nNH,OH 3200e3650 cmꢀ1. HRMS
(ESIþ) [M þ Na]þ calcd for C22H16N2O4Na 395.1008, found
1
395.1024. H NMR (400 MHz, DMSO-d6): 4.65 (2H, s), 5.12
(2H, s), 7.09 (1H, s), 7.27e7.42 (6H, m), 7.51 (1H, t,
J ¼ 7.5 Hz), 7.66 (1H, d, J ¼ 8.0 Hz), 8.28 (1H, d,
J ¼ 8.0 Hz), 11.76 (1H, s), 12.16 (1H, s, NH). 13C NMR
(100 MHz, DMSO-d6): 66.4, 70.2 (CH2O), 100.1, 111.8,
120.4, 122.6, 126.4, 127.5 (3C), 128.2 (2C) (C tert), 104.0,
115.8, 121.0, 131.4, 135.6, 137.8, 141.0, 159.4 (C quat),
167.3, 168.5 (C]O).
4.1.4. 2H,10H-5-Hydroxy-1,3-dihydropyrrolo[3,4-a]-
carbazole-1,3-dione 4
A solution of 3 (40 mg, 0.107 mmol) in THF/MeOH
(4/1 mL) was hydrogenated (1 bar) in the presence of 20%
Pd(OH)2/C (206 mg) for 3 h at room temperature. After filtra-
tion over Celite, the solvents were removed. The solid residue
was dissolved into THF/NH4OH (11/23 mL) and the mixture
was stirred at room temperature for 24 h. After evaporation,
AcOEt was added to the residue and the mixture was filtered
off to give 4 (9 mg, 0.036 mmol, 33% yield) as a yellow solid.
Mp > 280 ꢁC. IR (KBr) nC]C 1617, 1636 cmꢀ1, nC]O
1704, 1746 cmꢀ1, nNH,OH 3416, 3479 cmꢀ1. HRMS (FABþ)
[M þ H]þ calcd for C14H9N2O3 253.0613, found 253.0620.
1H NMR (400 MHz, DMSO-d6): 6.99 (1H, s), 7.28 (1H, t,
J ¼ 8.0 Hz), 7.47 (1H, t, J ¼ 8.0 Hz), 7.62 (1H, d,
J ¼ 8.0 Hz), 8.26 (1H, d, J ¼ 8.0 Hz), 10.88 (1H, s, NH),
11.56 (1H, s, NH), 12.01 (1H, s, NH). 13C NMR (100 MHz,
DMSO-d6): 99.5, 111.7, 120.1, 122.5, 126.1 (C tert arom),
105.2, 115.4, 121.1, 132.8, 135.3, 140.9, 159.0 (C quat
arom), 169.2, 170.1 (CO).
4.1.7. Ethyl 2-hydroxy-2-(N-phenylsulfonyl-indol-3-yl)-
propanoate 8
A 3 M solution of MeMgBr in Et2O (0.68 mmol, 249 mL)
was added dropwise to a solution of 7 (110 mg, 0.31 mmol)
in THF (4 mL). The mixture was stirred at ꢀ78 ꢁC for 6.5 h.
The mixture was allowed to reach room temperature, and
then water was added. After extraction with EtOAc, the or-
ganic phase was dried over MgSO4. The solvent was removed
and the residue was purified by flash chromatography (eluent:
cyclohexane/EtOAc 7:3) to give 8 (90 mg, 0.24 mmol, 77%
yield) as a yellow oil.
IR (NaCl film) nC]O 1725 cmꢀ1, nOH 3501 cmꢀ1. HRMS
(ESIþ) [M þ Na]þ calcd for C19H19NO5NaS 396.0882, found
396.0882. 1H NMR (400 MHz, DMSO-d6): 1.07 (3H, t,
J ¼ 7.0 Hz), 1.78 (3H, s), 4.08 (1H, q, J ¼ 7.0 Hz), 6.14 (1H,
s, OH), 7.27 (1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.37 (1H, dt,
J1 ¼ 7.5 Hz, J2 ¼ 1.0 Hz), 7.63 (2H, t, J ¼ 8.0 Hz), 7.71e
7.74 (2H, m), 7.75 (1H, s), 7.96 (1H, d, J ¼ 8.0 Hz), 8.03
(1H, d, J ¼ 9.0 Hz), 8.03 (1H, d, J ¼ 7.0 Hz). 13C NMR
(100 MHz, DMSO-d6): 13.8, 26.1 (CH3), 60.8 (CH2O),
4.1.5. 10H-4-Ethyloxycarbonyl-2-hydroxymethyl-
5-hydroxy-1,3-dihydropyrrolo[3,4-a]carbazole-1,3-dione 5
A solution of compound 1 (60 mg, 0.14 mmol) in THF/
MeOH (4.0/1.0 mL) was hydrogenated (1 bar) in the presence
of Pd(OH)2 (66 mg) for 5 h at room temperature. After

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E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
113.1, 121.8, 123.2 (2C), 124.7, 126.7 (2C), 129.8 (2C), 134.6
(C tert), 72.1, 126.1, 128.2, 136.1 (C quat), 173.5 (C]O).
Mp 260e261 ꢁC. IR (KBr) nSO Ph 1296, 1380, nC]O 1704,
2
1724 cmꢀ1, nNH 3329e3614 cmꢀ1. Mass (APCIþ) [M þ Na]þ
471, [M þ K]þ 487. 1H NMR (400 MHz, DMSO-d6): 1.43
(3H, t, J ¼ 7.0 Hz), 4.51 (2H, q, J ¼ 7.0 Hz), 7.40 (2H, t,
J ¼ 8.0 Hz), 7.44 (1H, t, J ¼ 8.0 Hz), 7.52 (2H, d,
J ¼ 8.0 Hz), 7.59 (1H, t, J ¼ 7.0 Hz), 7.64 (1H, t,
J ¼ 7.0 Hz), 8.01 (1H, d, J ¼ 8.0 Hz), 8.23 (1H, s), 8.34 (1H,
d, J ¼ 8.0 Hz), 11.63 (1H, s, NH). 13C NMR (100 MHz,
DMSO-d6): 13.9 (CH3), 62.2 (CH2O), 117.4, 120.8, 124.9,
125.7, 126.8 (2C), 129.1 (2C), 130.0, 134.4 (C tert), 124.7,
124.8, 130.4, 132.1, 132.8, 134.7, 135.8, 141.9 (C quat),
165.3, 165.8, 167.8 (C]O).
4.1.8. Ethyl 2-(N-phenylsulfonyl-indol-3-yl)prop-2-enoate 9
A solution of 8 (100 mg, 0.27 mmol) in toluene (4 mL) was
refluxed for 1.5 h in a DeaneStark apparatus in the presence
of catalytic amounts of p-TsOH (5 mg, 0.03 mmol). After
cooling to room temperature, and extraction with EtOAc, the
organic phase was washed with water and then dried over
MgSO4. Compound 9, a brown oil, was isolated as a mixture
of two conformers (91 mg, 0.26 mmol, 95% yield).
IR (NaCl film) nC]O 1723 cmꢀ1. Mass (APCIþ) [M þ H]þ
1
356. H NMR (400 MHz, CDCl3) of the major conformer:
1.24 (3H, t, J1 ¼ 7.0 Hz, J2 ¼ 2.0 Hz), 4.21 (2H, q,
J1 ¼ 7.0 Hz, J2 ¼ 2.0 Hz), 6.02 (1H, s), 6.45 (1H, s), 7.15
(1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.23 (1H, dt, J1 ¼ 8.0 Hz,
J2 ¼ 1.0 Hz), 7.31 (2H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.39
(1H, dd, J1 ¼ 7.5 Hz, J2 ¼ 2.0 Hz), 7.50 (1H, d, J ¼ 8.0 Hz),
7.81 (2H, d, J1 ¼ 7.5 Hz, J2 ¼ 1.0 Hz), 7.83 (1H, s), 7.93
(1H, d, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3) of the major
conformer: 14.2 (CH3), 61.3 (CH2O), 113.7, 120.4, 123.6,
124.8, 126.9, 127.0 (2C), 129.3 (2C), 133.9 (C tert), 126.7
(]CH2), 78.6, 117.9, 120.2, 132.7, 138.1 (C quat). The signal
of the carbonyl was not observed.
4.1.11. 2H,10H-5-Ethoxycarbonyl-1,3-dihydropyrrolo-
[3,4-a]carbazole-1,3-dione 12
A 1 M solution of TBAF in THF (1.40 mL) was added
dropwise to a solution of 10 (40 mg, 0.09 mmol) in THF
(4 mL). The mixture was refluxed for 48 h. The solvent was
removed, water was added to the residue and the mixture
was filtered off. The solid residue was washed with water to
give 12 (23 mg, 0.075 mmol, 83% yield) as a yellow solid.
Mp > 220 ꢁC (degradation). IR (KBr) nC]O 1704,
1767 cmꢀ1, nNH 3355 cmꢀ1. HRMS (ESIþ) [M þ H]þ calcd
for C17H13N2O4 309.0875, found 309.0888. 1H NMR
(400 MHz, DMSO-d6): 1.44 (3H, t, J ¼ 7.0 Hz), 4.52 (2H, q,
J ¼ 7.0 Hz), 7.29 (1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.59
(1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.72 (1H, d, J ¼ 8.0 Hz),
7.97 (1H, s), 8.64 (1H, d, J ¼ 8.0 Hz), 11.42 (1H, s, NH),
12.48 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6): 14.0
(CH3), 61.7 (CH2), 112.4, 114.1, 120.3, 124.9, 128.4 (C tert
arom), 116.2, 119.7, 127.2, 128.7, 129.0, 133.9, 143.3 (C
quat arom), 166.3, 168.9, 169.3 (C]O).
4.1.9. 2H-5-Ethoxycarbonyl-10-phenylsulfonyl-1,2,3,
4-tetrahydropyrrolo[3,4-a]carbazole-1,3-dione 10
A mixture of 9 (50 mg, 0.14 mmol) and maleimide (16 mg,
0.16 mmol) in toluene (4 mL) was refluxed for 48 h. The sol-
vent was removed and the residue was purified by flash chro-
matography (eluent: cyclohexane/EtOAc from 7:3 to 5:5) to
give 10 (21 mg, 0.046 mmol, 33% yield) as a pale yellow oil.
IR (NaCl film) nC]O 1724, 1782 cmꢀ1, nNH 3441 cmꢀ1
.
HRMS (ESIþ) [M þ H]þ calcd for C23H21N2O6S 453.1120,
4.1.12. 2H,6H-5-Ethoxycarbonyl-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3-dione 13
1
found 453.1132. H NMR (400 MHz, CDCl3): 1.24 (3H, t,
J ¼ 7.0 Hz), 2.11 (1H, m), 3.19 (1H, ddd, J1 ¼ 9.0 Hz,
J2 ¼ 7.0 Hz, J2 ¼ 1.0 Hz), 3.57 (1H, dd, J1 ¼ 16.0 Hz,
J2 ¼ 1.0 Hz), 4.07 (1H, dd, J1 ¼ 9.0 Hz, J2 ¼ 7.0 Hz), 4.17
(2H, dq, J1 ¼ 7.0 Hz, J2 ¼ 3.0 Hz), 4.42 (1H, dd,
J1 ¼ 7.0 Hz, J2 ¼ 1.0 Hz), 7.00 (1H, t, J ¼ 7.0 Hz), 7.33 (1H,
t, J ¼ 8.0 Hz), 7.38 (2H, t, J ¼ 8.0 Hz), 7.50 (1H, t,
J ¼ 8.0 Hz), 7.71 (1H, d, J ¼ 8.0 Hz), 7.79 (2H, d,
J ¼ 8.0 Hz), 8.02 (1H, s, NH), 8.45 (1H, d, J ¼ 8.0 Hz). 13C
NMR (100 MHz, CDCl3): 14.1 (CH3), 27.4, 61.3 (CH2),
37.5, 43.5, 63.5 (CH), 60.4 (C quat), 115.0, 124.2, 127.2
(2C), 128.4 (2C), 129.4, 132.8, 133.9 (C tert arom), 118.4,
124.6, 136.7, 144.6, 147.2 (C quat arom), 165.2, 173.5,
177.5 (C]O).
A mixture of 3-(indol-3-yl)maleimide (250 mg, 1.18 mmol)
and ethyl acrylate (16.6 mmol, 1.75 mL) in toluene (25 mL)
was refluxed for 36 h. After evaporation, the solid residue
was purified by flash chromatography (eluent: EtOAc/cyclo-
hexane 6:4) to give the DielseAlder cycloadduct as a mixture
of isomers (orange solid, 258 mg, 0.83 mmol, 70% yield).
The mixture of isomers (140 mg, 0.45 mmol) and DDQ
(224 mg, 0.90 mmol) in dioxane (15 mL) was refluxed for
24 h. After filtration, the filtrate was evaporated and the resi-
due was purified by flash chromatography (eluent: cyclohex-
ane/EtOAc 5:5) to give 13 (88 mg, 0.28 mmol, 64% yield)
as a yellow solid.
Mp > 300 ꢁC. IR (KBr) nC]O 1686, 1716, 1765 cmꢀ1, nNeH
3236, 3398 cmꢀ1. HRMS (ESIþ) [M þ H]þ calcd for
1
C17H13N2O4 309.0875, found 309.0888. H NMR (400 MHz,
4.1.10. 2H-5-Ethoxycarbonyl-10-phenylsulfonyl-1,
3-dihydro-pyrrolo[3,4-a]carbazole-1,3-dione 11
DMSO): 1.48 (3H, t, J ¼ 7.0 Hz), 4.52 (2H, q, J ¼ 7.0 Hz),
7.39 (1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 0.5 Hz), 7.63 (1H, dt,
J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.85 (1H, d, J ¼ 8.0 Hz), 8.23 (1H,
s), 8.85 (1H, d, J ¼ 8.0 Hz), 11.45 (1H, s, NH), 12.05 (1H, s,
NH). 13C NMR (100 MHz, DMSO): 14.2 (CH3), 61.3 (CH2),
112.8, 120.2, 121.0, 124.6, 128.7 (C tert), 114.7, 119.4,
A mixture of 10 (90 mg, 0.20 mmol) and DDQ (100 mg,
0.40 mmol) in dioxane (6 mL) was refluxed for three days.
The solvent was removed and the residue was purified by flash
chromatography (eluent: cyclohexane/EtOAc 7:3) to give 11
(18 mg, 0.04 mmol, 20% yield) as a yellow solid.

E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
291
120.6, 122.7, 129.7, 142.1, 143.0 (C quat), 164.4, 169.3, 169.4
(C]O).
(1H, t, J ¼ 8.0 Hz), 7.42 (1H, d, J ¼ 8.0 Hz), 7.64 (1H, d,
J ¼ 8.0 Hz), 11.44 (1H, s, NH), 11.54 (1H, s, NH). 13C
NMR (100 MHz, DMSO-d6): 39.3, 40.2, 40.5 (2C) (CH),
100.7, 125.2, 128.2, 137.0 (C quat), 111.7, 119.2, 119.5,
122.2 (C tert), 170.4, 171.2, 175.6, 177.2 (C]O).
4.1.13. 6H-2-N-Benzyloxymethyl-5-ethoxycarbonyl-
pyrrolo[3,4-c]carbazole-1,3-dione 14 and 6H-2-
N-hydroxymethyl-5-ethoxycarbonyl-pyrrolo-
[3,4-c]carbazole-1,3-dione 15
4.1.15. 5H,7H-1,3,4,6-Tetrahydrofuro[3,4-c]pyrrolo-
[3,4-a]carbazol-1,3,4,6-tetraone 17
To a mixture of 13 (50 mg, 0.16 mmol) in acetone (1 mL)
was added diisopropylethylamine (70 mL, 0.40 mmol). The
mixture was stirred for 15 min at room temperature before
dropwise addition of benzyloxymethylchloride (45 mL,
0.32 mmol). The mixture was stirred at room temperature
for 1 h, and then water was added. After extraction with
EtOAc, the organic phase was dried over MgSO4, the solvent
was removed and the residue was purified by flash chromatog-
raphy (eluent: EtOAc/cyclohexane from 3:7 to 1:0). Two com-
pounds were isolated: 14 (52 mg, 0.12 mmol, 76% yield) and
15 (9 mg, 0.028 mmol, 17% yield) as yellow solids.
A solution of anhydride 16 (196 mg, 0.63 mmol) and DDQ
(575 mg, 1.39 mmol) in dioxane (10 mL) was refluxed for
three days. After removal of the solvent, water was added to
the residue. The mixture was filtered off and the solid residue
was washed with water and EtOAc to give 17 (166 mg,
0.54 mmol, 86% yield) as a yellow solid.
Mp > 300 ꢁC. IR (KBr) nC]O 1701, 1776, 1847 cmꢀ1, nNH
3247, 3368 cmꢀ1. HRMS (ESIþ) [M þ H]þ calcd for
1
C16H7N2O5 307.0355, found 307.0360. H NMR (400 MHz,
DMSO-d6): 7.51 (1H, t, J ¼ 7.0 Hz), 7.75 (1H, t, J ¼ 7.0 Hz),
7.82 (1H, d, J ¼ 7.5 Hz), 8.83 (1H, d, J ¼ 7.5 Hz), 11.78 (1H,
s, NH), 13.09 (1H, s, NH). Due to its insolubility, the 13C
NMR spectrum could not be recorded.
4.1.13.1. Compound 14. Mp 153 ꢁC. IR (KBr) nC]C
1607 cmꢀ1, nC]O 1698, 1708, 1761 cmꢀ1, nNH 3355 cmꢀ1
.
HRMS (ESIþ) [M þ Na]þ calcd for C25H20N2O5Na
1
451.1270, found 451.1284. H NMR (400 MHz, DMSO-d6):
4.1.16. 5H,6H,8H-1,3,4,7-Tetrahydrofurano-
1.50 (3H, t, J ¼ 7.0 Hz), 4.56 (2H, q, J ¼ 7.0 Hz), 4.69
(2H, s), 5.21 (2H, s), 7.24e7.40 (5H, m), 7.45 (1H, t,
J ¼ 7.5 Hz), 7.68 (1H, t, J ¼ 8.0 Hz), 7.91 (1H, d,
J ¼ 8.0 Hz), 8.36 (1H, d, J ¼ 2.0 Hz), 8.91 (1H, d,
J ¼ 8.0 Hz), 12.21 (1H, s). 13C NMR (100 MHz, DMSO-d6):
14.2 (CH3), 61.5, 66.9, 70.5 (CH2O), 113.0, 120.7, 121.2,
124.6, 127.5 (2C), 128.2 (2C), 129.1 (C tert), 115.2, 119.3,
121.0, 121.4, 128.4, 137.7, 142.3, 143.2 (C quat), 164.8,
167.8, 167.9 (C]O).
[3,4-c]pyridazino[3,4-a]carbazole 18 and 2H,5H,6H,
8H-2-amino-1,3,4,7-tetrahydropyridazino[3,4-a]pyrrolo-
[3,4-c]carbazole-1,3,4,7-tetraone 19
A solution of 17 (150 mg, 0.49 mmol) in hydrazine hydrate
(5 mL) was stirred at 60 ꢁC for 24 h. Water (150 mL) was
added, then 12 N HCl (20 mL) was added dropwise. After stir-
ring for 30 min at 0 ꢁC, the precipitate was filtered off, and
then washed with water and EtOAc to give 19 (60 mg,
0.18 mmol, 37% yield) as an orange solid. The filtrate was
evaporated to give 18 (5 mg, 0.016 mmol, 3% yield) as a yel-
low solid.
4.1.13.2. Compound 15. Mp > 230 ꢁC (decomposition). IR
(KBr) nC]C1608 cmꢀ1, nC]O 1687, 1708, 1763 cmꢀ1, nNH,OH
3362, 3439 cmꢀ1. HRMS (ESIþ) [M þ Na]þ calcd for
C18H14N2O5Na 361.0800, found 361.0805. 1H NMR
(400 MHz, DMSO-d6): 1.49 (3H, t, J ¼ 7.0 Hz), 4.53 (2H, q,
J ¼ 7.0 Hz), 5.03 (2H, d, J ¼ 7.0 Hz), 6.45 (1H, t,
J ¼ 7.0 Hz, OH), 7.40 (1H, t, J ¼ 7.5 Hz), 7.64 (1H, t,
J ¼ 7.5 Hz), 7.86 (1H, d, J ¼ 8.0 Hz), 8.23 (1H, s), 8.82 (1H,
d, J ¼ 8.0 Hz), 12.09 (1H, s, NH). 13C NMR (100 MHz,
DMSO-d6): 14.1 (CH3), 60.1, 61.4 (CH2O), 112.9, 120.4,
121.1, 124.4, 128.9 (C tert), 115.0, 119.2, 120.8, 121.3,
128.3, 124.2, 143.1 (C quat), 164.7, 167.3, 167.4 (C]O).
4.1.16.1. Compound 18. Mp > 280 ꢁC. IR (KBr) nC]O 1720,
1782 cmꢀ1, nNH 3411 cmꢀ1. Mass (ESIþ) [M þ H]þ 322. H
1
NMR (400 MHz, DMSO-d6): 7.35 (1H, dt, J1 ¼ 7.0 Hz,
J2 ¼ 1.0 Hz), 7.61 (1H, dt, J1 ¼ 8.0 Hz, J2 ¼ 1.0 Hz), 7.99
(1H, d, J ¼ 8.0 Hz), 8.16 (1H, d, J ¼ 8.0 Hz), 11.90e12.60
(3H, br s, NH).
4.1.16.2. Compound 19. Mp > 300 ꢁC. IR (KBr) nC]O
1736 cmꢀ1
, , . HRMS
nN]N 2173 cmꢀ1 nNH 3445 cmꢀ1
(FABþ) [M þ H]þ calcd for C16H10N5O4 336.0732, found
336.0730. 1H NMR (400 MHz, DMSO-d6): 5.30 (2H, s,
NH2), 7.52 (1H, t, J ¼ 7.5 Hz), 7.73 (1H, t, J ¼ 7.5 Hz), 8.07
(1H, d, J ¼ 8.0 Hz), 8.99 (1H, d, J ¼ 8.0 Hz), 12.36 (1H, s,
NH), 12.46 (1H, s, NH), 12.90 (1H, s, NH). 13C NMR
(100 MHz, DMSO-d6): 113.2, 115.1, 118.8, 119.1, 119.2,
128.4, 141.4, 142.4 (C quat arom), 113.7, 121.9, 124.6,
129.3 (C tert arom), 148.5, 158.1, 166.5, 171.5 (C]O).
4.1.14. 5H,7H-1,3,3a,3b,4,6,6a,11c-Octahydrofuro-
[3,4-c]pyrrolo[3,4-a]carbazol-1,3,4,6-tetraone 16
A solution of 3-(indol-3-yl)furane-2,5-dione (150 mg,
0.70 mmol) and maleimide (82 mg, 0.84 mmol) in xylene
(15 mL) was refluxed for five days. After filtration, the solid
residue was washed with water and dried to give 16
(195 mg, 0.63 mmol, 90% yield) as an orange solid.
Mp 220e222 ꢁC. IR (KBr) nC]O 1719, 1782 cmꢀ1, nNH
4.2. Kinase inhibition assays
3411 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 4.03 (1H, t,
.
J ¼ 7.5 Hz), 4.21 (1H, t, J ¼ 8.0 Hz), 4.34 (1H, d,
Chk1inhibition assays: Human Chk1 full-length enzyme
with an N-terminal GST sequence was either purchased
J ¼ 7.5 Hz), 4.78 (1H, d, J ¼ 8.0 Hz), 7.06 (1H, m), 7.15

292
E. Conchon et al. / European Journal of Medicinal Chemistry 43 (2008) 282e292
from Upstate Biochemicals (No. 14-346) or purified from ex-
tracts of Sf9 cells infected with a baculovirus encoding GST-
Chk1. Assays for compound testing were based upon the
method described by Davies et al. [26].
References
´ ´
[1] H.J. Anderson, R.J. Andersen, M. Roberge, in: L. Meijer, A. Jezequel,
M. Roberge (Eds.), Prog. Cell Cycle Res., 5, 2003, pp. 423e430.
[2] M. Prudhomme, Recent Pat. Anti-Cancer Drug Discovery 1 (2006) 55e68.
[3] J. Bartek, J. Lukas, Cancer Cell 3 (2003) 421e429.
[4] Z.F. Tao, Anticancer Agents Med. Chem. 6 (2006) 377e388.
[5] R.G.S. Berlinck, R. Britton, E. Piers, L.Y. Lim, M. Roberge, R. Moreira
da Rocha, R.J. Andersen, J. Org. Chem. 63 (1998) 9850e9856.
[6] M. Roberge, R.G.S. Berlinck, L. Xu, H.J. Anderson, L.Y. Lim,
D. Curman, C.M. Stringer, S.H. Friend, P. Davies, I. Vincent,
S.J. Haggarty, M.T. Kelly, R. Britton, E. Piers, R.J. Andersen, Cancer
Res. 58 (1998) 5701e5706.
Src inhibition assays: Inhibitors were diluted with a Tecan
Evo150 robot. The kinase assay was performed with 4 mL of
inhibitor (10% dimethylsulfoxide (DMSO)), 10 mL of kinase
assay buffer 4ꢂ concentrated (80 mM MgCl2, 200 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
0.4 mM ethylenediaminetetraacetic acid (EDTA), 2 mM DL-di-
thiothreitol (DTT)), 10 mL substrate peptide (KVEKIGE-
GYYGVVYK, 370 nM) and 6 mL Src kinase (stock GTP
purified diluted with 1ꢂ kinase assay buffer to 200 nM).
10 mL co-substrate (40 mM ATP with 0.2 mCi P33-g-ATP)
was added with a Precision 2000 (Biotek Robotic). The assay
was incubated 20 min at 30 ꢁC then stopped by adding 200 mL
0.85% orthophosphoric acid, then transferred to a phosphocel-
lulose filter microplate (Whatman - P81). The plate was
washed three times with 200 mL 0.85% orthophosphoric acid
and dried with 200 mL acetone. The remaining activity was
measured on a Topcount with 25 mL scintillation solution
(Packard UltimaGold).
[7] R.J. Andersen, M. Roberge, J. Sanghera, D. Leung, PCT. Int. Appl.
WO99/47522, 1999. Chem. Abstr. 131:243451.
[8] X. Jiang, B. Zhao, R. Britton, L.Y. Lim, D. Leong, J.S. Sanghera,
B.B.S. Zhou, E. Piers, R.J. Andersen, M. Roberge, Mol. Cancer Ther.
3 (2004) 1221e1227.
[9] E. Piers, R. Britton, R.J. Andersen, J. Org. Chem. 65 (2000) 530e535.
[10] B. Zhao, M.J. Bower, P.J. McDevitt, H. Zhao, S.T. Davis, K.O. Johanson,
S.M. Green, N.O. Concha, B.B.S. Zhou, J. Biol. Chem. 277 (2002)
46609e46615.
[11] B. Hugon, B. Pfeiffer, P. Renard, M. Prudhomme, Tetrahedron Lett. 44
(2003) 3935e3937.
[12] B. Hugon, B. Pfeiffer, P. Renard, M. Prudhomme, Tetrahedron Lett. 44
(2003) 3927e3930.
´
[13] H. Henon, S. Messaoudi, B. Hugon, F. Anizon, B. Pfeiffer,
M. Prudhomme, Tetrahedron 61 (2005) 5599e5614.
´
[14] H. Henon, F. Anizon, B. Pfeiffer, M. Prudhomme, Tetrahedron 62 (2006)
4.3. Molecular modelling
1116e1123.
´
All molecular mechanics calculations were performed by
the Macromodel [27] molecular modelling software. We
used as model the complex structure of Chk1/staurosporine
[10] downloaded from the Protein Data Bank (1NVR file).
Energy minimisation was done with AMBER force field
[28,29] using the Truncated Newton Conjugate Gradient
method (TNCG).
[15] H. Henon, F. Anizon, N. Kucharczyk, A. Loynel, P. Casara, B. Pfeiffer,
M. Prudhomme, Synthesis 4 (2006) 711e715.
´
´
[16] H. Henon, F. Anizon, R.M. Golsteyn, S. Leonce, R. Hofmann,
B. Pfeiffer, M. Prudhomme, Bioorg. Med. Chem. 14 (2006) 3825e
3834.
´
´
[17] H. Henon, F. Anizon, B. Aboab, N. Kucharczyk, S. Leonce,
R.M. Golsteyn, B. Pfeiffer, M. Prudhomme, Eur. J. Pharmacol. 554
(2007) 106e112.
´
[18] E. Conchon, F. Anizon, R. Golsteyn, S. Leonce, B. Pfeiffer,
M. Prudhomme, Tetrahedron 62 (2006) 11136e11144.
4.4. Growth inhibition assays
´
[19] E. Conchon, B. Aboab, R. Golsteyn, S. Leonce, B. Pfeiffer,
M. Prudhomme, Eur. J. Med. Chem. 41 (2006) 1470e1477.
[20] H.J. Knolker, R. Hitzemann, Tetrahedron Lett. 35 (1994) 2157e2160.
Tumor cells were provided by American Type Culture Col-
lection (Frederik, MD, USA). They were cultivated in RPMI
1640 medium (Life Science technologies, Cergy-Pontoise,
France) supplemented with 10% fetal calf serum, 2 mM L-glu-
tamine, 100 units/mL penicillin, 100 mg/mL streptomycin, and
10 mM HEPES buffer (pH ¼ 7.4). Cytotoxicity was measured
by the microculture tetrazolium assay as described [30]. Cells
were continuously exposed to graded concentrations of the
compounds for four doubling times, then 15 mL of 5 mg/mL
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
were 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 mL of DMSO. Results are expressed as
IC50, concentration which reduced by 50% the optical density
of treated cells with respect to untreated controls.
¨
[21] E. Wenkert, P.D.R. Moeller, S.R. Piettre, J. Am. Chem. Soc. 110 (1988)
7188e7194.
[22] C. Bailly, X. Qu, F. Anizon, M. Prudhomme, J.F. Riou, J.B. Chaires, Mol.
Pharmacol. 55 (1999) 377e385.
[23] E. Rodrigues Pereira, S. Fabre, M. Sancelme, M. Prudhomme, M. Rapp,
J. Antibiot. 48 (1995) 863e868.
[24] (a) E. Rodrigues Pereira, L. Belin, M. Sancelme, M. Prudhomme,
`
M. Ollier, M. Rapp, D. Severe, J.F. Riou, D. Fabbro, T. Meyer, J. Med.
Chem. 39 (1996) 4471e4477;
(b) F. Anizon, PhD thesis, Universite de Clermont-Ferrand, France, DU
´
No 1162, 1999.
[25] See Belstein 24, 373.
[26] S.P. Davies, H. Reddy, M. Caivano, P. Cohen, Biochem. J. 351 (2000)
95e105.
[27] Macromodel 8.0, Shroedinger Inc, 1500 SW First Ave. Suite 1180, Port-
land, OR 97201, USA.
[28] S.J. Weiner, P.A. Kollman, A.D. Case, U.C. Singh, C. Ghio, G. Alagona,
S. Profeta, P. Weiner, J. Am. Chem. Soc. 106 (1984) 765e784.
[29] W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz,
D.M. Ferguson, D.C. Spellmeyer, T. Fox, J.M. Caldwell, P.A. Kollman,
J. Am. Chem. Soc. 117 (1995) 5179e5197.
Acknowledgement
´
The authors are grateful to Bertrand Legeret, University
Blaise Pascal, Clermont-Ferrand, for recording the mass
spectra.
´
´
`
[30] S. Leonce, V. Perez, M.R. Casabianca-Pignede, M. Anstett, E. Bisagni,
G. Atassi, Invest. New Drugs 14 (1996) 169e180.