Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of dipyrrolo[3,4-a:3,4-c]carbazole-triones

Article abstract of DOI:10.1016/j.tet.2006.09.027

The syntheses of dipyrrolo[3,4-a:3,4-c]carbazole-1,4,6-triones and dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-triones are reported. These compounds can be considered as granulatimide analogues in which a maleimide replaces the imidazole moiety and a five-membe

Full text of DOI:10.1016/j.tet.2006.09.027

Tetrahedron 62 (2006) 11136–11144
Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory
properties of dipyrrolo[3,4-a:3,4-c]carbazole-triones
a
Elisabeth Conchon, Fabrice Anizon, Roy M. Golsteyn, Stephane Leonce,
a
b
b
ꢀ
ꢀ
Bruno Pfeifferb and Michelle Prudhommea,
*
a
´
ꢁ
Laboratoire SEESIB, Universite Blaise Pascal, Clermont Ferrand, UMR 6504 du CNRS, 63177 Aubiere Cedex, France
b
´
Institut de Recherches SERVIER, Division Recherche Cancerologie, 125 Chemin de ronde, 78290 Croissy sur Seine, France
Received 23 June 2006; revised 5 September 2006; accepted 12 September 2006
Available online 5 October 2006
Abstract—The syntheses of dipyrrolo[3,4-a:3,4-c]carbazole-1,4,6-triones and dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-triones are reported.
These compounds can be considered as granulatimide analogues in which a maleimide replaces the imidazole moiety and a five-membered
lactam ring replaces the upper maleimide. The Chk1 inhibitory properties of the more soluble compounds have been evaluated and their in
vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, and human colon carcinoma HT29 and HCT116. Due
to their insolubility, the biological activities of the other compounds in this series could not be evaluated. All the tested compounds proved to
be potent Chk1 inhibitors.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
to kill selectively cancer cells without causing damage to
healthy cells. Granulatimide, isogranulatimide, staurospor-
ine, UCN-01, and SB-218078 were found to be efficient
Chk1 inhibitors. All of them possess a carbazole moiety,
with an upper heterocycle containing an imide or a lactam
function. Moreover, in staurosporine, UCN-01, and SB-
218078, a carbohydrate-like heterocycle is linked to both in-
dole nitrogens.
The carbazole framework is found in many biologically
active compounds. Some of them, such as rebeccamycin,
are topoisomerase I inhibitors. Others, such as staurosporine
and UCN-01, are inhibitors of kinases.1–3 Granulatimide and
isogranulatimide, natural compounds isolated from an ascid-
ian, as well as staurosporine and UCN-01, indolocarbazole
compounds isolated from cultures of Streptomyces, or syn-
thetic compounds such as SB-218078 have triggered consid-
erable interest as cell cycle G2 checkpoint inhibitors
(Fig. 1).4–7 In the cell division cycle, the G2 checkpoint is
activated in response to DNA damage. Its role consists in
blocking the cell cycle to allow time for DNA repair. In
more than 60% of cancer cells, the G1 checkpoint is lacking,
due to mutations of the p53 gene. In the p53-mutated cells,
only the G2 ckeckpoint provides cancer cells with an
opportunity to repair their DNA after damage. Accordingly,
combining a DNA damaging agent with a G2 checkpoint in-
hibitor will force selectively cancer cells into a premature
and lethal mitosis due to an accumulation of DNA le-
sions.8–10 The Chk1 kinase plays a major role in the G2
checkpoint regulation.11,12 Therefore, Chk1 inhibitors are
relevant targets for the conception of agents that are able
The crystal structures of SB-218078, staurosporine, UCN-
01,13 and isogranulatimide14 in complex with Chk1 kinase,
have been determined. These compounds are ATP-competi-
tive Chk1 inhibitors. In the structures of the four complexes,
H
N
H
N
H
N
O
R
O
O
O
O
NH
N
N
N
N
H
N
N
H
N
O
H3C
H3CO
granulatimide
isogranulatimide
H
N
NHCH3
O
O
staurosporine R = H
UCN-01 R = OH
N
N
O
Keywords: Granulatimide; Dipyrrolo[3,4-a:3,4-c]carbazole-1,4,6-trione;
Dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-trione; Antitumor agents; Chk1 in-
hibitors.
* Corresponding author. Tel.: +33 4 73 40 7124; fax: +33 4 73 40 7717;
e-mail: michelle.prudhomme@univ-bpclermont.fr
SB-218078
Figure 1.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.09.027

E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
11137
H
H
N
H
N
H
H
N
N
O
O
N
O
O
O
O
R
10
10
R
R
O
O
reduction
O
R
NH
NH
N
NH
N
N
H
N
H
N
H
H
O
H
1a R = H
O
O
A
2
7 R = H
13 R = H
1b R = CH3
1c R = OBn
1d R = OH
9 R = CH3
10 R = OBn
12 R = OH
14 R = CH3
16 R = OBn
+
H
H
H
H
N 1
N1
2
N1
N1
5
2
3
O
R
HO
OH
O
O
O
5
5
2
5
2
Figure 2.
4
3
3
4
4 R
+
3
4
4'
7'
R
R
3'
+
+
5'
6'
2'
N
H
N
H
N1'
H
N
H
two hydrogen bonds between the inhibitors and the ATP
binding site of the enzyme are conserved: the first one be-
tween the NH of the upper heterocycle and the carbonyl oxy-
gen of Glu85, and the second one between the oxygen of the
carbonyl group of the lactam or imide function of the drug
and the amide nitrogen of Cys87. Granulatimide and iso-
granulatimide isomers, and structurally related compounds
bearing modified heterocycles, have been recently synthe-
sized.15–24
3
4
5
6
Scheme 2.
dipyrrolo[3,4-a:3,4-c]carbazole-triones. The reduction of
the 3-(indol-3-yl)-maleimides intermediates led to lactams
and hydroxylactams, from which the synthesis was com-
pleted.
In this paper, we describe the syntheses of dipyrrolo[3,4-a:
3,4-c]carbazole-1,4,6-triones and dipyrrolo[3,4-a:3,4-c]
carbazole-3,4,6-triones (Fig. 2). Compared with granulati-
mide and isogranulatimide, the imidazole has been replaced
by a maleimide and the upper heterocycle contains a lactam
function, like in staurosporine, instead of the imide function
present in granulatimide and isogranulatimide. Moreover,
several substituents have been introduced in the 10-position
of the indole moiety. Some compounds in this series proved
to be extremely insoluble, therefore, their biological activi-
ties could not be evaluated. For the most soluble compounds,
the Chk1 inhibitory activities and the cytotoxicities toward
three tumor cell lines: murine leukemia L1210, and human
colon carcinoma HT29 and HCT116 were evaluated.
Hydroxy and methyl substituents were introduced in the 10-
position because in the bis-imide series, these substitutions
led to the most efficient Chk1 inhibitors.
3-(Indol-3-yl)-maleimides 1a–d (R¼H, OBn, OH, and CH3)
were prepared as previously described22,24 in two steps from
the corresponding substituted indoles via a Michael addition
with maleimide followed by dehydrogenation of the Michael
adduct using DDQ. The corresponding lactams and hydroxy-
lactams were obtained by reduction of the 3-(indol-3-yl)-
maleimides (Scheme 2).
Depending on the reducing agent (LiAlH4, NaBH4 or
DIBAL-H) and the substituent on the indole moiety, impor-
tant variations were observed in the yields of compounds
2–6 (Table 1). Indeed, Mase et al.25 showed that the regiose-
lectivity of the reduction of monosubstituted maleimides us-
ing NaBH4 was due to the approach of the hydride anion
from the less hindered carbonyl group. Therefore, the hy-
dride anion attacks the more hindered carbonyl group.
When using DIBAL-H, the inverted regioselectivity is ex-
plained by the complexation effect of the carbonyl group
with an aluminum atom preferably coordinated to the less
hindered carbonyl group. Therefore, the hydride anion ap-
proaches from the more hindered carbonyl group and attacks
2. Results and discussion
2.1. Chemistry
In previous works,22–24 we described the four-step synthesis
of bis-imides granulatimide analogues (Scheme 1). A simi-
lar synthetic scheme was applied for the synthesis of
H
N
H
N
H
N
O
O
O
O
O
O
Table 1. Percentages of compounds 2, 3, 4, 5, and 6 obtained by reduction of
1a–d using LiAlH4, NaBH4 or DIBAL-H and ratio for the reductions on Cl/
Cm (Cl: less hindered carbon, Cm: more hindered carbon)
DDQ
R
R
R
AcOH
N
H
N
H
N
O
Starting Reducing
agent
Compounds (%)
Reduction ratio
on Cl/Cm (%)
H
Michael addition
product
Diels-Alder
O
NH
2
3
4
5
6
O
1a
LiAlH4
NaBH4
DIBAL-H
LiAlH4
DIBAL-H
LiAlH4
DIBAL-H
LiAlH4
DIBAL-H
9
0
0
1
0
0
0
0
0
6
11
11 23 19
29 71
16 84
77 23
22 78
78 22
H
N
H
0
60
0
0
O
N
O
O
O
17 33 15
O
1b
1c
1d
0
11 17 21
54 11 19
24 40
O
oxidation
0
R
NH
0
6
8
92
R
NH
N
H
N
H
42 13 13
0
0
0
0
0
81 19
27 73
65 35
O
12 33
28 15
Scheme 1.

11138
E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
H
N
H
N
H
N
the less hindered carbonyl group (Scheme 3). In compounds
3 and 4, the less hindered carbonyl group has been reduced,
whereas in compounds 5 and 6, the more hindered carbonyl
group has been reduced. With the less bulky LiAlH4, the
complexation of both carbonyl groups with an aluminum
atom may occur, but the hydride anion approaches very
probably more quickly from the less hindered side. The
structure of compounds 4a was assigned from NMR NOESY
correlations between the two methylene protons and the NH
of the lactam function and the vicinal ethylenic proton
(Scheme 4). In compound 6a, no NOESY correlations
were observed between the protons of the methylene group
and the ethylenic proton. The H4 of compound 4a is shifted
at 7.43 ppm, whereas the H3 of compound 6a is shifted at
6.26 ppm. Based on these NMR data, the structures of com-
pounds 3a and 5a were assigned (3a: H4 at 7.14 ppm, 5a: H3
at 6.17 ppm). By analogy with the unsubstituted compounds
3a–6a, the structures of the analogues substituted in 50-posi-
tion on the indole moiety were assigned from the chemical
shifts of the ethylenic protons.
O
O
O
O
TFA, dioxane
NH
O
O
R
R
R
O
NH
NH
N
H
N
H
N
H
O
O
7 R = H
4a-d
R = H, OBn
9 R = CH3
10 R = OBn
12 R = OH
H
N
H
N
O
O
O
O
R
NH
R
NH
N
H
N
H
O
O
8 R = CH3
11 R = OH
H
H
N
O
N
O
O
HO
1)
NH
O
R
R
O
NH
N
2) DDQ, dioxane
or TFA, dioxane
N
H
H
O
5a-c
13 R = H
14 R = CH3
16 R = OBn
H-
H
N
H
R
H
H
O
R
N
O
O
O
HO
NaBH4
NH
O
OH
O
O
O
N
HO
N
H
O
HO
H
major isomer
NH
N
H
H-
N
H
5d
17
H
R
H
R
DIBAL-H
HO
M
Scheme 5.
O
O
O
N
H
N
H
major isomer
the presence of TFA gave lactams 7, 9, 10, and 12
(Scheme 5).
Scheme 3.
Diels–Alder reactions performed from hydroxy-lactams
3a–d did not lead to the cycloadducts, whereas from hy-
droxy-lactams 5a–d, the cycloaddition occurred with the
loss of a water molecule. With compounds 5c and 5d, the in-
dole intermediates 15 and 17 could be isolated. Oxidation of
the intermediates in dioxane, either in the presence of TFA or
with DDQ, led to the required lactams 13, 14, and 16. In spite
of various modifications of the oxidation procedure of inter-
mediate 17 (in dioxane in the presence of TFA from
6 to 20 equiv from 60 ꢀC to 80 ꢀC or with DDQ 2 equiv in
dioxane at room temperature), the required aromatized
compound could not be obtained. Concomitant with the aro-
matization, oxidation of the lactam heterocycle to imide was
observed.
(8.44 ppm)
H
N
O
(4.06 ppm)
CH2
H
(7.43 ppm)
N
H
4a
Scheme 4.
The next step was a Diels–Alder cycloaddition with male-
imide. In previous studies, it was observed that the Diels–
Alder cycloaddition carried out between 3-indolyl-maleimide
and maleimide could lead, according to the treatment, filtra-
tion or chromatography on silicagel, to indoline or indole
isomers.22,26 When the Diels–Alder reaction was performed
from 4a and 4c, the mixture of isomers could not be sepa-
rated. The oxidation yielding to the final aromatic com-
pounds 7 and 10 was carried out on the isomeric mixture.
With lactams 4b and 4d, the indole intermediate 8 and the
indoline intermediate 11 were isolated. The position of the
Cycloadditions between maleimide and lactams 6 did not
lead to the required cycloadducts. With lactam 6 in which
R¼H, depending on the solvents used, either a double
Diels–Alder reaction or a Diels–Alder reaction followed
by a Michael addition with a second molecule of maleimide
occurred.
1
double bond was determined from H NMR data. Indeed,
an indole and an imide NH are usually shifted at about
11–12 ppm, whereas an indoline and a lactam NH are shifted
at about 7–9 ppm. In compounds 8, two exchangeable
protons are shifted at 11.12, and 11.52 ppm whereas in
compound 11, only one exchangeable proton was shifted
at 10.94 ppm. Oxidation of the intermediates in dioxane in
2.2. Chk1 inhibitory activities
The Chk1 inhibitory activities could only be determined
with compounds 7, 10, 12, 13, and intermediate 17 and
were compared with those of granulatimide, isogranula-
timide, and bis-imide analogue A (Fig. 2) (Table 2).27 Due

E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
11139
Table 2. Percentages of Chk1 inhibition at a drug concentration of 10 mM,
IC50 values (mM) toward Chk1; in vitro antiproliferative activities against
three tumor cell lines: murine leukemia L1210, and human HT29 and
HCT116 colon carcinoma (IC50 mM)
related to the Chk1 inhibitor granulatimide. Their upper het-
erocycle contains a lactam function like in staurosporine. All
the new compounds are potent Chk1 inhibitors suggesting
that the orientation of the carbonyl group of the upper hetero-
cycle, either toward the indole moiety or toward the male-
imide unit, could modify the positioning of the drug in the
active site of the kinase. Their weak cytotoxicity could be
due to a limited penetration into the cells or to a degradation
in the biological media. This hypothesis is currently under
investigation.
Compound
% of Chk1 IC50
inhibition
at 10 mM
L1210 HCT116 HT29
Chk1
(mM)
Granulatimide 93.9
Isogranulatimide 89.7
0.08
0.44
0.02
0.05
1
0.01
0.37
2.83
2.8
10
32.7
nd
25.9
54.5
47.0
>50
6.1
13
nd
nd
28.3
63.5
58.9
43.7
5.7
13.7
9.7
A
94.4
85.4
69.8
95.7
71.7
78.6
7
nd
10
12
13
17
36.4
41.8
>100
49.7
4. Experimental
4.1. Chemistry
to the insolubility of compounds 9, 14, and 16, their Chk1
inhibitory activities could not be evaluated. Compounds 7,
unsubstituted at the 10-position, and compound 12 bearing
a hydroxy group, are stronger Chk1 inhibitors than granula-
timide and isogranulatimide. Interestingly, when the carbonyl
of the lactam heterocycle is oriented toward the indole
moiety, the compounds seem to be more efficient Chk1
inhibitors than those in which the carbonyl of the lactam het-
erocycle is oriented toward the imide heterocycle (compare
7 and 13). These results are not completely surprising since,
in the crystal structures of staurosporine, UCN-01, and iso-
granulatimide in complex with Chk1, the carbonyl on the
left of the upper heterocycle accepts a hydrogen bond
from the amide nitrogen of Cys87, whereas no hydrogen
bond is formed with the carbonyl located on the right. How-
ever, the strong Chk1 inhibitory activity of compound 13
could be due to a different position of the molecule in the
ATP binding site allowing the formation of the two funda-
mental hydrogen bonds with Glu85 and Cys87. In this orien-
tation, the imide heterocycle would be positioned on the left
and the indole moiety would lie on the right. No significant
differences are observed between the Chk1 inhibitory activ-
ities of lactam 7 and imide A. Compared with unsubstituted
compound 7, compound 12 substituted with a hydroxy group
is a stronger Chk1 inhibitor.
IR spectra were recorded on a Perkin–Elmer 881 spectro-
meter (n in cmꢁ1). NMR spectra were performed on a Bruker
AVANCE 400 and AVANCE 500 (chemical shifts d in parts
per million, the following abbreviations are used: singlet (s),
broad singlet (br s), doublet (d), doubled doublet (dd), triplet
(t), doubled triplet (dt), multiplet (m), pseudo quadruplet
(pq), tertiary carbons (C tert), and quaternary carbons
1
(C quat). The signals were assigned from H–1H COSY,
HSQC, and HMBC NMR correlations. Low-resolution mass
spectra (ESI+ and APCI+) and HRMS were determined on a
MS Hewlett Packard instrument. Chromatographic purifica-
tions were performed by flash silicagel Geduran SI 60
(Merck) 0.040–0.063 mm column chromatography.
4.2. Typical procedure for the reduction using LiAlH4
To
a solution of 3-(indol-3-yl)-maleimide (200 mg,
0.94 mmol) in THF (40 mL) was added dropwise a 1 M so-
lution of LiAlH4 in Et2O (6 mL) at room temperature. The
mixture was stirred for 3 days. After cooling to 0 ꢀC, water
(14 mL) was added. The mixture was acidified to pH 2 with
2 M HCl (2 mL). After extraction with EtOAc, the organic
phase was washed with saturated aqueous NaHCO3. The
organic phase was dried over MgSO4 and the solvent was
removed. The residue was purified by flash chromatography
(eluent: from EtOAc/cyclohexane 1:1 to EtOAc/MeOH 9:1).
2.3. In vitro antiproliferative activities
4.3. Typical procedure for the reduction using NaBH4
The cytotoxicities of the soluble compounds were evaluated
toward three tumor cell lines: murine leukemia L1210, and
human colon carcinoma HT29 and HCT116 and compared
with those of granulatimide, isogranulatimide, and com-
pound A (Table 2). Compared with granulatimide and iso-
granulatimide, all the lactams tested are considerably less
active, their cytotoxicities are in the same range as those
of imide A. A checkpoint inhibitor is not expected to be cy-
totoxic by itself. However, the weak in vitro antiproliferative
activities of the new compounds described in this paper
suggests a possible instability of these compounds in the bio-
logical medium.
To a solution of 3-(indol-3-yl)-maleimide (2.4 mmol) in
THF (50 mL) was added sodium borohydride (90 mg,
2.4 mmol) in portions. The mixture was stirred at room tem-
perature for 24 h. After cooling to 0 ꢀC, 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 chromatography (eluent: from cyclohex-
ane/EtOAc 1:1 to EtOAc/MeOH 9:1).
4.4. Typical procedure for the reduction using DIBAL-H
To a solution of 3-(indol-3-yl)-maleimide (0.94 mmol) in
THF (70 mL) at ꢁ78 ꢀC was added dropwise a 1 M solution
of DIBAL-H in toluene (2.3 mL). The mixture was stirred
for 1.5 h at ꢁ78 ꢀC, then a 1 M solution of DIBAL-H in tol-
uene (2.3 mL) was added. The mixture was stirred at ꢁ78 ꢀC
for 6 h. Then the mixture was warmed to 0 ꢀC and saturated
aqueous NaHCO3 was added dropwise. After extraction
3. Conclusion
In conclusion, this work reports the synthesis of pyrrolo[3,4-
a:3,4-c]carbazole-1,4,6-tetraones and pyrrolo[3,4-a:3,4-c]-
carbazole-3,4,6-tetraones. These compounds are structurally

11140
E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
with EtOAc, the organic phase was dried over MgSO4, the
solvent was removed and the residue was purified by flash
chromatography (eluent: from EtOAc/cyclohexane 1:1 to
EtOAc/MeOH 9:1).
13C NMR (100 MHz, DMSO-d6): 80.5 (CHOH), 108.0,
125.3, 136.6, 154.4 (C quat), 112.1, 113.2, 120.1, 120.6,
122.1, 128.5 (C tert), 172.8 (C]O).
4.4.5. 4-(1H-Indol-3-yl)-1H-2,5-dihydro-pyrrol-2-one
(6a). Brown solid. Mp 255 ꢀC. IR (KBr) nC]C 1610 cmꢁ1
,
4.4.1. 3-(1H-Indol-3-yl)-1H-pyrrole (2a). Yellow solid. Mp
65 ꢀC. IR (KBr) nNH 2924 cmꢁ1. HRMS (ESI+) [M+H]+
calcd for C12H11N2 183.0922, found 183.0926.
nC]O 1649 cmꢁ1
,
nNH 3255 cmꢁ1
.
HRMS (ESIꢁ)
[MꢁH]ꢁ calcd for C12H9N2O 197.0715, found 197.0722.
1H NMR (400 MHz, DMSO-d6): 6.38 (1H, s), 6.79 (1H, s),
7.04 (1H, dt, J1¼7.0 Hz, J2¼1.0 Hz), 7.09–7.10 (2H, m),
7.37 (1H, d, J¼8.0 Hz), 7.40 (1H, d, J¼2.5 Hz), 7.77 (1H,
d, J¼8.0 Hz), 10.73 (1H, br s), 10.91 (1H, s).
13C NMR (100 MHz, DMSO-d6): 106.1, 111.4, 113.4,
117.8, 118.5, 119.5, 120.6, 120.8 (C tert), 111.6, 117.4,
125.4, 136.5 (C quat).
1H NMR (400 MHz, DMSO-d6): 4.42 (2H, s, CH2), 6.26
(1H, d, J¼1.0 Hz, H3), 7.15 (1H, dt, J1¼8.0 Hz,
0
0
J2¼1.0 Hz, H5 ), 7.19 (1H, dt, J1¼8.0 Hz, J2¼1.0 Hz, H6 ),
0
0
0
7.46 (1H, d, J¼8.0 Hz, H7 ), 7.84 (3H, m, H2 , H4 , NH1),
11.69 (1H, d, J¼1.0 Hz, NHindole).
13C NMR (100 MHz, DMSO-d6): 48.3 (CH2), 109.0, 124.8,
136.9, 152.7 (C quat), 112.1, 114.4, 119.9, 120.6, 122.1,
126.7 (C tert), 175.0 (C]O).
4.4.2. 3-(1H-Indol-3-yl)-1H-5-hydroxy-2,5-dihydro-pyr-
rol-2-one (3a). Orange-brown solid. Mp >300 ꢀC. IR
(KBr) nC]C 1632 cmꢁ1, nC]O 1705 cmꢁ1, nNH–OH 3200–
3500 cmꢁ1. Mass (ESI+) [M+Na]+ 237.
4.4.6. 3-(5-Methyl-1H-indol-3-yl)-1H-pyrrole (2b). Green
solid. Mp 110 ꢀC. IR (KBr) nNH 3423 cmꢁ1. HRMS (ESI+)
[M+H]+ calcd for C13H13N2 197.1079, found 197.1084.
1H NMR (400 MHz, DMSO-d6): 5.59 (1H, d, J¼9.0 Hz,
H5), 5.99 (1H, d, J¼9.0 Hz, OH), 7.14 (1H, s, H4), 7.18
(1H, dt, J1¼8.0 Hz, J2¼1.0 Hz), 7.21 (1H, dt, J1¼8.0 Hz,
J2¼1.0 Hz), 7.51 (1H, d, J¼8.0 Hz), 7.94 (1H, d,
J¼8.0 Hz), 8.28 (1H, d, J¼2.5 Hz), 8.65 (1H, s), 11.46
(1H, s).
1H NMR (400 MHz, DMSO-d6): 2.44 (3H, s), 6.39 (1H, pq,
J¼2.0 Hz), 6.82 (1H, pq, J¼2.0 Hz), 6.94 (1H, dd,
J1¼8.0 Hz, J2¼1.5 Hz), 7.11 (1H, pq, J¼2.0 Hz), 7.28
(1H, d, J¼8.0 Hz), 7.37 (1H, d, J¼2.5 Hz), 10.80 (1H, s,
NH), 10.74 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 21.3 (CH3), 106.1, 111.0,
113.3, 117.7, 119.1, 120.7, 122.3 (C tert), 111.1, 117.6,
125.7, 126.9, 134.9 (C quat).
13C NMR (100 MHz, DMSO-d6): 78.2 (CHOH), 106.0,
125.5, 130.6, 136.1 (C quat), 112.0, 119.7, 119.9, 121.7,
126.5, 133.8 (C tert), 171.7 (C]O).
4.4.7. 3-(5-Methyl-1H-indol-3-yl)-1H-5-hydroxy-2,5-di-
hydro-pyrrol-2-one (3b). Off-white solid. Mp >300 ꢀC.
IR (KBr) nC]C 1633 cmꢁ1, nC]O 1697 cmꢁ1, nNH,OH
3200–3500 cmꢁ1. HRMS (ESI+) [M+HꢁH2O]+ calcd for
C13H11N2O 211.0871, found 211.0871.
4.4.3. 3-(1H-Indol-3-yl)-1H-2,5-dihydro-pyrrol-2-one
(4a). Orange-brown solid. Mp 205–207 ꢀC. IR (KBr) nC]C
1628 cmꢁ1, nC]O 1678 cmꢁ1, nNH 3284 cmꢁ1. HRMS
(ESI+) [M+H]+calcd for C12H11N2O 199.0871, found
199.0880.
1H NMR (400 MHz, DMSO-d6): 2.48 (3H, s), 5.59 (1H, d,
J¼7.5 Hz), 5.99 (1H, d, J¼8.5 Hz, OH), 7.04 (1H, t,
J¼8.0 Hz), 7.14 (1H, s, H4), 7.38 (1H, d, J¼8.0 Hz), 7.73
(1H, s), 8.25 (1H, d, J¼3.0 Hz), 8.65 (1H, s, NH), 11.32
(1H, s, NH).
1H NMR (400 MHz, DMSO-d6): 4.06 (2H, s, CH2), 7.15
0
(1H, dt, J1¼8.0 Hz, J2¼1.0 Hz, H5 ), 7.24 (1H, dt,
0
J1¼8.0 Hz, J2¼1.0 Hz, H6 ), 7.43 (1H, d, J¼1.5 Hz, H4),
0
0
7.49 (1H, d, J¼8.0 Hz, H7 ), 7.93 (1H, d, J¼8.0 Hz, H4 ),
0
8.28 (1H, d, J¼2.5 Hz, H2 ), 8.44 (1H, s, NH1), 11.36 (1H,
s, NHindole).
13C NMR (100 MHz, DMSO-d6): 21.3 (CH3), 78.3 (CHOH),
105.6, 125.8, 128.7, 130.8, 134.5 (C quat), 111.6, 119.5,
123.3, 126.5, 133.5 (C tert), 171.8 (C]O).
13C NMR (100 MHz, DMSO-d6): 46.0 (CH2), 106.8, 125.5
(C quat), 130.6, 136.0 (C quat), 111.8, 119.3, 119.6, 121.5,
125.5 (C tert arom), 131.8 (CH lactam), 173.1 (C]O).
4.4.8. 3-(5-Methyl-1H-indol-3-yl)-1H-2,5-dihydro-pyr-
rol-2-one (4b). Ochre solid. Mp >300 ꢀC. IR (KBr) nC]C
4.4.4. 4-(1H-Indol-3-yl)-1H-5-hydroxy-2,5-dihydro-pyr-
rol-2-one (5a). Orange-brown solid. Mp 207 ꢀC. IR (KBr)
1606 cmꢁ1 nC]O 1649 cmꢁ1 nNH 3100–3300 cmꢁ1
, , .
HRMS (ESI+) [M+Na]+ calcd for C13H12N2ONa
235.0847, found 235.0853.
nC]C 1608 cmꢁ1, nC]O 1662 cmꢁ1, nNH 3271 cmꢁ1
.
HRMS (ESIꢁ) [MꢁH]ꢁ calcd for C12H9N2O2 213.0664,
found 213.0667.
1H NMR (400 MHz, DMSO-d6): 2.47 (3H, s), 4.05 (2H, s),
7.02 (1H, dd, J1¼8.0 Hz, J2¼1.0 Hz), 7.36 (1H, d,
J¼8.0 Hz), 7.41 (1H, d, J¼1.5 Hz, H4), 7.71 (1H, br s),
8.22 (1H, d, J¼2.5 Hz), 8.40 (1H, s, NH), 11.21 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 21.3 (CH3), 45.9 (CH2N),
106.3, 125.7, 128.3, 130.8, 134.4 (C quat), 111.5, 119.3,
123.1, 125.5, 131.5 (C tert), 173.2 (C]O).
1H NMR (400 MHz, DMSO-d6): 5.80 (1H, dd, J1¼9.5 Hz,
J2¼1.0 Hz, H5), 6.17 (1H, d, J¼1.0 Hz, H3), 6.22 (1H, d,
J¼9.5 Hz, OH), 7.17 (1H, dt, J1¼8.0 Hz, J2¼1.0 Hz), 7.19
(1H, dt, J1¼8.0 Hz, J2¼1.0 Hz), 7.47 (1H, d, J¼8.0 Hz),
7.84 (1H, d, J¼3.0 Hz), 7.85 (1H, d, J¼8.0 Hz), 8.16 (1H,
s), 11.69 (1H, s).

E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
11141
4.4.9. 4-(5-Methyl-1H-indol-3-yl)-1H-5-hydroxy-2,5-di-
hydro-pyrrol-2-one (5b). Off-white solid. Mp 104 ꢀC. IR
(KBr) nC]C 1615 cmꢁ1, nC]O 1682 cmꢁ1, nNH 3274,
.
C13H12N2O2Na 251.0796, found 251.0805.
4.4.13. 4-(5-Benzyloxy-1H-indol-3-yl)-1H-5-hydroxy-2,5-
dihydro-pyrrol-2-one (5c). Off-white solid. Mp 215–
216 ꢀC. IR (KBr) nC]C 1613 cmꢁ1, nC]O 1681 cmꢁ1
,
3398 cmꢁ1
HRMS (ESI+) [M+Na]+ calcd for
nNH,OH 3040–3663 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for
C19H17N2O3 321.1239, found 321.1242.
1H NMR (400 MHz, DMSO-d6): 2.47 (3H, s), 5.83 (1H, dd,
J1¼9.5 Hz, J2¼1.5 Hz), 6.21 (1H, d, J¼1.0 Hz, H3), 6.23
(1H, d, J¼9.5 Hz), 7.05 (1H, dd, J1¼8.0 Hz, J2¼1.0 Hz),
7.39 (1H, d, J¼8.0 Hz), 7.70 (1H, br s), 7.82 (1H, d,
J¼2.5 Hz), 8.16 (1H, s, NH), 11.59 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 21.2 (CH3), 80.5 (CHOH),
107.6, 125.6, 129.4, 134.9, 154.5 (C quat), 111.7, 113.0,
119.8, 123.6, 128.4 (C tert), 172.9 (C]O).
1H NMR (400 MHz, DMSO-d6): 5.23 (2H, s), 5.81 (1H, d,
J¼9.5 Hz), 6.22 (1H, s), 6.23 (1H, d, J¼9.0 Hz), 6.95 (1H,
d, J¼9.0 Hz), 7.34–7.46 (5H, m), 7.54 (2H, d, J¼7.5 Hz),
7.83 (1H, s), 8.17 (1H, s), 11.61 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 69.7 (CH2OBn), 80.5
(CHOH), 103.5, 111.4, 111.9, 112.8, 127.6 (3C), 128.3
(2C), 129.0 (C tert), 107.9, 125.8, 131.7, 137.7, 153.6,
154.3 (C quat), 173.0 (C]O).
4.4.10. 4-(5-Methyl-1H-indol-3-yl)-1H-2,5-dihydro-pyr-
rol-2-one (6b). Off-white solid. Mp 140 ꢀC. IR (KBr)
4.4.14. 4-(5-Benzyloxy-1H-indol-3-yl)-1H-2,5-dihydro-
pyrrol-2-one (6c). Off-white solid. Mp 235–237 ꢀC. IR
(KBr) nC]C 1606 cmꢁ1, nC]O 1651 cmꢁ1, nNH 3200–
3430 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C19H17N2O2
305.1290, found 305.1297.
nC]C 1605 cmꢁ1, nC]O 1649 cmꢁ1, nNH 3035–3430 cmꢁ1
.
HRMS (ESI+) [M+H]+ calcd for C13H13N2O 213.1028,
found 213.1027.
1H NMR (400 MHz, DMSO-d6): 2.46 (3H, s), 4.43 (2H, s),
6.28 (1H, d, J¼1.0 Hz, H3), 7.05 (1H, dd, J1¼8.0 Hz,
J2¼1.0 Hz), 7.37 (1H, d, J¼8.0 Hz), 7.69 (1H, s), 7.81
(1H, s), 7.82 (1H, s), 11.58 (1H, s, NH).
1H NMR (400 MHz, DMSO-d6): 4.41 (2H, s), 5.23 (2H, s),
6.29 (1H, s, H3), 6.94 (1H, d, J¼9.0 Hz), 7.34–7.46 (5H, m),
7.54 (2H, d, J¼7.5 Hz), 7.82 (2H, s), 11.59 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 48.3, 69.7 (CH2), 103.2,
103.5, 112.9, 104.0, 114.0, 127.2, 127.6 (2C), 128.3 (2C),
129.0 (C tert), 108.9, 125.3, 132.0, 137.7, 152.7, 153.6 (C
quat), 175.2 (CO).
13C NMR (100 MHz, DMSO-d6): 21.2 (CH3), 48.3 (CH2N),
108.6, 125.1, 129.4, 135.1, 152.8 (C quat), 111.8, 114.1,
119.6, 123.6, 126.7 (C tert), 175.1 (C]O).
4.4.11. 3-(5-Benzyloxy-1H-indol-3-yl)-1H-5-hydroxy-2,5-
dihydro-pyrrol-2-one (3c). Off-white solid. Mp >200 ꢀC
4.4.15. 4-(5-Hydroxy-1H-indol-3-yl)-1H-5-hydroxy-2,5-
dihydro-pyrrol-2-one (5d). Brown solid. Mp 102–104 ꢀC.
IR (KBr) nC]C 1614 cmꢁ1, nC]O 1678 cmꢁ1, nNH,OH
2981–3599 cmꢁ1. HRMS (ESI+) [M+Na]+ calcd for
C12H10N2O3Na 253.0589, found 253.0595.
(decomposition). IR (KBr) nC]C 1631 cmꢁ1
, nC]O
1706 cmꢁ1
, . HRMS (ESI+)
nNH,OH 3100–3500 cmꢁ1
[M+H]+ calcd for C19H17N2O3 321.1239, found 321.1256.
1H NMR (400 MHz, DMSO-d6): 5.22 (2H, s, CH2), 5.58
(1H, dt, J1¼9.0 Hz, J2¼2.0 Hz, H5), 5.98 (1H, d,
J¼9.0 Hz, OH), 6.94 (1H, dd, J1¼9.0 Hz, J2¼2.0 Hz),
7.12 (1H, t, J¼1.5 Hz), 7.36–7.46 (5H, m), 7.55 (2H, d,
J¼7.0 Hz), 8.25 (1H, d, J¼3.0 Hz), 8.63 (1H, s, NH),
11.33 (1H, d, J¼2.5 Hz, NH).
13C NMR (100 MHz, DMSO-d6): 69.9 (CH2O), 78.3
(CHOH), 103.4, 112.3, 112.6, 127.1, 127.6 (3C), 128.3
(2C), 133.5 (C tert), 105.9, 125.9, 130.6, 131.3, 137.8,
153.2 (C quat), 171.8 (C]O).
1H NMR (400 MHz, DMSO-d6): 5.81 (1H, d, J¼10.0 Hz),
5.93 (1H, s), 6.23 (1H, d, J¼10.0 Hz, OH), 6.75 (1H, d,
J¼9.0 Hz), 7.14 (1H, s), 7.31 (1H, dd, J1¼9.0 Hz,
J2¼1.0 Hz), 7.78 (1H, s), 8.16 (1H, s), 8.97 (1H, s), 11.49
(1H, s, NHindole).
13C NMR (100 MHz, DMSO-d6): 80.5 (CHOH), 104.2,
111.9, 112.0, 112.6, 128.8 (C tert), 107.2, 126.3, 130.9,
152.1, 154.9 (C quat), 172.8 (C]O).
4.4.16. 3-(5-Hydroxy-1H-indol-3-yl)-1H-2,5-dihydro-
pyrrol-2-one (4d). Brown solid. Mp 180 ꢀC. IR (KBr)
4.4.12. 3-(5-Benzyloxy-1H-indol-3-yl)-1H-2,5-dihydro-
pyrrol-2-one (4c). Brown solid. Mp 185 ꢀC. IR (KBr)
nC]C 1618 cmꢁ1, nC]O 1672 cmꢁ1, nNH 3100–3550 cmꢁ1
.
Mass (APCI+) [M+H]+ 215.
nC]C 1620 cmꢁ1, nC]O 1680 cmꢁ1, nNH 3100–3500 cmꢁ1
.
Mass (ESI+) [M+K]+ 343, [M+Na]+ 327.
1H NMR (400 MHz, DMSO-d6): 4.04 (2H, s), 6.72 (1H, d,
J¼9.0 Hz), 7.20 (1H, s), 7.21 (1H, s), 7.27 (1H, d,
J¼9.0 Hz), 8.18 (1H, s), 8.39 (1H, s), 8.84 (1H, br s),
11.08 (1H, s, NH).
1H NMR (400 MHz, DMSO-d6): 4.05 (2H, s), 5.20 (2H, s),
6.92 (1H, dd, J1¼9.0 Hz, J2¼2.0 Hz), 7.33–7.47 (6H, m),
7.54 (2H, d, J¼7.5 Hz), 8.23 (1H, d, J¼2.5 Hz), 8.41 (1H,
s, NH), 11.22 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 45.9 (CH2), 103.9, 111.6,
112.1, 125.8, 130.5 (C tert), 106.0, 126.3, 130.4, 131.0,
151.4 (C quat), 173.2 (C]O).
13C NMR (100 MHz, DMSO-d6): 46.0 (CH2N), 69.9
(CH2OBn), 103.4, 112.0, 112.4, 126.1, 127.6, 127.7 (2C),
128.3 (3C), 131.3 (C tert), 106.7, 125.8, 130.6, 131.2,
137.8, 153.0 (C quat), 173.1 (C]O).
4.4.17. 2H,5H,7H-1,3,4,6-Tetrahydro-dipyrrolo[3,4-
a:3,4-c]carbazole-1,4,6-trione (7). A mixture of maleimide

11142
E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
(244 mg, 2.51 mmol) and compound 4a (100 mg,
0.505 mmol) in xylene (14 mL) was refluxed for 3 days.
After cooling, the yellow precipitate was filtered off
washed with xylene and dried. The yellow solid (144 mg,
0.491 mmol, 97% yield) corresponds to an isomeric mixture
of Diels–Alder adducts. The mixture of isomers (84 mg,
0.286 mmol) in dioxane (22 mL) was refluxed for 36 h in
the presence of trifluoroacetic acid (293 mL). After evapora-
tion, EtOAc was added to the residue. The mixture was
filtered off. The solid was successively washed with satu-
rated aqueous NaHCO3, brine, and EtOAc to give compound
7 as a yellow-brown solid (22 mg, 0.075 mmol, 27% yield).
1H NMR (400 MHz, DMSO-d6): 2.50 (3H, s), 4.75 (2H, s),
7.43 (1H, d, J¼8.0 Hz), 7.61 (1H, d, J¼8.0 Hz), 9.05 (2H, s),
11.36 (1H, s), 12.23 (1H, s).
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
4.4.20. 10-Benzyloxy-2H,5H,7H-1,3,4,6-tetrahydro-di-
pyrrolo[3,4-a:3,4-c]carbazole-1,4,6-trione (10). A mix-
ture of maleimide (122 mg, 1.25 mmol) and compound 4c
(74 mg, 0.25 mmol) in xylene (6 mL) was refluxed for
18 h. After cooling, the mixture was filtered off and the solid
residue was washed with CH2Cl2 then was dried to give an
orange solid as a mixture of isomers (97 mg, 0.25 mmol,
quantitative yield).
Mp >310 ꢀC. IR (KBr) nC]O 1690, 1700, 1720 cmꢁ1, nNH
3387 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C16H10N3O3
292.0722, found 292.0745.
The mixture of isomers (90 mg, 0.23 mmol), dioxane (6 mL)
and trifluoroacetic acid (2.2 mmol, 172 mL) was stirred at
80 ꢀCfor48 h. After evaporation, water was addedtotheresi-
due, the mixture was filtered off and the solid residue was
washed repeatedly with water and small amounts of EtOAc
to give 10 (61 mg, 0.15 mmol, 65% yield) as a dark red solid.
1H NMR (400 MHz, DMSO-d6): 4.77 (2H, s), 7.34 (1H, t,
J¼7.5 Hz), 7.61 (1H, t, J¼8.0 Hz), 7.73 (1H, d,
J¼7.5 Hz), 9.08 (1H, s), 9.25 (1H, d, J¼8.0 Hz), 11.40
(1H, s), 12.37 (1H, s).
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
Mp >300 ꢀC. IR (KBr) nC]C 1617 cmꢁ1, nC]O 1722,
1774 cmꢁ1, nNH 3100–3550 cmꢁ1. HRMS (ESI+) [M+Na]+
calcd for C23H15N3O4Na 420.0960, found 420.0974.
4.4.18. 10-Methyl-2H,5H,7H-1,3,3a,3b,4,6,6a,11c-octa-
hydro-dipyrrolo[3,4-a:3,4-c]carbazole-1,4,6-trione (8).
A mixture of maleimide (179 mg, 1.84 mmol) and com-
pound 4b (78 mg, 0.37 mmol) in xylene (10 mL) was re-
fluxed for 3 days. After cooling, the mixture was filtered
off and the solid residue was washed with xylene then dried
to give 8 as an off-white solid (116 mg, 0.37 mmol, 100%
yield).
1H NMR (400 MHz, DMSO-d6): 4.76 (2H, s), 5.21 (2H, s),
7.33–7.65 (7H, m), 8.95 (1H, d, J¼2.0 Hz), 9.07 (1H, s),
11.37 (1H, s), 12.21 (1H, s).
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
4.4.21. 10-Hydroxy-2H,5H,7H-1,3,3a,3b,4,6,6a,11c-octa-
hydro-dipyrrolo[3,4-a:3,4-c]carbazole-1,4,6-trione (11).
A mixture of maleimide (40 mg, 0.48 mmol) and 4d
(92 mg, 0.43 mmol) in xylene (5 mL) was refluxed for
24 h. After cooling, the mixture was filtered off and the solid
residue was washed with CH2Cl2 then was dried to give 11
(40 mg, 0.13 mmol, 30% yield) as a dark orange solid.
Mp >295 ꢀC. IR (KBr) nC]O 1715, 1777 cmꢁ1, nNH 3061–
3684 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C17H16N3O3
310.1192, found 310.1178.
1H NMR (400 MHz, DMSO-d6): 2.37 (3H, s), 2.82 (1H, t,
J¼9.5 Hz), 3.11 (1H, t, J¼9.0 Hz), 3.25 (1H, m), 3.51
(1H, t, J¼7.0 Hz), 3.56 (1H, d, J¼7.0 Hz), 4.38 (1H, d,
J¼8.5 Hz), 6.92 (1H, d, J¼8.0 Hz), 7.28 (1H, d,
J¼8.5 Hz), 7.58 (1H, s, NH), 7.75 (1H, s), 11.12 (1H, s,
NH), 11.52 (1H, s, NH).
Mp >300 ꢀC. IR (KBr) nC]O 1691, 1763 cmꢁ1, nNH,OH
3300–3550 cmꢁ1. Mass (ESI+) [M+H]+ 312.
1H NMR (400 MHz, DMSO-d6): 2.94 (1H, m), 3.27 (1H, t,
J¼8.0 Hz), 3.47 (1H, t, J¼9.0 Hz), 3.61 (1H, t, J¼10.0 Hz),
4.23–4.29 (2H, m), 6.28 (1H, s), 6.59 (1H, d, J¼8.0 Hz),
6.63 (1H, d, J¼8.0 Hz), 7.83 (1H, s), 8.06 (1H, s), 8.67
(1H, s), 10.94 (1H, s).
13C NMR (100 MHz, DMSO-d6): 21.3 (CH3), 41.3 (CH2N),
35.6, 38.6, 40.3, 40.7 (CH), 110.7, 120.2, 122.8 (C tert),
102.2, 125.9, 126.3, 126.7, 135.3 (C quat), 175.1, 177.0,
178.5 (C]O).
4.4.19. 10-Methyl-2H,5H,7H-1,3,4,6-tetrahydro-dipyr-
rolo[3,4-a:3,4-c]carbazole-1,4,6-trione (9). A solution of
8 (104 mg, 0.34 mmol) in dioxane (11 mL) and trifluoroace-
tic acid (266 mL) was stirred at 80 ꢀC for 48 h. After evapo-
ration, water was added to the residue, the mixture was
filtered off, the solid was washed with water and with small
amounts of EtOAc to give 9 as an orange solid (80 mg,
0.263 mmol, 78% yield).
13C NMR (100 MHz, DMSO-d6): 41.1 (CH2N), 36.2, 42.7,
60.5, 70.0 (CH), 110.4, 113.2, 119.8 (C tert arom), 119.5,
122.8, 143.1, 148.8, 149.8 (C quat arom), 168.4, 175.7,
178.4 (C]O).
4.4.22. 10-Hydroxy-2H,5H,7H-1,3,4,6-tetrahydro-dipyr-
rolo[3,4-a:3,4-c]carbazole-1,4,6-trione (12). A solution of
11 (80 mg, 0.257 mmol) in dioxane (8 mL) was stirred at
80 ꢀC for 48 h in the presence of trifluoroacetic acid
(2.56 mmol, 200 mL). After evaporation, water was added
to the residue, the mixture was filtered off and the solid resi-
due was washed successively with water and small amounts
Mp >300 ꢀC. IR (KBr) nC]O 1716, 1758 cmꢁ1, nNH 3208–
3664 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C17H12N3O3
306.0879, found 306.0894.

E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
11143
of EtOAc to give 12 (52 mg, 0.169 mmol, 66% yield) as
a red solid.
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
Mp >300 ꢀC. IR (KBr) nC]O 1711 cmꢁ1, nNH,OH 3200–
3672 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C16H10N3O4
308.0671, found 308.0672.
4.4.25. 10-Benzyloxy-2H,5H,7H-1,3,3a,3b,4,6,6a,11c-oc-
tahydro-dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-trione
(15). A mixture of 5c (90 mg, 0.28 mmol) and maleimide
(34 mg, 0.35 mmol) in xylene (7 mL) was refluxed for
48 h. After filtration, the solid was washed with water and
dried to give 15 (102 mg, 0.26 mmol, 91% yield) as
a brown-orange solid.
1H NMR (400 MHz, DMSO-d6): 4.75 (2H, s), 7.11 (1H, dd,
J1¼9.0 Hz, J2¼2.0 Hz), 7.54 (1H, d, J¼9.0 Hz), 8.66 (1H, d,
J¼2.0 Hz), 9.02 (1H, s), 9.27 (1H, s), 11.33 (1H, s), 12.06
(1H, s).
Mp >300 ꢀC. IR (KBr) nC]O 1719, 1778 cmꢁ1, nNH 3100–
3550 cmꢁ1. HRMS (ESI+) [M+H]+ calcd for C23H18N3O4
400.1297, found 400.1285.
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
4.4.23. 2H,5H,7H-1,3,4,6-Tetrahydro-dipyrrolo[3,4-a:
3,4-c]carbazole-3,4,6-trione (13).
1H NMR (400 MHz, DMSO-d6): 4.33–4.39 (2H, m), 4.59
(1H, d, J¼11.0 Hz), 4.64 (1H, d, J¼17.5 Hz), 5.16 (2H, s),
6.89 (1H, dd, J1¼9.0 Hz, J2¼2.0 Hz), 7.13 (1H, d,
J¼2.0 Hz), 7.36–7.52 (6H, m), 7.97 (1H, s, NH), 11.72
(1H, s, NH), 11.73 (1H, s, NH).
A mixture of 5a
(241 mg, 1.126 mmol) and maleimide (131 mg, 1.35 mmol)
in xylene (20 mL) was refluxed for 4 days. After filtration,
the solid residue was washed with water to give an isomeric
mixture of the Diels–Alder adducts as a brown solid
(254 mg, 0.86 mmol, 77% yield).
13C NMR (100 MHz, DMSO-d6): 45.2, 69.6 (CH2), 42.3,
46.7 (CH), 102.4, 111.2, 113.1, 127.6 (2C), 127.7, 128.3
(2C) (C tert), 105.2, 115.7, 123.1, 132.1, 133.0, 137.4,
148.3, 153.4 (C quat), 172.3, 177.3, 177.5 (CO).
A solution of the isomeric mixture (60 mg, 0.205 mmol) in
dioxane (7 mL) was refluxed for 21 days in the presence
of trifluoroacetic acid (900 mL). After evaporation, EtOAc
was added to the residue. The mixture was filtered off, and
the solid was successively washed with saturated aqueous
NaHCO3, water, and EtOAc to give 13 as a brown solid
(32 mg, 0.110 mmol, 54% yield).
4.4.26. 10-Benzyloxy-2H,5H,7H-1,3,4,6-tetrahydro-
dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-trione (16). A mix-
ture of compound 15 (50 mg, 0.12 mmol) and DDQ
(28 mg, 0.12 mmol) in dioxane (5 mL) was stirred over-
night. After removal of the solvent, the residue was washed
successively with water, EtOAc, and small amounts of THF.
Compound 16 (19 mg, 0.05 mmol, 40% yield) was isolated
as a brown solid.
Mp >300 ꢀC. IR (KBr) nC]O1710, 1720, 1780 cmꢁ1, nN–H
3000–3500 cmꢁ1. Mass (ESI+) [M+H]+ 292. HRMS
(ESI+) [M+Na]+ calcd for C16H9N3O3Na 314.0542, found
314.0556.
Mp >300 ꢀC. IR (KBr) nC]C 1617 cmꢁ1, nC]O 1722,
1774 cmꢁ1, nNH 3100–3500 cmꢁ1. Mass (ESI+) [M+H]+
398. HRMS (ESI+) [M+Na]+ calcd for C23H15N3O4Na
420.0960, found 420.0955.
1H NMR (400 MHz, DMSO): 5.03 (2H, s), 7.41 (1H, t,
J¼7.5 Hz), 7.63 (1H, t, J¼7.5 Hz), 7.78 (1H, d, J¼8.0 Hz),
8.12 (1H, d, J¼8.0 Hz), 8.76 (1H, s, NH), 11.21 (1H, s, NH),
12.46 (1H, s, NH). Due to its insolubility, the 13C NMR spec-
trum could not be recorded.
1H NMR (400 MHz, DMSO-d6): 5.03 (2H, s), 5.29 (2H, s),
7.36–7.70 (8H, m), 8.79 (1H, s, NH), 11.19 (1H, s, NH),
12.33 (1H, s, NH).
4.4.24. 10-Methyl-2H,5H,7H-1,3,4,6-tetrahydro-dipyr-
rolo[3,4-a:3,4-c]carbazole-3,4,6-trione (14). A mixture of
compound 5b (120 mg, 0.53 mmol) and maleimide
(61 mg, 0.62 mmol) in xylene (5 mL) was refluxed for 3
days. After filtration, the solid was washed with water and
dried to give an isomeric mixture of the Diels–Alder adducts
(161 mg, 0.52 mmol, 100% yield) as a brown solid.
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
4.4.27. 10-Hydroxy-2H,5H,7H-1,3,3b,4,6,6a-hexahydro-
dipyrrolo[3,4-a:3,4-c]carbazole-3,4,6-trione (17). A mix-
ture of 5d (120 mg, 0.52 mmol) and maleimide (61 mg,
0.62 mmol) in xylene (14 mL) was refluxed for 48 h. After
filtration, the solid was washed with water and dried to
give 17 (129 mg, 0.42 mmol, 81% yield) as an off-white
solid.
A solution of the isomeric mixture (150 mg, 0.49 mmol) in
dioxane (16 mL) was refluxed for 48 h in the presence of tri-
fluoroacetic acid (2.2 mL). After evaporation, EtOAc was
added to the residue. The mixture was filtered off to give
14 (75 mg, 0.24 mmol, 46% yield) as a brown solid.
Mp >300 ꢀC. IR (KBr) nC]O 1708, 1778 cmꢁ1, nNH,OH
3200–3550 cmꢁ1. Mass (ESI+) [M+H]+ 310, [M+Na]+
332. HRMS (ESI+) [M+Na]+ calcd for C16H11N3O4Na
332.0647, found 332.0664.
Mp >300 ꢀC. IR (KBr) nC]O 1719, 1770 cmꢁ1, nNH 3100–
3550 cmꢁ1
C17H11N3O3Na 328.0698, found 328.0681.
.
HRMS (ESI+) [M+Na]+ calcd for
1H NMR (400 MHz, DMSO-d6): 5.01 (2H, s), 7.45 (1H, d,
J¼8.5 Hz), 7.66 (1H, d, J¼8.0 Hz), 7.92 (1H, s), 8.76 (1H,
s, NH), 11.18 (1H, s, NH), 12.34 (1H, s, NH).
1H NMR (400 MHz, DMSO-d6): 4.32 (1H, d, J¼18.0 Hz),
4.36 (1H, d, J¼11.0 Hz), 4.54 (1H, d, J¼19.0 Hz), 4.57
(1H, d, J¼11.0 Hz), 6.69 (1H, dd, J1¼9.0 Hz, J2¼2.0 Hz),

11144
E. Conchon et al. / Tetrahedron 62 (2006) 11136–11144
5. Roberge, M.; Berlinck, R. G. S.; Xu, L.; Anderson, H. J.; Lim,
L. Y.; Curman, D.; Stringer, C. M.; Friend, S. H.; Davies, P.;
Vincent, I.; Haggarty, S. J.; Kelly, M. T.; Britton, R.; Piers,
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Cancer Res. 2000, 60, 2108–2112.
6.84 (1H, d, J¼2.0 Hz), 7.29 (1H, d, J¼9.0 Hz), 7.88 (1H, s,
NH), 8.96 (1H, s), 11.57 (1H, s, NH), 11.71 (1H, s, NH).
Due to its insolubility, the 13C NMR spectrum could not be
recorded.
7. Jackson, J. R.; Gilmartin, A.; Imburgia, C.; Winkler, J. D.;
Marshall, L. A.; Roshak, A. Cancer Res. 2000, 60, 566–572.
8. Vermeulen, K.; Van Bockstaele, D. R.; Berneman, Z. N. Cell
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10. McGowan, C. H. Mutat. Res. 2003, 532, 75–84.
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2004, 3, 1177–1181.
4.5. Chk1 inhibitory assays
Human Chk1 full-length enzyme with an N-terminal GST
sequence was either purchased from Upstate Biochemicals
(No. 14-346) or purified from extracts of Sf9 cells infected
with a baculovirus encoding GST-Chk1. Assays for com-
pound testing were based upon the method described by
Davies et al.28
13. Zhao, B.; Bower, M. J.; McDevitt, P. J.; Zhao, H.; Davis, S. T.;
Johanson, K. O.; Green, S. M.; Concha, N. O.; Zhou, B. B. S.
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4.6. Growth inhibition assays
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-glutamine, 100 units/mL penicillin, 100 mg/mL strepto-
mycin, and 10 mM HEPES buffer (pH¼7.4). Cytotoxicity
was measured by the microculture tetrazolium assay as
described.29 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 was added to each well and
the plates were incubated for 4 h at 37 ꢀC. The medium
was then aspirated and the formazan solubilized by 100 mL
of DMSO. Results are expressed as IC50, concentration,
which reduced by 50% the optical density of treated cells
with respect to untreated controls.
16. Piers, E.; Britton, R.; Andersen, R. J. J. Org. Chem. 2000, 65,
530–535.
17. Yoshida, T.; Nishiyachi, M.; Nakashima, N.; Murase, M.;
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18. Yoshida, T.; Nishiyachi, M.; Nakashima, N.; Murase, M.;
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19. Terpin, A.; Winklhofer, C.; Schumann, S.; Steglich, W.
Tetrahedron 1998, 54, 1745–1752.
20. Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M.
Tetrahedron Lett. 2003, 44, 3927–3930.
21. Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M.
Tetrahedron Lett. 2003, 44, 4607–4611.
Acknowledgements
22. Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M.
Tetrahedron Lett. 2003, 44, 3935–3937.
ꢀ
The authors are grateful to Bertrand Legeret, University
ꢀ
23. Henon, H.; Messaoudi, S.; Hugon, B.; Anizon, F.; Pfeiffer, B.;
Prudhomme, M. Tetrahedron 2005, 61, 5599–5614.
Blaise Pascal, Clermont-Ferrand, for recording the mass
spectra.
ꢀ
24. Henon, H.; Anizon, F.; Kucharczyk, N.; Loynel, A.; Casara, P.;
Pfeiffer, B.; Prudhomme, M. Synthesis 2006, 711–715.
25. Mase, N.; Nishi, T.; Hiyoshi, M.; Ichihara, K.; Bessho, J.; Yoda,
H.; Takabe, K. J. Chem. Soc., Perkin Trans. 1 2002, 707–709.
26. Bergman, J.; Janosik, T.; Koch, E.; Pelcman, B. J. Chem. Soc.,
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