Synthesis and?biological activity of?6H-isoindolo[2,1-a]indol-6-ones, analogues of?batracylin, and?related compounds

Article abstract of DOI:10.1016/j.ejmech.2005.10.008

Closely related to batracylin, 6H-isoindolo[2,1-a]indol-6-ones including 2-nitro- 13a, 2-amino- 14, and 2-diethylaminopropionamide derivative 16 as well as D-ring substituted 13b, 13c or A-ring substituted 13d and 20 analogues, were synthesised and evaluated against L1210 leukaemia. Subsequent treatment of 13b and 13c with N,N-diethylethylenediamine at 180?°C, led to compounds 17a and 17b arising from an unexpected opening of the pyrrolidinone ring and amidification of the keto group. Under the same conditions, the dichloro derivative 13d led to the monoalkyl compound 20 which was the most cytotoxic of the series.

Full text of DOI:10.1016/j.ejmech.2005.10.008

European Journal of Medicinal Chemistry 41 (2006) 379–386
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and biological activity of 6H-isoindolo[2,1-a]
indol-6-ones, analogues of batracylin, and related compounds
Jean Guillaumel a,*, Stéphane Léonce b, Alain Pierré b, Pierre Renard c, Bruno Pfeiffer c,
Paola B. Arimondo d, Claude Monneret a,*
a Service de pharmacochimie, UMR 176 CNRS-institut Curie, 26, rue d’Ulm, 75248 Paris cedex 5, France
b Institut de recherches Servier, 11, rue des Moulineaux, 92450 Suresnes, France
c ADIR, 1, rue Carle-Hébert, 92415 Courbevoie, France
d UMR5153 CNRS-MNHN USM0503, Inserm UR565, 43, rue Cuvier, 75231 Paris cedex 5, France
Received 21 January 2005; received in revised form 23 September 2005; accepted 11 October 2005
Available online 25 January 2006
Abstract
Closely related to batracylin, 6H-isoindolo[2,1-a]indol-6-ones including 2-nitro- 13a, 2-amino- 14, and 2-diethylaminopropionamide deriva-
tive 16 as well as D-ring substituted 13b, 13c or A-ring substituted 13d and 20 analogues, were synthesised and evaluated against L1210
leukaemia. Subsequent treatment of 13b and 13c with N,N-diethylethylenediamine at 180 °C, led to compounds 17a and 17b arising from an
unexpected opening of the pyrrolidinone ring and amidification of the keto group. Under the same conditions, the dichloro derivative 13d led to
the monoalkyl compound 20 which was the most cytotoxic of the series.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Batracylin; Isoindolo[2,1-a]indole; Indole; Amino-basic chain; Intramolecular Wittig reaction
1. Introduction
but also in human liver [8] and the high dose required for anti-
neoplastic activity were the principal adverse effects of batra-
cylin.
Batracylin 1 [8-amino-isoindolo[1,2-b]quinazolin-12(10H)-
one] is a water-insoluble compound synthesised by Kabbe
[1], acting as an ATP-insensitive topoisomerase II poison [2].
Originally investigated in vivo against murine tumours, this
drug displays an original spectrum of activity. Although inac-
tive against L1210 leukaemia, B16 melanoma and marginally
active against P388 leukaemia, it inhibits tumour growth of the
subcutaneous implanted colon 38 adenocarcinoma [3]. In addi-
tion, it possesses collateral sensitivity against, both adriamycin
and cisplatin-resistant leukaemia P-388 [4]. Preclinical toxici-
ties studies revealed that batracylin was much more toxic when
administered orally to rats than to mice. The responsible meta-
bolite was identified to be N-acetylbatracylin 2 [5] which is a
potent inducer of unscheduled DNA synthesis [6], and cyto-
chrome P450 3A was probably involved into this transforma-
tion [7]. The systemic toxicity, which occurs especially in rats,
Taking these data into account, several studies have been
undertaken to find compounds endowed with decreased toxi-
city. The structural modifications, applied to batracylin, were
undertaken in order to examine the influence of additional ni-
trogen atom in “A” or “D” ring (3 [9], 4 [6]), or increasing the
size of the polycyclic system (5) [9]. Contraction of the “C”
ring from a six-membered into a five-membered ring providing
the benzimidazol analogues of batracylin (6a–b) was also re-
ported [6] (Fig. 1). It must be noticed than none of these com-
pounds was found to act as a topoisomerase inhibitor.
For our part, in a general program within discovering new
potential antitumour heterocycles [10], we designed and synthe-
sised new isoindolo[2,1-a]indole-6-ones, of type A (Fig. 2),
which can be considered as C-pyrrole-containing analogues of
batracylin. The influence of nitrogen introduction on rings A or
D, as a free amino group, a diethylamino ethylamide or a diethy-
lamino ethylamine side-chain, such as in 14, 16 and 20, respec-
tively, was studied. This is the subject of the present report.
* Corresponding author. Tel.: +33 1 42 34 66 55; fax: +33 1 42 34 66 31.
E-mail address: claude.monneret@curie.fr (C. Monneret).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.10.008

380
J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
Fig. 1. Chemical structures of batracylin 1 and related compounds.
mino)propionic acid hydrochloride, in the presence of DCC
and DMAP afforded 16 (46% yield) (Fig. 3).
As described by Krapcho et al. [11,12] in anthracene-9,10-
dione series, we were also planning to obtain the [[(diethylami-
no)ethyl]amino]isoindolo[2,1-a]indol analogues, by substitu-
tion of either the fluorine or chlorine group of 13b or 13c by
N,N-diethylethylenediamine. In order to be efficient, the reac-
tion must be performed at 180 °C for 4 days under pressure in
steel vessel, and without solvent [13–15]. Under these drastic
conditions, instead of the expected isoindoloindole derivatives,
this reaction led to the opened derivatives, the fluoro and
chloro derivatives of N-[2-(diethylamino)ethyl]-2-(indol-2-yl)
benzenecarboxamide 17a, 17b (Fig. 4). These structures were
assigned on the basis of mass spectrometry and X-ray spectra
of 17b (Fig. 5). The relative fragility of the lactam bond was
also observed during the reduction of the 2-methoxyisoindolo
[2,1-a]indol-6-one 18 with LiAlH4 which provided the alcohol
19 (Fig. 6).
In contrast, treatment of the dichloro derivative 13d by N,N-
diethylethylenediamine, under the afore-mentioned conditions,
readily afforded the mono-substituted product 20. It can be hy-
pothesised that the halogen displacement (ipso substitution) is
favoured in the latter case at C-7 by the presence of the adja-
cent carbonyl group in peri position [11]. This was not the case
for 13b and 13c or for the chlorine present at C-10 in 13d
(Fig. 7).
Fig. 2. Structure of type A.
2. Chemistry
The synthesis of the tetracyclic system was carried out via
an intramolecular Wittig reaction, using the protocol that we
had already reported [10]. Diversely substituted phthalimides
9 were obtained by reaction of the corresponding phthalic an-
hydrides 7 with various aniline derivatives 8 in acetic acid.
Next, the derivatives 10 were prepared by bromination of 9
with N-bromosuccinimide (NBS) under radical conditions.
This led to the inseparable mixtures of the expected monobro-
mo compounds 10, along with the dibromo compounds 11 and
the recovered starting materials 9 (Table 1).
Therefore, the crude mixtures were engaged in the follow-
ing Wittig step and the resulting phosphonium salts 12, when
treated with a base in toluene under reflux, provided the ex-
pected isoindolo[2,1-a]indol-6-one derivatives 13. Catalytic
hydrogenation of the 2-nitro-6H-isoindolo[2,1-a]indol-6-one
13a furnished 14 along with 15 resulting from reduction of
the double-bond of the indole ring (ratio of 55:45, as estimated
1
by H NMR). After these compounds had been separated by
column chromatography, condensation of 14 with 3-(diethyla-
3. Biological results and discussion
Table 1
Bromination of 2-(2-methylphenyl)-1H-isoindole-1,3(2H) dione 9
Compounds 14, 15, 16, 17a, 17b and 20 were tested in vitro
against the murine L1210 leukaemia and the human HT-29
colon carcinoma cell lines (Table 3). Although these com-
pounds did not show properties comparable to that of the re-
ference drugs (adriamycin and camptothecin), it should be
noted that the most active were the basic chain-containing de-
rivatives 16 and 20, followed by the parent compound 14.
Despite the reported [4] inhibition of adenocarcinoma 38
growth in mice (but at 400 mg kg–1) by batracylin, the relative
lack of in vitro activity against L1210 and HT-29 was not sur-
prising, since the same relative inactivity in vitro was already
demonstrated against the leukaemia cell line [3] but also the
Compounds
NBS equivalenta
Ratiob
CH2Br
Starting
Material
38
31
25
12
10
11
CHBr2
11
14
16
21
24
24
23
10
48
53
54
64
66
66
10a/11a
1.1
1.25
1.5
1.1
1.1
1.1
10b/11b
10c/11c
10d/11d
a
All experiments were conducted in CCl4 at 76 °C for 8 h except in the case
of 10d/11d, 1 h.
b
1
The ratio were calculated from an average of H NMR signals integrations
of each component.

J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
381
Fig. 5. X-ray crystallography of 17b.
Fig. 6. Reduction of 2-methoxyisoindolo[2,1-a]indol-6-one (18).
Fig. 7. Synthesis of 10-chloro-7-[[(diethylamino)ethyl]amino]-6H-isoindol[2,1-
a]indol-6-one (20).
cancer lung (H-125), human colon (CX1) and human intestinal
(HCT-8) adenocarcinoma [19].
A tetracyclic aromatic scaffold is a critical feature to retain
antiproliferative activity as demonstrated by the dramatic de-
crease in activity when partial reduction occurred (see 15) or
when the ring B was cleaved (see 17a and 17b).
Fig. 3. Synthesis of 2-amino-6H-isoindolo[2,1-a]indol-6-one (14) and related
derivatives.
Blockade of the cellular cycle in G2 + M was observed in
the case of 14 and 20, the active compound 16 being too toxic
to observe a specific effect.
These amino compounds were also tested for topoisome-
rases I and II inhibition. None of the tested compounds was a
specific topoisomerase I inhibitor; only a non-specific effect
was observed with compounds 14, 16 and 20. Indeed these
three compounds bind to DNA and interfere non-specifically
with the topoisomerase I catalytic cycle. Noteworthy, these
compounds are planar aromatic molecules and when a dou-
ble-bond is lost likely in compound 15, the molecule was de-
void of the DNA-binding properties. The conformation of
Fig. 4. Synthesis of N-[2-(diethylamino)ethyl]-2-(indol-2-yl)benzenecarboxa-
mides (17).

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J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
Table 2
displayed the most relevant cytotoxicity against HT-29 and
L1210 cell lines.
Crystal data of compound 17b for C21H24ClN3O, CH3OH
Empirical formula
Crystal class
Shape
C22H28ClN3O2
Triclinic
Parallelepiped
Colourless
401.94
P–1
4. Conclusion
Colour
M (g mol−1
)
The synthesis of isoindolo analogues of batracylin was un-
dertaken. Contrary to what was observed with stricto sensu
five-membered ring analogues, some of these new compounds
displayed a topoisomerase II inhibition comparable to that of
etoposide. They also displayed modest but slightly superior
antiproliferative effect against HT-29 and L1210 cell lines ver-
sus batracylin itself. Dihydro derivatives and B-ring opened
analogues were completely devoid of activity underlining the
importance of the planar tetracyclic aromatic system in this
class of compounds.
Space group
Temperature (K)
Wavelength (Å)
a (Å)
b (Å)
c (Å)
295
0.710690
11.837 (3)
13.505 (4)
13.717 (2)
89.74 (2)
86.42 (1)
84.08 (2)
2177 (5)
4
α (°)
β (°)
γ (°)
Volume (A3)
Z
Density
1.23
Crystal size (mm)
Scan type
0.5 × 0.4 × 0.3
2 θ/ω
5. Experimental protocols
θ
min, θmax
1–25
0.197
μ (mm–1
)
5.1. Chemistry
Radiation type
MoKα
Reflections measured
Independent reflections
Reflections used
Number of parameters
Goodness of fit
7993
7641
3184
507
5.1.1. General remarks
Uncorrected melting points were determined on a Kofler hot
stage or an electrothermal capillary melting apparatus. The H
1
1.09
NMR spectra were recorded at 90 MHz using a Varian EM
390, and at 300 MHz on a Brucker AM-300 spectrometer with
TMS as internal standard (s = singlet, bs = broad singlet,
d = doublet, dd = doublet of doublet, m = multiplet); coupling
constants (J) were given in hertz (Hz). Electronic impact (EI,
70 eV) and chemical ionisation (CI) mass spectra (MS) were
recorded on a Nermag R10-10-C spectrometer. Elemental ana-
lyses were performed by the “Service de Microanalyses du
CNRS” (Vernaison-Lyon, France); all compounds had analyti-
cal results within 0.4% of their theoretical values. The thin-
layer chromatographic analyses were performed using pre-
coated silica gel (Merck, 60F254). Column chromatographies
were carried out on Merck silica gel 60 (70–230 mesh).
R = Σ IIFoI–IFcII/Σ IFoI
Rw = [Σw (IIFoI–IFcII)2/Σ IFoI2]1/2
compounds 17a and 17b allowed even less DNA binding.
Furthermore, the presence of the amino side-chains in 16 and
20 increased DNA affinity as compared to 14.
On the other hand, compounds 14 and 16 showed an inhi-
bitory potency of topoisomerase II comparable to that of etopo-
side (50% of linear DNA in the presence of 20 μM of drug).
However, at high concentrations, compound 16 proved to be
highly toxic. Despite the fact that the chloro-derivative 20
was less active for inhibiting topoisomerase II, this compound
Table 3
Cytotoxicity, effect on the cellular cycle and activity against topoisomerase I and II of 14, 15, 16, 17a, 17b, 20
Compounds
HT-29
L1210
% of L1210
in G2 + Ma
Conc.
TOPO I
TOPO II
b
c
IC50
(μM)
0.068
0.008
> 50
0.83
15.1
80
NE
NE
NE
3.2
IC50
%
MIC
linDNA (%)
(at 20 μM)
(μM)
0.022
0.025
> 50
(μM)
0.1
0.25
(μM)
Adriamycin
Camptothecin
Batracylin
Etoposide
14
15
16
17a
17b
20
80%
78%
10
76%
71%
NE
2.5
25
50
50
0
50
0
10.2
> 50
6.7
42.9
14.6
4.4
20
> 100
50
> 100
> 100
50
d
NS
20
10
0
30
52%
NE: not evaluated; NS: non-specific.
a
Distribution of control cells in the cycle: 37% (G1), 39% (S), 24% (G2 + M).
MIC: minimal concentration of drug for which an inhibition of relaxation in the presence of topoisomerase I is detected.
linDNA (%): percentage of linear DNA measured in a DNA relaxation assay in the presence of topoisomerase II reported at 20 μM of drug.
Toxic at 25 μM and inactive at lower concentrations.
b
c
d

J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
383
5.1.2. Synthesis of 2-(2-methylphenyl)-1H-isoindole-1,3(2H)-
diones 9
yethane (100 ml), the gummy residue crystallised. The precipi-
tate was filtered, carefully washed with dimethoxyethane to
eliminate the impurities, and dried at 80 °C in vacuo.
General procedure: the 3,6-dichlorophthalic anhydride was
prepared from tetrachlorophthalic anhydride [13,15].
A mixture of phthalic anhydride 7 (10 mmol) and aniline 8
(10.5 mmol) was refluxed for 6 h in 20 ml of acetic acid. The
mixture was poured into water, the resulting precipitate was
filtered off, washed with water, dried and recrystallised.
5.1.4.1. [[2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)-5-nitro-
phenyl]methyl] triphenyl phosphonium bromide (12a). Yield
1
(with regard to 9a) 35%, m.p. 272–274 °C (dec.); H NMR
(90 MHz, CDCl3): δ 5.55 (d, 2H, CH2–P, J = 15 Hz), 7.26–
8.00 (m, 20H arom), 8.16–8.26 (m, H3′), 8.56 (m, 1H, H5′).
Anal. C33H24BrN2O4P (C, H, N, Br).
5.1.2.1. 2-(2-Methyl-4-nitrophenyl)-1H-isoindole-1,3(2H)-
dione (9a). This compound was synthesised from 7a and 8b:
yield 91%; m.p. 200–202 °C (EtOH); 1H NMR (90 MHz,
CDCl3) δ 2.33 (s, 3H, CH3), 7.40 (d, 1H, H6′, J = 9 Hz),
7.76–8.06 (m, 4H arom.), 8.16–8.33 (m, H3′, H5′). Anal.
C15H10N2O4 (C, H, N).
5.1.4.2. [[2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)-4-fluoro-
phenyl]methyl] triphenyl phosphonium bromide (12b). Yield
1
(with regard to 9b) 67%, m.p. 218–220 °C (dec.); H NMR
(300 MHz, CDCl3): δ 5.03 (d, 2H, CH2–P, J = 13.8 Hz), 6.82
(d, 2H, H3′, H4′, J = 8.4 Hz), 7.33–7.48 (m, 15 H arom.), 7.56
(m, 2H, H5, H6), 7.68 (m, 1H, H6′), 7.78 (m, 2H, H4, H7).
Anal. C33H24BrFNO2P, C4H10O2, dimethoxyethane (C, H,
Br, F, N).
5.1.2.2. 2-(5-Fluoro-2-methylphenyl)-1H-isoindole-1,3(2H)-
dione (9b). This compound was synthesised from 7a and 8c:
yield 95%; m.p. 158–160 °C (EtOH); 1H NMR (90 MHz,
CDCl3) δ 2.16 (s, 3H, CH3), 6.86–7.46 (m, H3′, H4′, H6′),
7.73–8.00 (m, 4H arom.). Anal. C15H10FNO2 (C, H, N, F).
5.1.4.3. [[2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)-4-chlor-
ophenyl]methyl] triphenyl phosphonium bromide (12c). Yield
(with regard to 9c) 66%, m.p. 204–206 °C (dec.); H NMR
(90 MHz, CDCl3): δ 5.26 (d, 2H, CH2–P, J = 15 Hz), 7.10 (s,
1H, H3′), 7.20–8.06 (m, 21 H arom.). Anal. C33H24BrClNO2P
(C, H, Br, Cl, N).
5.1.2.3. 2-(5-Chloro-2-methylphenyl)-1H-isoindole-1,3(2H)-
1
dione (9c). This compound was synthesised from 7a and 8d:
1
yield 75%; m.p. 170–172 °C (EtOH); H NMR (300 MHz,
CDCl3) δ 2.17 (s, 3H, CH3), 7.22–7.34 (m, 3H, H3′, H4′, H6′),
7.81 (m, 2H, H5, H6), 7.96 (m, 2H, H4, H7); IR (CDCl3)
1709 cm−1 (C=O). Anal. C15H10ClNO2 (C, H, N, Cl).
5.1.4.4. [[2-(4,7-Dichloro-1,3-dihydro-1,3-dioxo-2H-isoindol-
2-yl)phenyl]methyl] triphenyl phosphonium bromide (12d).
Yield (with regard to 9d) 51%, m.p. 220–222 °C (dec.); H
5.1.2.4. 4,7-Dichloro-2-(2-methylphenyl)-1H-isoindole-1,3
(2H)-dione (9d). This compound was synthesised from 7b and
8a: yield 91%; m.p. 201–203 °C (EtOH); 1H NMR (300 MHz,
CDCl3) δ 2.16 (s, 3H, CH3), 7.17 (d, 1H, H3′, J = 7.64), 7.30–
7.39 (m, 3H, H4′, H5′, H6′), 7.64 (s, 2H, H5, H6). Anal.
C15H9Cl2NO2 (C, H, N, Cl).
1
NMR (300 MHz, CDCl3):
δ 5.19 (d, 2H, CH2–P,
J = 13.9 Hz), 7.10 (d, 1H, H3′, J = 7.8 Hz), 7.22 (m, 2H, H4′,
H5′), 7.35–7.67 (m, 15 H arom.), 7.74 (s, 2H, H5, H6), 7.87 (d,
1H, H6′, J = 8.2 Hz). Anal. C33H23BrCl2NO2P (C, H, Br, Cl,
N).
5.1.3. Synthesis of bromo compounds 10
5.1.5. Synthesis of 6H-isoindol[2,1-a]indol-6-one derivatives
13, by intramolecular Wittig reaction
General procedure for bromination:
A suspension of methylated derivatives 9 (10 mmol), NBS
(according to the quantity indicated on Table 1) and benzoyl
peroxide (50 mg) in dry carbon tetrachloride (80 ml) was re-
fluxed under stirring, under exposure of a 150 W tungsten
lamp, for the indicated time. The reaction mixture was poured
into water, extracted with dichloromethane, and the solvent
was evaporated.
General procedure:
To a suspension of phosphonium salts 12 (10 mmol) in to-
luene (125 ml) was added the under-mentioned quantity of
triethylamine (NEt3) or 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU). The mixture was refluxed for the indicated time. After
cooling, the insoluble was filtered and the mother liquor was
directly chromatographed on the silica column using toluene as
eluent.
The dosage of each component, monobromo 10 and dibro-
mo compound 11 of the crude reaction mixture was analysed
1
by H NMR spectroscopy.
5.1.5.1. 2-Nitro-6H-isoindolo[2,1-a]indol-6-one (13a). This
compound was prepared, from phosphonium salt 12a with
1.1 equivalent of DBU for 24 h: yield 89%, m.p. 268–270 °C
5.1.4. Synthesis of phosphonium salts 12
General procedure:
1
To a solution of crude mixture of bromo derivatives (3 g) in
CHCl3 (25 ml) was added triphenylphosphine (1.05 equivalent
with regard to the theoretical quantity of mono bromo com-
pound present in the mixture). After heating at reflux for
18 h, the solvent was evaporated. Under agitation in dimethox-
(chlorobenzene); H NMR (300 MHz, CDCl3): δ 7.18 (s, 1H,
H11), 7.53 (m, 1H, H8 or H9), 7.74 (m, 1H, H8 or H9), 7.84 (d,
1H, H7, J = 7.5 Hz), 7.92 (m, 2H, H4 and H10, J = 9 Hz), 8.21
(dd, 1H, H3, J = 2.3 Hz, J = 8.8 Hz), 8.56 (d, 1H, H1,
J = 2.2 Hz). Anal. C15H8N2O3 (C, H, N).

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J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
5.1.5.2. 3-Fluoro-6H-isoindolo[2,1-a]indol-6-one (13b). This
compound was prepared, from phosphonium salt 12b with 2
equivalents of NEt3 for 5 h: yield 86%, m.p. 193–195 °C (to-
5.1.7. N-(6-oxo-6H-isoindolo[2,1-a]indol-2-yl)-3-
diethylaminopropionamide (16)
To a solution of the amino compound 14 (117 mg,
0.5 mmol) in CH2Cl2 were added 3-(diethyamino)propionic
acid hydrochloride (181.5 mg, 1 mmol), dicyclohexyl carbodii-
mide (DCC, 1 M in solution in CH2Cl2, 1.05 ml, 1 mmol), and
dimethylamino-pyridine (DMAP, 244 mg, 2 mmol). The mix-
ture was allowed to stir for 16 h at room temperature under
argon atmosphere. The resulting white precipitate of dicyclo-
hexylurea was removed by filtration. The solvent was evapo-
rated in vacuo and the residue was purified on silica gel (eluent
CH2Cl2/MeOH 98:2). Compound 16 was obtained in 46%
1
luene/cyclohexane); H NMR (300 MHz, CDCl3): δ 6.58 (s,
1H, H11), 6.89 (m, 1H, H2, J = 2.3 Hz, J = 9.2 Hz), 7.32–
7.39 (m, 2H, H1, H8), 7.51–7.53 (m, 2H, H9, H10), 7.61 (dd,
1H, H4, J = 2.3 Hz, J = 8.8 Hz), 7.77 (d, 1H, H7, J = 7.9 Hz).
Anal. C15H8FNO (C, H, F, N).
5.1.5.3. 3-Chloro-6H-isoindolo[2,1-a]indol-6-one (13c). This
compound was prepared, from phosphonium salt 12c with 1.5
equivalents of NEt3 for 4 h: yield 92%, m.p. 203–205 °C (to-
1
1
luene/cyclohexane); H NMR (300 MHz, CDCl3): δ 6.58 (s,
yield, m.p. 92–94 °C (toluene-C6H12); H NMR (300 MHz,
1H, H11), 7.12 (dd, 1H, H2, J = 1.9 Hz, J = 8.2 Hz), 7.35 (d,
1H, H1, J = 8.3 Hz), 7.37 (m, 1H, H8), 7.53 (m, 2H, H9, H10),
7.77 (d, 1H, H7, J = 6.8 Hz), 7.90 (d, 1H, H4). MS (EI) m/z
253–255 (M+, 100%). Anal. C15H8ClNO (C, H, Cl, N).
CDCl3): δ 1.16 (t, 6H, 2CH3), 2.53 (t, 2H, Hb), 2.69 (q, 4H,
Hc, Hd), 2.80 (t, 2H, Ha), 6.59 (d, 1H, H11, J = 1.1 Hz), 7.12 (d,
1H, H1, J = 8.7 Hz), 7.33 (m, 1H, H3), 7.51 (m, 2H, H8, H9),
7.75 (m, 2H, H7, H10), 8.00 (s, 1H, H4), 11.37 (s, 1H, NH); MS
(CI–NH3) m/z 362 (M + H)+. Anal. C22H23N3O2,H2O (C, H,
N).
5.1.5.4. 7,10-Dichloro-6H-isoindolo[2,1-a]indol-6-one (13d).
This compound was prepared, from phosphonium salt 12d with
2 equivalents of NEt3 for 6 h: yield 82%, m.p. 206–208 °C
5.1.8. Synthesis of 17 and 20
1
(toluene); H NMR (300 MHz, CDCl3): δ 6.89 (s, 1H, H11),
General procedure:
7.18 (m, 2H, H2, H3), 7.34 (m, 2H, H1, H8), 7.48 (d, 1H, H9,
J = 7.3 Hz), 7.89 (d, 1H, H4, J = 8.1 Hz). Anal. C15H7Cl2NO
(C, H, Cl, N).
A mixture of halogenoisoindolo[2,1-a]indol-6-one 13 (1 g)
and freshly distilled N,N-diethylethylenediamine (20 ml) was
heated in a steel vessel at 180 °C for 4 days. Excess of diamine
was removed under reduced pressure and the residue was di-
5.1.6. Reduction of 2-nitro-6H-isoindolo[2,1-a]indol-6-
one (13a)
rectly chromatographed on
CH2Cl2/MeOH 99:1).
a
silica column (eluent:
Hydrogenation was accomplished at air-pressure using 1.5 g
(5.68 mmol) of nitro compound and 10% Pd–C (335 mg) in
dioxane, for 5 h. The reaction mixture was filtered off over
celite and the solvent was evaporated. The residue, dissolved
in dichloromethane, was chromatographed on silica (increasing
the eluent polarity from CH2Cl2 to MeOH: 99.5–0.5 to 98–2)
to give the following:
5.1.8.1. N-[2-(diethylamino)ethyl]-2-(6-fluoroindol-2-yl)benze-
necarboxamide 17a. This compound was synthesised from
13b: yield 88%, m.p. 93–95 °C (hexane): 1H NMR (300 MHz,
CDCl3): δ 0.85 (t, 6H, 2 CH3), 2.38 (q, 4H, Hc, Hd), 2.47 (t,
2H, Hb), 3.42 (q, 2H, Ha), 6.58 (bs, 1H exchangeable, NH–
CO), 6.72 (s, 1H, H3), 6.86 (m, 1H, H5, J = 2.1 Hz,
J = 9.2 Hz), 7.11 (dd, 1H, H7, J = 1.9 Hz, J = 9.7 Hz), 7.36
(m, 1H, H4′, J = 1.3 Hz, J = 7.8 Hz), 7.47–7.54 (m, 3H, H4,
H3′, H5′), 7.75 (d, 1H, H6′, J = 1.4 Hz, J = 7.7 Hz), 10.20 (bs,
NH-indole). MS (CI–NH3) m/z 354 (M + H)+. Anal.
C21H24FN3O (C, H, F, N).
5.1.6.1. 2-Amino-6H-isoindolo[2,1-a]indol-6-one (14). 0.62 g
1
(yield 46%), m.p. 189–191 °C (toluene); H NMR (300 MHz,
CDCl3): δ 3.65 (bs, 2H exchangeable, NH2), 6.46 (s, 1H, H11),
6.66 (dd, 1H, H3, J = 2.2 Hz, J = 8.2 Hz), 6.75 (d, 1H, H1,
J = 2.2 Hz), 7.31 (m, 1H, H9), 7.47 (m, 2H, H8, H10), 7.67
(d, 1H, H4, J = 8.3 Hz), 7.72 (d, 1H, 1H, H7, J = 7.9 Hz);
MS (EI) m/z 234 (M+). Anal. C15H10N2O (C, H, N).
5.1.8.2. N-[2-(diethylamino)ethyl]-2-(6-chloroindol-2-yl)benze-
necarboxamide 17b. This compound was synthesised from
13c: yield 70%, m.p. 107–109 °C (hexane); 1H NMR
(300 MHz, CDCl3): δ 0.85 (t, 6H, 2 CH3), 2.38 (q, 4H, Hc,
Hd), 2.47 (t, 2H, Hb), 3.41 (q, 2H, Ha), 6.50 (bs, 1H exchange-
able, NH–C=O), 6.71 (d, 1H, H3, J = 0.9 Hz), 7.06 (dd, 1H,
H5, J = 1.9 Hz, J = 8.5 Hz), 7.36 (m, 1H, H4′), 7.42 (m, 1H,
H7), 7.47–7.52 (m, 3H, H4, H3′, H5′), 7.75 (d, 1H, H6′,
J = 7.9 Hz), 10.25 (bs, NH-indole). MS (CI–NH3) m/z 370–
372 (M + H)+. Anal. C21H24ClN3O (C, H, N, Cl).
5.1.6.2. Hydrochloride. The free base was dissolved in dry di-
chloromethane and dry HCl gas was bubbled through the solu-
tion. The hydrochloride was collected by filtration, m.p. >
260 °C. Anal. C15H10N2O, HCl (C, H, Cl, N).
5.1.6.3. 2-Amino-6H-isoindolo[2,1-a]indolin-6-one
(15).
0.47 g (yield 35%), m.p. 165–167 °C (toluene); 1H NMR
(300 MHz, CDCl3): δ 2.97 and 3.35 (2 m, 2H, H11a, H11b),
3.43 (bs, 2H exchangeable, NH2), 5.56 (t, 2H, H10b), 6.60
(m, 2H, H1, H3), 7.45–7.52 (m, 3H, H4, H8, H10), 7.58 (t, 1H,
H9), 7.90 (m, 1H, H7). MS (EI) m/z 236 (M+, 100%). Anal.
C15H12N2O (C, H, N).
5.1.8.3. 10-Chloro-7-[[(diethylamino)ethyl]amino]-6H-isoin-
dol[2,1-a]indol-6-one 20. This compound was synthesised
1
from 13d: yield 64%, m.p. 118–120 °C (hexane); H NMR
(90 MHz, CDCl3): δ 1.13 (t, 6H, 2 CH3), 2.60–2.86 (m, 6H,

J. Guillaumel et al. / European Journal of Medicinal Chemistry 41 (2006) 379–386
385
CH2–N(CH2)2), 3.33 (q, 2H, NH–CH2), 6.46 (d, 1H, H8,
J = 9 Hz), 6.80 (s, 1H, H11), 7.00–7.60 (m, 4H, H1, H2, H3,
H9), 7.83 (d, 1H, H4, J = 8.7 Hz); MS (CI–NH3) m/z 368–
370 (M + H)+. Anal. C21H22ClN3O (C, H, N).
methylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma) were added to each well and the plates were incubated
for 4 h at 37 °C. The medium was aspirated and the formazan
was solubilised by 100 μl of DMSO. The IC50 concentration,
reducing by 50% the optical density at 540 nm, was calculated
by a linear regression performed on the linear zone of the
dose–response curve. All the measurements were performed
in triplicate.
5.1.9. 2-(5-Methoxyindol-2-yl)benzenemethanol (19)
To a solution of 2-methoxy-6H-isoindol[2,1-a]indol-6-one
18 [10] (1 g, 4.01 mmol) in THF (40 ml) stirred at room tem-
perature, LiAlH4 (0.305 g, 8.02 mmol) was added portion-wise
and the stirring maintained for 3 h. Then the excess of reagent
was quenched carefully with water and the resulting mixture
was extracted with CH2Cl2.The alcohol was obtained with
For the cell cycle analysis, L1210 cells (2.5 × 105 cells
ml–1) were incubated for 21 h with various concentrations of
the compounds. Cells were then fixed by 70% ethanol (v/v),
washed and incubated in PBS containing 100 μg ml–1 RNAse
and 25 μg ml–1 propidium iodide for 30 min at 20 °C. For each
sample, 104 cells were analysed on an Epics XL/MCL flow
cytometer (Beckman Coulter, France). PI fluorescence was col-
lected through a 630 nm band-pass filter. Results are expressed
as the percentage of cells in each phase of the cell cycle.
1
87% yield; H NMR (90 MHz, CDCl3): δ 2.23 (bs, 1H ex-
changeable, OH), 4.73 (s, 2H, CH2OH), 6.66 (d, 1H, H3,
J = 2.1 Hz), 6.86 (dd, 1H, H6, J = 2.4 Hz, J = 9 Hz), 7.10 (d,
1H, H4, J = 9 Hz), 7.23–7.53 (m, 4H, H3′, H4′, H5′, H6′), 7.75
(d, 1H, H7, J = 9 Hz), 10.06 (bs, 1H, NH-indole); MS
(CI–NH3) m/z 254 (M + H)+. Anal. C16H15NO2 (C, H, N).
5.3.2. Topoisomerase I-mediated DNA relaxation assay
Supercoiled pBS DNA (0.1 μg) (Stratagene, France) was
incubated for 15 min at 30 °C, in a 50 mM Tris–HCl buffer,
pH 7.5, containing 1 mM ATP, 60 mM KCl, 10 mM MgCl2,
0.5 mM DTT, 0.1 mM EDTA and 30 mg BSA, in the presence
of the drug at the indicated concentration (total reaction vo-
lume 10 ml). 2 units of human DNA topoisomerase I (Topo-
GEN, Inc., USA) were added to the duplex, preincubated as
described, and incubated for 30 min at 30 °C. The DNA–topoi-
somerase II cleavage complexes were dissociated by addition
of SDS (final concentration 0.25%) and of proteinase K
(Sigma) to 250 mg ml–1, followed by incubation for 30 min at
55 °C. DNA samples were then added to the electrophoresis
dye mixture (5 ml) and electrophoresed (35 V cm–1) for 2 h
in a 1% agarose gel in TBEx1 at room temperature. Gels were
stained with SYBR® Green I (Molecular Probes), washed and
photographed under UV light.
5.2. X-ray diffraction analysis of N-[2-(diethylamino)ethyl]-2-
(6-chloroindol-2-yl)benzenecarboxamide (17b)
Crystal of 17b, suitable for X-ray analysis, was obtained by
slow recrystallisation from methanol. The carboxamide crystal-
lised with a MeOH molecule.
The data were collected on a Enraf-Nonius CAD-4 diffract-
ometer using graphite-monochromated MoKα radiation
(Table 2). The structure was solved by direct methods and sub-
sequent Fourier maps. Refinements were carried out by full-
matrix least-squares techniques. Non-hydrogen atoms were an-
isotropically refined. All hydrogen atoms’ positions were
found on difference maps, their coordinates were not refined
and they were given an overall isotropic thermal parameter.
An absorption correction was applied with the DIFABS pro-
gram [16] from CRYSTALS [17] which was used to carry
out all the calculations.
5.3.3. Topoisomerase II-mediated DNA cleavage assay
The assay was performed as described in [10].
5.3. Biopharmacological methods (bioevaluations)
Acknowledgments
5.3.1. Cell culture and cytotoxicity
The human HT29 and murine L1210 cell lines were ob-
tained from the American Type Culture Collection (ATCC,
Rockville, MD). Cells were maintained in RPMI 1640 medium
supplemented with 10% decomplemented fetal calf serum
(FCS), 2 mM L-glutamine, 100 U ml–1 penicillin, 100 μg
ml–1 streptomycin, and 10 mM HEPES, pH 7.4. Cells were
grown at 37 °C in 5% CO2/95% air. All media and supple-
ments were from Life Technologies (Cergy-Pontoise, France)
except FCS which was purchased from Sigma. Cytotoxicity
was measured by the microculture tetrazolium assay as de-
scribed in [18]. Briefly, adherent cells were seeded in 96 well
microplates and incubated for 2 days. The tested compounds
were then added and the plates were incubated for four dou-
bling times (continuous exposure). The non-adherent L1210
cells were directly incubated for 48 h with the compounds.
At the end of this period, 15 μl of 5 mg ml–1 3-(4,5 di-
This work was supported by the Servier-Adir Group.
Authors express their gratitude to Morgane Marcou for the to-
poisomerases I and II relaxation assays. We thank Dr. Bois and
J. Vaissermann for obtaining the X-ray crystal structure of
compound 17b.
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