Synthesis and antiproliferative activity of benzocyclobutacarbazol derivatives. A new class of potential antitumor agents

Article abstract of DOI:10.1016/S0960-894X(00)00532-1

Several benzocyclobutacarbazol derivatives were synthesized and evaluated for their potential cytotoxic properties. A number of these compounds exhibited significant antiproliferative activity with concomitant interaction with the cell cycle and represent a new class of potential anticancer agents. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00532-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2589±2591
Synthesis and Antiproliferative Activity of
Benzocyclobutacarbazol Derivatives. A New Class of Potential
Antitumor Agents
S. Graf-Christophe,a C. Kuehm-Caubere,b P. Renard,b B. Pfei€er,b
A. Pierre,c S. Leoncec and P. Cauberea,*
a
Â
 Â
Institut de Chimie Organique et Analytique, associe au CNRS, Universite d'Orleans, BP 6759, rue de Chatres,
Â
Â
F-45067 Orleans cedex 2, France
Â
 Â
Institut de Recherche Servier, Division de Cancerologie Experimentale, 11 rue des Moulineaux, 92150 Suresnes, France
b
Â
ADIR, 1 rue Carle Hebert, F-92415 Courbevoie cedex, France
c
Received 15 May 2000; revised 4 September 2000; accepted 5 September 2000
AbstractÐSeveral benzocyclobutacarbazol derivatives were synthesized and evaluated for their potential cytotoxic properties. A
number of these compounds exhibited signi®cant antiproliferative activity with concomitant interaction with the cell cycle and
represent a new class of potential anticancer agents. # 2000 Elsevier Science Ltd. All rights reserved.
Numerous anticancer agents are planar or partially
planar p-electron-containing molecules.1 Accordingly
we thought that benzocyclobutene derivatives, easily
obtained by arynic reaction2 could own cytotoxic prop-
erties. Actually we found3 that 1 and 2 strongly inter-
acted with phagic double stranded DNA but not with
single stranded DNA.4 These results could be indicative of
some intercalating properties and/or DNA interaction
with the strained reactive unsaturation of the central rings.
Here we describe the synthesis and antiproliferative activ-
ity of benzocyclobutacarbazoles 4, 5, 7, 8, 10 (Scheme 1).
Hydroxyl groups were unprotected in order to maintain
hydrophilicity. For the sake of comparison, activities of
1 and of two non-hydroxylated derivatives are also
mentioned.
The synthesis of 1 was previously reported.6 The other
compounds were prepared following Scheme 1.7 Tetra-
hydrobiphenylenones 3a±d and tetrahydrocarbazolones
6a±e, 9a±c were respectively obtained by arynic reac-
tions from appropriate starting materials according to
procedures previously published.5,8 Benzocyclobuta-
carbazoles 4a±e and 5 were synthesized with about 20%
yields from the corresponding starting material 3a±d.
Compounds 7a±h were obtained with yields varying
from 15 to 40%. The main by-products of the arynic
condensations were the corresponding aryl tetra-
hydrocarbazolones. Finally, yields of 10a±c varied from
20 to 57%. All products were obtained by ¯ash chro-
matography. Spectroscopic properties and elemental
analysis are in agreement with the structures.
Although these preliminary results were promising,
structural alterations soon appeared to be necessary.
Indeed, 1 and 2 are devoid of in vitro cytotoxicity
against human lymphoblasts3 and L1210 murine leuke-
mia cell line (present work). These drawbacks were
attributed to a lack of hydrophilicity as well as to badly
suited structures. Taking into account that indole rings,
also easily obtained by arynic reactions,5 are part of
numerous anticancer agents,1 we felt that joining indole
and benzocyclobutene units, could lead to a new class of
antitumor agents.
The derivatives were evaluated in vitro for their anti-
proliferative activity using the murine L1210 leukemia
cell line.10 The results expressed as IC50 are reported in
Table 1 as well as the cell cycle perturbations induced by
the most active compounds (IC50ꢀ15 mM).
The structure±activity relationships were ®rst established
within the benzo[3,4]cyclobuta[1,2-c]carbazol-3a-ol series.
*Corresponding author. Tel.: +33-5-5953-7090; fax: +33-5-5953-
5373; e-mail: paul.caubere@wanadoo.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00532-1

2590
S. Graf-Christophe et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2589±2591
It soon appeared that N-methylated compounds (7f
R1=OH, IC50=45.8 mM; 7a R1=OMe, IC50>5 0 mM)
were signi®cantly less active than their unsubstituted
counterpart 4e (IC50=0.6 mM) and 4a (Z1=H,
IC50=9.7 mM). Removal of the 9-methoxy or hydroxy
substituent (7b R1=H, R2=Me, IC50=40.4 mM) had
no clear e€ect on the cytotoxicity.The important part
played by the 3a-hydroxy group was illustrated by the
inactivity of 5 and 8 devoid of such a substituent.
phase of the cycle (68% at 100 mM versus 24% for
untreated control cells).
With the above results in hand, we investigated the
in¯uence of various substituents on the benzene ring of
the benzocyclobutene unit. Substrates substituted by a
methoxy group on the para position of the nitrogen
atom and with unsubstituted nitrogen, were chosen for
this study.
Surprisingly, 4e, despite its good cytotoxicity, possessed
no speci®c interaction with the cell cycle whilst 4a
induced a clear accumulation of the cell in the G2+M
With reference to 4a (IC50=9.7 mM) it appeared that a
6-hydroxy group induced a total loss of activity (4d
IC50>5 0 mM). On the contrary, a 4-hydroxy substituent
Scheme 1. (i) DHP, PTSA, CH2Cl2, 30 ꢁC; (ii) 3-Cl,4-MeOC6H3NH2, C6H6 re¯ux; (iii) NaNH2:t-BuONa (5:2), THF, 0 ꢁC±rt5 then H2O, HCl 10%
Me2CO; (iv) twice PhCH2SH:AlCl3 (20:1.5), 0 ꢁC;9 (v) POCl3, pyridine, 0 ꢁC±rt, then H2±Pd:C (5%); (vi) NaNH2:t-BuONa (9:4), THF 0±10 ꢁC, then
2-ZC6H4Br (Z=H, CH(OMe)2,6,8 then HCl 10% MeOH; (vii) H2 (50 Pa), Pd(OH)2/C (20%), AcOEt±AcOH, rt; (viii) H2N(CH2)2 NMe2, CH2Cl2,
0 ꢁC, then NaBH4, MeOH; (ix) CuSO4/SiO2, MeC6H5, re¯ux; (x) DDQ, C6H6, re¯ux.
Table 1. Antiproliferative activity
F (ꢁC)
IC50 L1210
(mM)
% of L1210
cells in the G2+M phaseb
(mM)
Compounds
F (ꢁC)
IC50 L1210
(mM)
% of L1210
cells in the G2+M phaseb
(mM)
a
Compounds
VP 16
Adriamycin
1
4e
4a
7f
7a
7b
5
0.23
0.025
312.2
0.6
83% (0.5 mM)
80% (0.1 mM)
220
88
88
118
144
93
162
109
189
nec
No speci®c
4d
4b
7g
7c
7d
7h
10a
10b
10c
159
98
>50
24.9
36.5
10.1
5.3
16.7
17
50
34.1
ne
ne
9.7
66% (100 mM)
ne
105
104
108
143
186
97
ne
40% (25 mM)
40% (50 mM)
45.8
>50
40.4
>50
>50
16.6
ne
ne
ne
ne
ne
ne
ne
ne
8
4c
68% (50 mM)
119
2
aA solution of product 10 M in DMSO was diluted to 100 mM (1% DMSO) in the culture medium. Then cells were exposed to graded con-
centrations of the compound for 48 h. Care was taken that no precipitation took place.
b24% of untreated control L1210 cells were in the G2M phase of the cell cycle.
cne, not evaluated.

S. Graf-Christophe et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2589±2591
2591
only slightly decreased the cytotoxicity (4c IC50=16.6
References and Notes
mM) and seemed to improve the cellular cycle interac-
tion (68% of the L1210 cells in the G2+M phase at 50
mM compared to 66% at 100 mM for 4a). Replacement
of a 4-hydroxy by a 4-methoxy (4b IC50=24.9 mM) or a
4-hydroxymethyl group (7g IC50=36.5 mM) resulted in
a decrease of the cytotoxicity. Surprisingly, while a 4-
formyl group had no e€ect on the IC50 when R1=OMe
and R2=H (7c IC50=10.1 mM), it substantially
improved the activity when R1=OMe and R2=Me (7d
IC50=5.3 mM compared to >50 mM for the unformylated
counterpart 7a).
1. See, for example: Pratt, W. B.; Ruddon, R. W.; Ensminger,
W. D.; Maybaum, J. The Anticancer Drugs, 2nd ed.; Oxford
University Press: Oxford, 1994. Ohashi, M.; Oki, T. Exp.
Opin. Ther. Patents 1996, 6, 1285. Prudhomme, M. Curr.
Pharm. Design 1997, 3, 265. Wang, H.-K.; Morris-Natsche, S.
L.; Lee, K.-H. Med. Res. Rev. 1997, 17, 367. Pierre, A.; Atassi,
G.; Devissaguet, M.; Bisagni, E. Drugs Fut. 1997, 22, 53.
Boger, D. L.; Boyce, C. W.; Garbaccio, R. M.; Goldberg, J. A.
Chem. Rev. 1997, 97, 787. Menta, E.; Palumbo, M. Exp. Opin.
Ther. Patents 1998, 8, 1627.
2. For reviews, see: Caubere, P. Rev. Heteroatom. Chem. 1991,
4, 78; Caubere, P. Chem. Rev. 1993, 93, 2317.
In the hope of reinforcing the interactions with DNA,
the formyl group was used to introduce a 4-N,N-di-
methylaminoethylaminomethyl unit. Unfortunately, the
resulting compound 7h was less active than 7d with an
IC50 of only 16.7 mM.
3. Caubere, C. Research Training Report; Ecole Superieure
des Biotechnologies: Strasbourg, France, 1989.
4. Evaluated by the rate of transformation and sequences
analysis in Escherichia coli after preincubation of nucleic acid
(bluscript M13 double or single strand) with the product. With
double-stranded DNA, rate of transformants was a€ected from
45 to 80%; no e€ect was detected with single-stranded DNA.
5. Caubere, C.; Caubere, P.; Ianelli, S.; Nardelli, M.; Jamart-
Gregoire, B. Tetrahedron 1994, 50, 11903. Kuehm-Caubere,
C.; Rodriguez, I.; Pfeifer, B.; Renard, P.; Caubere, P. J. Chem.
Soc., Perkin Trans. 1 1997, 2857.
Finally, from the data reported in Table 1, it appears
that the position of the benzocyclobutene unit plays a
substantial part since 10a and 10c were found to be less
active than 4a and 7d, respectively.
6. Zouaoui, M. A.; Mouaddib, A.; Jamart-Gregoire, B.;
Ianelli, S.; Nardelli, M.; Caubere, P. J. Org. Chem. 1991, 56,
4078.
7. Typical experimental procedure may be found in references
cited in the caption to Scheme 1.
8. Carre, M.-C.; Gregoire, B.; Caubere, P. J. Org. Chem.
1984, 49, 2050. Gregoire, B.; Carre, M.-C.; Caubere, P. J. Org.
Chem. 1986, 51, 1419.
9. Caubere, C.; Caubere, P.; Renard, P.; Bizot-Zspiart, J.-G.;
Ianelli, S.; Nardelli, M.; Jamart-Gregoire, B. Tetrahedron
1994, 50, 13433.
In conclusion, we have presently described the synthesis
and the in vitro antiproliferative activity of new ben-
zo[3,4]cyclobuta[1,2-a] and [1,2-c]carbazol derivatives.
Some of these compounds exhibit signi®cant cytotoxi-
city with concomitant accumulation of the L1210 cells
in the G2+M phase of the cell cycle, opening the door
to promising pharmacomodulations.
Acknowledgements
10. For experimental protocol, see: Leonce, S.; Perez, V.;
Casabianca-Pignede, M. R.; Bisagni, E.; Atassi, G. Invest.
New Drugs 1996, 14, 169.
Warm thanks are expressed to ADIR for ®nancial support.