Novel 1,4-benzodiazepine derivatives with antiproliferative properties on tumor cell lines

Article abstract of DOI:10.1016/j.bmcl.2007.02.016

Novel 1,4-benzodiazepine compounds were synthesized and evaluated for their ability to inhibit the proliferation of tumor cells. Some compounds revealed activities in the micromolar range and were more efficient than reference compound Ro 5-4864. Prelimin

Full text of DOI:10.1016/j.bmcl.2007.02.016

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2527–2530
Novel 1,4-benzodiazepine derivatives with antiproliferative
properties on tumor cell lines
Jennifer Dourlat, Wang-Qing Liu, Nohad Gresh and Christiane Garbay*
Universite´ Paris Descartes, UFR Biome´dicale, Laboratoire de Pharmacochimie Mole´culaire et Cellulaire, Paris F-75006, France
INSERM, U648, Paris F-75006, France
Received 15 December 2006; revised 2 February 2007; accepted 7 February 2007
Available online 9 February 2007
Abstract—Novel 1,4-benzodiazepine compounds were synthesized and evaluated for their ability to inhibit the proliferation of
tumor cells. Some compounds revealed activities in the micromolar range and were more efficient than reference compound Ro
5-4864. Preliminary SAR helped to identify critical motifs for antiproliferative activity and led to the discovery of a compound selec-
tive for a melanoma cell line, known for its resistance to chemotherapy.
Ó 2007 Elsevier Ltd. All rights reserved.
Benzodiazepines are widespread compounds used for
the treatment of mental disorders. A few years ago,
some of these compounds showed antiproliferative
properties against some tumor cell lines. This highlights
them as potential anticancer agents.1,2
We are presently resorting to the 1,4-benzodiazepine
scaffold, a suitable template for combinatorial chemis-
try,3 to design small inhibitors of STAT3 dimerization.
STAT3 (Signal Transducer and Activator of Transcrip-
tion 3) plays a key role in cancer, by regulating as a di-
mer the expression of anti-apoptotic or pro-survival
genes.4 Dimerization occurs upon phosphorylation on
its Tyr705, through reciprocal interaction between the
SH2 (Src Homology 2) domain of STAT3 and the pTyr
residue.
Figure 1. Structure of the 1,4-benzodiazepine compounds.
The compounds were evaluated by an ELISA for their
ability to disrupt in vitro STAT3 dimerization but
showed no significant activity. Nevertheless, they were
further evaluated for their possible antiproliferative
activities on two STAT3-dependent cell lines, NIH
3T3/v-Src cells6 and melanoma cells A2058,7 as well as
on HeLa cells, a cervical carcinoma tumor cell line,
whose growth is not dependent on STAT3.8 Here, we re-
port the synthesis and biological evaluation of our
derivatives.
On the basis of preliminary molecular modeling based
on the X-ray crystal structure of STAT3,5 the N-1 posi-
tion on the 1,4-benzodiazepine structure (Fig. 1) was
used to substitute phosphate or phosphonate analogs,
mimicking the role of pTyr in its SH2 domain interac-
tion. The C-3 position was used to introduce a basic sub-
stituent to probe the effect of a possible ionic interaction
with the SH2 domain.
The synthesis of the diazepine core is described in
Scheme 1. Peptidic coupling of 2-aminobenzophenone
with suitably protected alanine or lysine could be
achieved by the in situ formation of acyl-fluoride9 com-
pounds with the use of tetramethylfluoroformamidini-
um hexafluorophosphate (TFFH) to give 1 and 2 in
69–85% yields. Boc- and Cbz-protecting groups were
Keywords: Proliferation; 1,4-Benzodiazepines; Cancer; Melanoma.
*
Corresponding author. Tel.: +33 142 864 080; fax: +33 142 864
082; e-mail: christiane.garbay@univ-paris5.fr
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.016

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J. Dourlat et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2527–2530
lphosphonomethyl)benzylbromide used to synthesize
10 and 13 was prepared as previously described.12
Alkylation of diazepines 3 and 4 (Scheme 3) with benzyl
bromide derivatives in the presence of the mild base cesi-
um carbonate13 afforded 9–13 in 80–90% yields. Final
compounds 16, 17, and 20 were obtained in quantitative
yield, by treatment with 50% TFA in methylene chloride
for 3 h. Under such conditions, compounds 9 and 12
gave monobenzyl-protected compounds 14 and 18,
and the unprotected analogs 15 and 19 in about equal
proportions.
The lack of inhibitory effects of these benzodiazepines
against STAT3 dimerization led us to investigate if alter-
natively, and in line with the findings reported in the lit-
erature,14 they could inhibit Src kinase activity.
However, no inhibitory effect was found in the case of
the present compounds.
Scheme 1. Reagents and conditions: (a) TFFH, DIEA, CH2Cl2, rt; (b)
i—20% TFA/CH2Cl2 for 3 or H2/Pd–C, MeOH for 4; ii—5% AcOH in
CH2Cl2, reflux.
Nevertheless, in cell assays, some of the benzodiazepine
derivatives demonstrated efficient antiproliferative activ-
ities, inhibiting cell growth with IC50 in the micromolar
range.
completely removed, respectively, by treatment with
20% trifluoroacetic acid (TFA) in methylene chloride
and by Pd-catalyzed hydrogenolytic cleavage.
Cell growth inhibition was first evaluated on v-Src trans-
formed mouse fibroblasts.15 The cytotoxic activity of all
the compounds was then tested against two human
tumor cell lines, A2058 and HeLa cells, using a WST-1
colorimetric test.16 Results are given in Table 1. In addi-
tion, the benzodiazepine Ro 5-4864 (40-chlorodiaze-
pam), whose antiproliferative activity has already been
Cyclization to obtain the diazepine core was achieved
using 5% of acetic acid in methylene chloride to give 3
and 4 in quantitative yield.
The preparation of two brominated derivatives neces-
sary for the N-alkylation of the core diazepine is de-
scribed in Scheme 2. 4-hydroxybenzylalcohol was
treated with dibenzylphosphite10 to give dibenzyl-pro-
tected phosphate derivative 5 in 70% yield. Transforma-
tion of the benzyl alcohol 5 to the bromide derivative 6
was achieved in 63% yield using triphenylphosphine and
carbon tetrabromide.11 Bromide 8 was obtained by Boc-
protecting p-cresol followed by N-bromosuccinimide
(NBS) bromination in 65% yield. The 4-(di-tert-buty-
Scheme 2. Reagents and conditions: (a) Dibenzylphosphite, DIEA,
CCl4, DMAP, CH2Cl2, ꢀ15 °C; (b) CBr4, PPh3, CH2Cl2, rt; (c) Boc2O,
Na2CO3, THF, rt; (d) NBS, dibenzoylperoxide, CCl4, reflux.
Scheme 3. Reagents and conditions: (a) Cs2CO3, CH2Cl2, rt; (b) 50%
TFA/CH2Cl2, rt.

J. Dourlat et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2527–2530
Table 1. Antiproliferative activity against tumor cell lines
2529
Compound
R4
R5
IC50 SEMa (lM)
NIH 3T3/v-Srcb
A2058c
HeLac
Ro 5-486417
—
CH3
—
N-1 unsubstituted
31.9 4.4
n.ad
P100
n.a
P100
n.a
3
14
15
CH3
CH3
49.5 3.2
36.6 5.9
61.5 7.9
28.9 1.5
40.5 0.5
21.3 1.7
16
17
19
CH3
CH3
n.a
n.a
n.a
38.6 3.5
P80
27.2 0.3
33.8 0.5
29.3 0.6
P80
20
n.a
n.a
n.a
a IC50 values expressed with standard error.
b Cell growth determined by counting cells with a particle counter.15
c Cytotoxic activity determined by a WST-1 test.16
d Showed no activity at 150 lM.
demonstrated against some tumor cell lines,17–19 was
also evaluated as the reference compound.
with phosphonate abrogated biological activity in each
cell type. The higher pKa value of phosphonate deriva-
tive may be the cause for the loss of activity.20 The pres-
ence of an oxygen atom at the p-position on the benzyl
ring may also be important.
These data indicate that whilst the unsubstituted benzo-
diazepine 3 is inactive toward any of the three cell types,
some of our substituted benzodiazepines are more effi-
cient than reference compound Ro 5-4864, on both hu-
man tumor cell lines.
We therefore investigated if a hydroxyl group could be
sufficient to retain activity. 4-Hydroxylated compound
17 was accordingly synthesized and was found to be as
efficient as the phosphate analog 15. These data indicate
that the presence of an oxygen atom seems critical for
antiproliferative activity. Moreover, they suggest that
it may be possible to optimize potency with other
O-substituted compounds. On the other hand, the most
potent compounds (14, 15, and 17) were also active on
HeLa cells, indicating that they probably do not target
the STAT3 pathway.
N-1 unsubstituted compound 3 showed no antiprolifer-
ative activity toward any of the three cell types, indicat-
ing that further substitutions are necessary for biological
activity.
For the C-3 methyl series (R4 = CH3), N-substitution
with a p-phosphorylated benzyl moiety improved activ-
ity in each cell type with IC50 in the micromolar range
(15). Monobenzyl-protected phosphate derivative 14,
initially synthesized to improve cell penetration, was less
potent than the free phosphate analog 15 against the
three cell types. Interestingly, phosphate replacement
For the C-3 aminobutyl series (R4 = (CH2)4NH2), the
phosphonate derivative 20 was also inactive. These data
confirmed the importance of a phenolic group or a

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J. Dourlat et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2527–2530
2. Boitano, A.; Ellman, J. A.; Glick, G. D.; Opipari, A. W.,
Jr. Cancer Res. 2003, 63, 6870.
3. Lamb, M. L.; Burdick, K. W.; Toba, S.; Young, M. M.;
Skillman, A. G.; Zou, X.; Arnold, J. R.; Kuntz, I. D.
Proteins 2001, 42, 296.
4. Yu, H.; Jove, R. Nat. Rev. Cancer 2004, 4, 97.
5. Becker, S.; Groner, B.; Muller, C. W. Nature 1998, 394, 145.
6. Cao, X.; Tay, A.; Guy, G. R.; Tan, Y. H. Mol. Cell Biol.
1996, 16, 1595.
7. Niu, G.; Bowman, T.; Huang, M.; Shivers, S.; Reintgen,
D.; Daud, A.; Chang, A.; Kraker, A.; Jove, R.; Yu, H.
Oncogene 2002, 21, 7001.
8. Hess, S.; Smola, H.; Sandaradura De Silva, U.; Hadasc-
hik, D.; Kube, D.; Baldus, S. E.; Flucke, U.; Pfister, H. J.
Immunol. 2000, 165, 1939.
9. Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995,
117, 5401.
p-phosphorylated benzyl moiety. Monobenzyl-protected
phosphate derivative 18 could not be tested because of
low solubility. Surprisingly, introduction of a basic alkyl
chain in C-3 position led to a compound (19) rather
selective of melanoma cells, with only weak potency
on the two other cell types.
In the literature, other benzodiazepines, such as Ro
5-4864, have been shown to have antiproliferative activ-
ity, most of the time via an undefined or uncertain mech-
anism.17–19 Whilst PBR (Peripheral Benzodiazepine
Receptor) has been reported to be a possible target,18,19
benzodiazepine derivative Bz-423 has recently been
found to induce antiproliferative activity independent
of the PBR.21
10. Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron
Lett. 1996, 37, 771.
11. Nishizawa, S.; Cui, Y. Y.; Minagawa, M.; Morita, K.;
Kato, Y.; Taniguchi, S.; Kato, R.; Teramae, N. J. Chem.
Soc., Perkin Trans. 2 2002, 866.
Thus, to our knowledge, the present work reports one of
the first SAR studies, regarding the effects of substitu-
tion on both N-1 and C-3 position, on antiproliferative
properties of 1,4-benzodiazepines.
12. Baczko, K.; Liu, W. Q.; Roques, B. P.; Garbay-Jauregui-
berry, C. Tetrahedron 1996, 52, 2021.
Although our compounds did not inhibit STAT3 dimer-
ization in the Src–STAT3 pathway, they displayed inter-
esting antiproliferative activities against different tumor
cell lines. Benzodiazepines 14, 15, and 17 were even
more efficient than reference compound Ro 5-4864 on
both human tumor cell lines.
13. Nadin, A.; Lopez, J. M.; Owens, A. P.; Howells, D. M.;
Talbot, A. C.; Harrison, T. J. Org. Chem. 2003, 68, 2844.
14. Ramdas, L.; Bunnin, B. A.; Plunkett, M. J.; Sun, G.;
Ellman, J.; Gallick, G.; Budde, R. J. Arch. Biochem.
Biophys. 1999, 368, 394.
15. NIH 3T3/v-Src fibroblasts (105/well) were seeded into
6-well dishes and incubated overnight at 37 °C in 5%
CO2–95% air. After exposure to compounds (at concentra-
tions between 0 and 150 lM) for 72 h, cells were washed
with PBS, then trypsinized and counted in a particle counter
(Coulter Counter, Coulter Electronics, Luton, UK). The
results are expressed as means of at least two independent
experiments performed in duplicate. IC50 was determined
from a sigmoidal dose–response curve using GraphPad
Prism (GraphPad Software, San Diego, CA).
Preliminary SAR helped us to identify critical motifs for
activity and led to the discovery of a compound selective
for a melanoma cell line, known for its resistance to
chemotherapy.22
In conclusion, these two series of benzodiazepines are
promising leads for the development of anticancer
agents. Further experiments will be done to identify
their physiological target and improve the potency and
the selectivity of both series of compounds.
16. The cytotoxic effect of compounds was determined using the
Cell Proliferation Reagent WST-1 assay (Roche Diagnos-
tics, Mannheim, Germany). This colorimetric assay is based
on the cleavage of the tetrazolium salt WST-1 by mito-
chondrial dehydrogenases in viable cells, leading to forma-
zan formation. A2058 (4000 cells/well, 100 ll) and HeLa
cells (3000 cells/well, 100 ll) were seeded in 96-well plates
and incubated overnight at 37 °C in 5% CO2–95% air. After
exposure to compounds (at concentrations between 0 and
150 lM) for 72 h, cells were incubated with WST-1 (10 ll)
for 2 h, and the absorbance of the samples against a
background control was read at 450 nm using a microplate
reader. Results are expressed as means of at least two
independent experiments performed in triplicate. IC50 was
determined from a sigmoidal dose–response curve using
GraphPad Prism (GraphPad Software, San Diego, CA).
17. Zisterer, D. M.; Hance, N.; Campiani, G.; Garofalo, A.;
Nacci, V.; Williams, D. C. Biochem. Pharmacol. 1998, 55,
397.
Acknowledgments
This work was supported by La Ligue Nationale contre
´
le Cancer, Equipe Labellisee 2006. Jennifer Dourlat
benefited from a grant by La Ligue Nationale contre
le Cancer. We are very grateful to Pr. Vidal for helpful
discussions. We thank the Oncology Department of
Servier Institute for NIH 3T3/v-Src and A2058 cells gift
(Dr. Cruzalegui), and for technical support for Src
kinase activity assay.
Supplementary data
18. Carmel, I.; Fares, F. A.; Leschiner, S.; Scherubl, H.;
Weisinger, G.; Gavish, M. Biochem. Pharmacol. 1999, 58,
273.
19. Camins, A.; Diez-Fernandez, C.; Pujadas, E.; Camarasa,
J.; Escubedo, E. Eur. J. Pharmacol. 1995, 272, 289.
20. Mikol, V.; Baumann, G.; Keller, T. H.; Manning, U.;
Zurini, M. G. J. Mol. Biol. 1995, 246, 344.
Analytical data are available on-line. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2007.02.016.
References and notes
21. Sundberg, T. B.; Ney, G. M.; Subramanian, C.; Opipari,
A. W., Jr.; Glick, G. D. Cancer Res. 2006, 66, 1775.
22. Herlyn, M.; Clark, W. H.; Rodeck, U.; Mancianti, M. L.;
Jambrosic, J. Lab. Invest. 1987, 56, 461.
1. Nordenberg, J.; Fenig, E.; Landau, M.; Weizman, R.;
Weizman, A. Biochem. Pharmacol. 1999, 58, 1229.