Design and synthesis of novel chrysene-linked pyrrolo[2,1-c][1,4]-benzodiazepine hybrids as potential DNA-binding agents

Article abstract of DOI:10.1016/S0960-894X(03)00743-1

Chrysene-linked pyrrolobenzodiazepine hybrids have been prepared that posses cytotoxicity in some cancer cell lines. They also exhibit promising DNA-binding affinity and this is supported by molecular modeling studies.

Full text of DOI:10.1016/S0960-894X(03)00743-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3451–3454
Design and Synthesis of Novel Chrysene-Linked Pyrrolo[2,1-c]
[1,4]-benzodiazepine Hybrids as Potential DNA-Binding Agents
Ahmed Kamal,* G. Ramesh, P. Ramulu, O. Srinivas, Tasneem Rehana and G. Sheelu
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received 10 March 2003; revised 15 July 2003
Abstract—Chrysene-linked pyrrolobenzodiazepine hybrids have been prepared that posses cytotoxicity in some cancer cell lines.
They also exhibit promising DNA-binding affinity and this is supported by molecular modeling studies.
# 2003 Elsevier Ltd. All rights reserved.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
family of DNA interactive antitumour antibiotics
derived from Streptomyces species.1 The PBD class of
compounds that include the naturally occurring
anthramycin and DC-81, owes its DNA-interactive
ability and resultant biological effects to an N10–C11
carbinol amine/imine moiety in the central B-ring,
which is capable of covalently binding to the N2 of the
guanine residues in the minor groove of DNA. X-ray
and DNA foot printing studies on covalent adducts
have demonstrated a high sequence specificity for G-C
rich DNA regions, in particular for Pu-G-pu triplets.2
Recently, chrysene derivatives have been synthesized
and evaluated for their antitumour activity and DNA
intercalating potential. Among them, some of the of
2-[(arylmethyl)amino]-1,3-propanediol derivatives such
as 770U82 (crisnatol), 773U82 and 502U83 are cur-
rently in different phases of clinical trials as potential
antitumour agents.7 Therefore, based on these findings,
it has been considered of interest to link the polycyclic
aromatic rings like chrysene at C8 position of PBD ring
system through an alkylamide spacer. The design of
such hybrids of PBD linked to highly lipophilic aro-
matic amines could result in primarily, selective inter-
actions with cancer cells as a major affect in cell killing.
We have been engaged in the last few years in the
structural modifications8 and the development of new
synthetic strategies9 for the PBD-based ring systems. In
continuation of these efforts, we herein report the
design, synthesis, DNA-binding affinity and anticancer
potential of chrysene linked PBD hybrids.
A large number of PBD conjugates have been prepared
and evaluated for their biological activity, particularly
their antitumour potential.3 Thurston and co-workers
have synthesized novel C2-aryl pyrrolobenzodiazepines
(PBDs) as potential antitumour agents.4 Recently, we
have designed and synthesized non-cross-linking mixed
imine–amide PBD dimers that have significant DNA-
binding ability and potent antitumour activity.5 Many
compounds that have planar ring systems possess
chemotherapeutic activity by intercalating with DNA.6
Synthesis of the novel PBD hybrids has been carried
out employing the amino chrysene 2 have been obtained
by the reduction of the nitro chrysene 1 by SnCl2.2H2O
in EtOAc (Scheme 1). The (2S)-N-(4-benzyloxy-5-
methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde
diethylthioacetal 3 has been prepared by literature
method,10 which upon debenzylation gives 4. Etherifi-
cation of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-
pyrrolidine-2-carboxaldehyde diethylthioacetal
4 by
methyl bromoalkanoates provides 5a–b. Basic hydrolysis
of these esters 5a–b gives the desired precursor acids
6a–b. Amidation of these with chrysene amine 2 in
presence of isobutyl chloroformate triethyl amine
affords the corresponding nitrothioacetal intermediates
*Corresponding author. Tel.: +91-40-2719-3157; fax: +91-40-2719-
3189; e-mail: ahmedkamal@iict.ap.nic.in
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00743-1

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3451–3454
Table 1. In vitro one dose primary anticancer assaya chrysene linked
PBD hybridsa
PBD hybrids
Growth percentages
(Lung)
NCI-H460
(Breast)
MCF7
(CNS)
SF-268
.
Scheme 1. (i) SnCl2 2H2O, EtOAc, 3 h, 80%.
9a
9b
105
0
56
1
121
12
7a–b in moderate yields. Further, these upon reduction
by SnCl2.2H2O in EtOAc and followed by deprotection of
aminothioacetal precursors 8a–b using HgCl2/CaCO3
affords the chrysene liked PBD hybrids 9a–b (Scheme 2).11
aOne dose of 9a–b at 10ꢀ4 molar concentration.
mM), one melanoma cancer (UACC-62, 4.07 mM) and
one renal cancer (A498, 6.29 mM) in the 60-cell line
panel. The in vitro cytotoxicity (IC50) for the naturally
occurring DC-8112 is 0.38 and 0.33 mM in L1210 and
PC6 cell lines, respectively.
Compounds 9a–b have been evaluated for the primary
anticancer activity in the standard three-cell line panel
consisting of the MCF7 (breast), NCI-H460 (lung) and
SF-268 (CNS). The results are described in Table 1.
Amongst these hybrids 9b have promising anticancer
activity and further, the PBD hybrid 9b has shown to
possess <10 micro molar potency (at the LC50 level)
against one non-small cell lung cancer (NCI-H226, 4.5
The DNA binding activity for these novel chrysene
linked PBD hybrids has been examined by thermal
denaturation studies using calf thymus (CT) DNA.5
Melting studies show that these compounds stabilize the
Scheme 2. (i) EtSH–BF3OEt2, CH2Cl2, 12 h, rt, 75%; (ii) methyl bromoalkanoate, K2CO3, DMF, 24 h, rt, 89–91%; (iii) 1 N LiOH, THF–MeOH–
.
H2O, 12 h, rt, 74–78%; (iv) isobutyl chloroformate, Et3N, compound 2, 12 h, rt, 59–62%; (v) SnCl2 2H2O, EtOAc, reflux, 3 h, 72–72%; (vi) HgCl2–
CaCO3, CH3CN–H2O, 12 h, rt, 50–52%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3451–3454
Table 2. Thermal denaturation data for chrysene linked PBD hybrids
with CT-DNA
3453
PBD hybrids
[PBD]/[DNA]
molar ratiob
ÁTm (ꢁC)a after incubation
at 37 ꢁC for
0 h
18 h
9a
9b
DC-81
1:5
1:5
1:5
2.6
1.2
0.3
2.8
1.8
0.7
aFor CT-DNA alone at pH 7.00ꢂ0.01, Tm=69.2 ꢁCꢂ0.01 (mean value
from 10 separate determinations), all ÁTm values areꢂ0.1–0.2 ꢁC.
bFor a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concen-
tration=100 mM and ligand concentration=20 mM in aqueous
sodium phosphate buffer [10 mM sodium phosphate+1 mM EDTA,
pH 7.00ꢂ0.01].
Table 3. The values of energy of interactions calculated for the
DNA-PBD hybrid complexes
Complex
Energy of interaction
(Eint) in kcal molꢀ1
DNA–9a
DNA–9b
ꢀ65.6
ꢀ62.4
Figure 1. Projection diagram showing the DNA-9a complex, side-on
view.
Figure 2. Schematic representation of the preferred DNA binding sites of chrysene linked PBD hybrid 9a.
thermal helix-coil or melting stabilization (ÁTm) for the
CT-DNA duplex at pH 7.0, incubated at 37 ꢁC, where
PBD/DNA molar ratio is 1:5. Interestingly, in this
assay, one of the chrysene-linked PBD hybrid (9a) ele-
vates the helix melting temperature of CT-DNA by a
2.8 ꢁC after incubation for 18 h at 37 ꢁC. On the other
hand, the naturally occurring DC-81 exhibits a ÁTm of
0.7 ꢁC. This demonstrates the hybrid 9a has moderate
DNA-binding affinity, as illustrated in Table 2.
that in these compounds as the carbon chain number
reduces from three to two the stability of their DNA
complexes decreases (calculated Eint values of chrysene-
linked PBD hybrids for the two carbon chain from
molecular modeling are 57.4 kcal molꢀ1). From the
above data it appears that there is no clear relationship
between anticancer activity and DNA-binding affinity
for these compounds while membrane interaction as the
primary effect for anticancer activity cannot be ruled
out. This aspect of membrane interaction has been dis-
cussed in detail by Banik et al.7 on the mechanism of
antitumour activity of polyaromatic hydrocarbons.
The difference of DNA binding affinity with respect to
the linker length in this class of hybrids has been inves-
tigated by molecular modeling studies. Energetically
favorable models of the DNA–PBD hybrid complexes
for 9a–b molecules were built using the systematic pro-
cedure as described in our previous report,5 involving
molecular modeling and docking followed by detailed
molecular dynamic stimulations. The DNA sequence
that has been used for this study is 50-CGCA-
GATCTGCG. The energy of interaction (Eint) between
the DNA and the PBD-hybrid molecule in a complex
was calculated as a measure of stability of that complex.
The values of Eint obtained for the DNA–PBD com-
plexes are shown in Table 3. The lowest value of Eint
corresponds to the DNA-9a complex indicating that the
molecule 9a forms energetically the most stable complex
with DNA (Figs 1 and 2). Further, it has been observed
In conclusion, new chrysene-linked PBD hybrids have
been synthesized that exhibit cytotoxic activity in some
cancer cell lines. Some of these hybrids have promising
DNA-binding ability that has been supported by the
molecular modeling studies. The detailed mechanistic
studies of these PBD hybrids is in progress.
Acknowledgements
We thank the National Cancer Institute, Maryland for
the in vitro anticancer assay in human cell lines. We are
also grateful to CSIR, New Delhi for the award of
research fellowships to G.R., P.R. and O.S.

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3451–3454
Peverley, A. D.; Robinson, R. G.; Corbett, T. H.; Jones, J. L.;
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