Design, synthesis, and evaluation of mixed imine-amine pyrrolobenzodiazepine dimers with efficient DNA binding affinity and potent cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2004.07.045

Synthesis of mixed imine-amine pyrrolobenzodiazepine (PBD) dimers that are comprised of DC-81 and secondary amine (N10) of DC-81 subunits tethered to their C8 positions through alkanedioxy linkers (comprised of three and five carbons) is described. These

Full text of DOI:10.1016/j.bmc.2004.07.045

Bioorganic& Medicinal Chemistry 12 (2004) 5427–5436
Design, synthesis, and evaluation of mixed imine–amine
pyrrolobenzodiazepine dimers with efficient DNA binding
affinity and potent cytotoxicity
Ahmed Kamal,a,* G. Ramesh,a O. Srinivas,a P. Ramulu,a N. Laxman,a Tasneem Rehana,a
M. Deepak,b M. S. Acharyb and H. A. Nagarajaramb
aBiotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bLaboratory of Computational Biology and Bioinformatics Facility, Centre for DNA Fingerprinting and Diagnostics,
Hyderabad 500 076, India
Received 24 June 2004; revised 21 July 2004; accepted 21 July 2004
Available online 20 August 2004
Abstract—Synthesis of mixed imine–amine pyrrolobenzodiazepine (PBD) dimers that are comprised of DC-81 and secondary amine
(N10) of DC-81 subunits tethered to their C8 positions through alkanedioxy linkers (comprised of three and five carbons) is
described. These noncross-linking unsymmetrical molecules exhibit significant DNA minor groove binding ability and one of them
5b linked through the pentanedioxy chain exhibits efficient DNA binding ability (DTm = 11.0ꢀC) when compared to naturally occur-
ring DC-81, 1 (DTm = 0.7ꢀC). The imine–amine PBD dimers exhibit promising in vitro antitumor activity in a number of human
cancer cell lines.
ꢁ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
moiety to the C2-amino position of a guanine residue
within the minor groove of DNA.6 Molecular modeling,
solution NMR, fluorimetry, and DNA foot printing
experiments have shown that these molecules have a
preferred selectivity for Pu-G-Pu sequences7,8 and can
be oriented with their A-rings pointed either toward
the 30- or 50 end of the covalently bonded DNA strand
as in the case of naturally occurring anthramycin, to-
maymycin, and DC-81 (1). The reduction of the imine
functionality at N10–C11 of the DC-81 (1) provides
the secondary amine form of DC-81 (2), which loses
the covalent binding capability and could exhibit nonco-
valent interactions alone. The PBDs have been used to
attach ethylenediaminetetraacetic acid (EDTA),9 epox-
ide,10 polyamide,11 oligopyrrole,12 and cyclopropyl-
pyrroloindole (CPI)13 moieties leading to novel hybrids
of PBD, which have exhibited sequence selective
DNA-cleaving and cross-linking properties. Further-
more, there have been attempts to develop more potent
compounds that could enhance the sequence selectivity
as in case of C8 diether-linked PBD dimers DSB-120
(3),14 SJG-136,15 DRG-16,16 and C2–C3/C20–C30 endo
unsaturated PBD dimer.17 It has been observed that
there has been extensive improvement in the biological
activity due to the cross-linking property by the presence
Recently, the design and development of DNA interac-
tive ligands that are capable of binding to DNA in a
sequence selective manner1–3 has received considerable
attention. Inspite of rational design of syntheticDNA
intercalators, there are few such compounds, which
exhibit sequence selectivity that are in current clinical
usage. These compounds with the ability to target and
then down regulate individual genes have potential use
as drugs for therapy of geneticbased diseases including
some cancers,4 and as research tools for using functional
genomicstudies.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
well known class of sequence selective DNA binding
antitumor antibiotics derived from Streptomyces spe-
cies.5 They exert their biological activity through cova-
lent binding via their N10–C11 imine/carbinolamine
Keywords: Pyrrolobenzodiazepines; DNA binding affinity; Cytotoxi-
city.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.045

5428
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
of two imine functionalities. We have been involved in
10
N
B
5
H
N
9
11a
11
HO
8
7
the design and synthesis of PBD dimers with one imine
functionality alone for exploring their DNA binding
ability and cytotoxicity as noncross-linking agents and
have also been interested in the design and synthesis of
new PBD hybrids.18–27 In continuation of these efforts,
it was considered significant to synthesize and evaluate
mixed dimers of PBD that contain an imino functional-
ity in one of the PBD rings and a secondary amine (N10)
group in the other, which are linked at the C8 position
by a suitable alkane spacer. Therefore, we herein report
the synthesis and biological evaluation and molecular
modeling studies of mixed imine–amine PBD dimers
(Fig. 1).
HO
H
H
1
A
C
N
N
H3CO
H3CO
2
6
4
3
O
1
O
2
N
O
N
H
O
H
N
OCH3
N
H3CO
O
O
3
H
N
O
O
N
H
O
H
H
(CH2)5
2. Results and discussion
2.1. Synthesis
N
N
OCH3
N
N
H3CO
O
O
4
Synthesis of the mixed imine–amine PBD dimers has
been carried out employing methyl vanillate10 followed
by benzylation, nitration, and ester hydrolysis to
H
N
O
N
H
O
provide 4-benzyloxy-5-methoxy-2-nitrobenzoicaicd
9.
(CH2)n
L-Proline methyl ester hydrochloride has been coupled
to compound 9 to afford compound 10, which upon
reduction followed by oxidation gives compound 12.
While compound 2 has been prepared from compound
12 by hydrogenation over Pd/C at 275.76 · 103 Pa
(Scheme 1). The other precursors, (2S)-N-[n-(bromo-
alkyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carb-
oxaldehyde diethyl thioacetals 13a–b have been
prepared by our previous method.19 The key intermedi-
ates 14a–b have been prepared by linking 2 and 13a–b,
OCH3
H3CO
O
O
5a n = 3
5b n = 5
Figure 1. Structures of the PBD monomer DC-81 (1), PBD secondary
amine (2), and PBD dimers, DSB-120 (3), mixed imine–amide PBD
dimer (4) and 5.
BnO
HO
BnO
NO2
OCH3
ii
i
OCH3
OCH3
H3CO
H3CO
H3CO
O
O
O
7
8
6
iii
NO2
NO2
BnO
NO2
BnO
H3CO
BnO
H3CO
CH2OH
COOMe
v
iv
OH
N
N
H3CO
O
O
O
11
9
10
vi
H
N
NO2
HO
BnO
H3CO
H
CHO
vii
N
N
H3CO
O
O
2
12
Scheme 1. Reagents and conditions: (i) C6H5CH2Br, K2CO3, CH3COCH3, reflux, 12h, 92%; (ii) HNO3–SnCl4, CH2Cl2, À78%; (iii) 1N LiOH, THF–
H2O–MeOH (3:1:1), room temperature, 12h, 83%; (iv) SOCl2, C6H6, L-proline methylester hydrochloride, Et3N, H2O, THF, 0ꢀC, 1h, 85%;
(v) LiBH4, THF, 0ꢀC, 2.5h, 85%; (vi) (COCl)2/DMSO, TEA, CH2Cl2, À70ꢀC, 90%; (vii) Pd/C, H2, MeOH, room temperature, 8h, 62%.

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
5429
Table 1. Thermal denaturation data for DC-81 (1), DSB-120 (3),
mixed imine–amide PBD dimer (4), and compounds 5a–b with calf
thymus DNA
NO2
Br(CH2)nO
H3CO
CH(SEt)2
N
PBD
compound
[PBD]:[DNA]
molar ratiob
Induced DTm (ꢀC)a after
incubation at 37ꢀC for
O
13a-b
0h
18h
5a
5b
4
1:5
1:5
1:5
1:5
1:5
8.7
9.7
11.0
17.0
15.4
0.7
i
10.6
14.0
10.2
0.3
3
H
N
1
(CH2)n
NO2
N
O
O
H
CH(SEt)2
a For CT-DNA alone at pH7.00 0.01, Tm = 69.2ꢀC 0.01 (mean
value from 30 separate determinations), all DTm values are 0.1–
0.2ꢀC.
N
OCH3
H3CO
O
O
b For a 1:5 molar ratio of [ligand]/[DNA], where CT-DNA con-
centration = 100lM and ligand concentration = 20lM in aqueous
sodium phosphate buffer [10mM sodium phosphate + 1mM EDTA,
pH7.00 0.01].
14a-b
ii
H
N
(CH2)n
NH2
O
noncovalent interactions. However, mixed imine–amide
PBD dimer, 4 (five carbon chain linker) gives a DTm of
17.0ꢀC. It is interesting to note that the change of PBD
dilactam (amide) subunit (4) to PBD secondary amine
subunit in compound (2) slightly lowers the DTm value.
These results further exhibit that there is a decrease in
the DTm value for these mixed imine–amine PBD dimers
(5a, 5b) in comparison to the other noncross-linking
mixed imine–amide PBD dimers (4) and this could be
explained due to the absence of the carbonyl group in
the noncovalent subunit part (2). Therefore, this investi-
gation shows the importance of the carbonyl group for
noncovalent interaction. As generally observed for the
PBD dimers, compounds 5a–b exert most of the effect
upon the GC rich or high temperature regions of the
DNA melting curves.
O
H
CH(SEt)2
N
N
OCH3
15a-b
H3CO
O
O
iii
H
N
(CH2)n
N
O
O
H
H
N
N
OCH3
H3CO
O
O
5a-b
n = 3 and 5
Scheme 2. Reagents and conditions: (i) compound 2, K2CO3,
CH3COCH3, reflux, 70–72%; (ii) SnCl2Æ2H2O, MeOH, reflux, 78–
80%; (iii) HgCl2, CaCO3, CH3CN–H2O (4:1), 50–51%.
2.3. Cytotoxicity
Compounds 5a–b have been evaluated in vitro against
60 human tumor cell lines derived from 9 cancer types
(leukemia, nonsmall cell lung cancer, colon cancer, cen-
tral nervous system cancer, melanoma, ovarian cancer,
renal cancer, prostate cancer, and breast cancer). For
each compound, dose response curves for each cell line
have been measured at a minimum of five concentra-
tions at 10-fold dilutions. A protocol of 48h continuous
drug exposure is used, and a sulforhodamine B (SRB)
protein assay has been used to estimate cell viability or
growth. The concentration causing 50% cell growth
inhibition (GI50), total cell growth inhibition (TGI, 0%
growth), and 50% cell death (LC50, À50% growth) as
compared with the control has been calculated. The
mean graph midpoint (MG MID) values of log TGI
and log LC50 as well as log GI50 for 5a–b are listed in
Table 2. As demonstrated by mean graph pattern (Fig.
2), compound 5a exhibits an interesting profile of activ-
ity and selectivity for various cell lines. The MG MID of
log TGI and log LC50 showed similar pattern to the log
GI50 MG MID.
there upon reduction gives 15a–b. Finally, the deprotec-
tion of thioacetal functionality affords the desired mixed
imine–amine PBD dimers 5a–b, respectively (Scheme 2).
2.2. DNA interactions: thermal denaturation studies
The DNA binding affinity of the novel noncross-linking
imine–amine PBD dimers (5a–b) has been investigated
by thermal denaturation using calf thymus (CT)-
DNA.28 The studies for these compounds (5a–b) have
been carried out at DNA/PBD molar ratios of 5:1.
The increase in melting temperature (DTm) for each
compound is examined at 0 and 18h incubation at
37ꢀC (Table 1). It is observed from the data that com-
pound 5b is an efficient stabilizing agent for double-
stranded CT-DNA. This compound elevates the helix
melting temperature of the CT-DNA by 11.0ꢀC after
incubation at 37ꢀC for 18h. In the same experiment
the naturally occurring DC-81 1 gives a DTm of 0.7ꢀC
and whereas syntheticDC-81 dimer 3 (DSB-120) gives
a DTm of 15.4ꢀC. These results clearly demonstrate that
compound 5b containing a single imino functionality
has a significant DNA binding affinity mainly due to
The GI50 values of compound 5a (Table 3) against
leukemia cancer CCRF-CEM, SR, and MOLT-4 cell

5430
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
Table 2. Log GI50, log TGI, and log LC50 MG MID of in vitro
cytotoxicity data for compounds 5a–b against human tumor cell linesa
lines are <0.01, <0.01, and 0.01lM, respectively. In the
nonsmall cell lung cancer panel, the growth of NCI-H
522 cell line is affected by compound 5a with GI50 value
as <0.01lM. Compound 5a exhibits a cytotoxic potency
in CNS cancer panel, in which SNB-75 and U251 cell
lines are affected, with GI50 values of 0.02lM. Com-
pound 5b (Table 3) exhibited cytotoxic potency against
leukemia cancer cell lines CCRF-CEM and SF, with
GI50 values of 0.2 and 0.3lM, respectively, and it also
exhibited cytotoxicity against renal cancer SN12C cell
line, with GI50 value of 0.5lM.
Compound
Log GI50
Log TGI
Log LC50
5a
5b
À6.80
À5.29
À5.73
À4.55
À4.48
À4.15
a GI50, drug molar concentration causing 50% cell growth inhibition;
TGI, drug concentration causing total cell growth inhibition (0%
growth); LC50: drug concentration causing 50% cell death (À50%);
MG MID, mean graph midpoints, the average sensitivity of all cell
lines toward the test agent.
Figure 2. Log10 GI50 data from the NCI 60 cell line screen for PBD compound 5a.

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
Table 3. In vitro cytotoxicity of compound 5a and 5b in selected
human cancer cell lines
5431
Cancer panel/cell line
GI50 (lM)
5a
5b
Leukemia
CCRF-CEM
SR
<0.01
<0.01
0.01
0.2
0.3
––
MOLT-4
Nonsmall cell lung
NCI-H22
<0.01
––
––
Central nervous system
SNB-75
U251
0.02
0.02
Renal
SN12C
0.9
0.5
The comparison of the data in Table 3 reveals the
importance of alkane spacer. Surprisingly, as the alkane
spacer increases from three to five the cytotoxic activity
moderately decreases and therefore, the DNA binding
ability of these compounds could not be correlated to
the cytotoxicity.
2.4. Molecular modeling studies
Modeling of the complexes of PBD dimers 1, 4, 5a, and
5b with the B-DNA duplex structure has been carried
out as described in the methods section. A B-DNA du-
plex structure has been considered with a sequence 50-
GGGGAGAGAGAGGGG-30––a symmetricsequenec
about the central triplet AGA, which is the most pre-
ferred site for PBD binding.5 Each of the DNA–PBD
complexes has been subjected to molecular dynamics
(MD) simulations followed by energy minimization of
ꢀsnap shotsꢁ collected at regular time intervals during
the MD simulations. After energy minimization all the
energy minima obtained have been compared with each
other and the one with the lowest energy has been
picked as the representative of the DNA–PBD complex
(Fig. 3). The least energy complex thus picked up has
been selected to calculate the interaction energy as a
measure of stability of complex.
The interaction energies between DNA and PBDs are gi-
ven in Table 4. It can be seen from this table that the
imine–amide PBD dimer 4 renders more stability to
the complex compared to the imine–amine PBD dimers
(5a and 5b), and the PBD monomer 1. This property
correlates with the experimentally determined values
of the DNA melting temperature in these complexes
(Fig. 4).
The complexes are characterized by the presence of a
number of nonbonded interactions (Table 4) formed be-
tween DNA and PBD molecules in addition to the cova-
lent linkage formed between the imine PBD subunit and
exocyclic C2-amino group of the guanine (G8). The di-
mericPBDs ( 4, 5a, and 5b) offer more favorable non-
bonded interactions as compared to the monomeric
Figure 3. Projection diagram showing the DNA-5b complex; (a) side-
on view; (b) down the helix axis.
DC-81 (1) and this is evident from their respective non-
bonded energy terms (Table 4), which are about

5432
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
Table 4. The values of energy of interactions calculated for the DNA–PBD dimer complexes
PBD
compound
Total interaction
energy (kcalmolÀ1
Bonded interaction
energy (kcalmolÀ1
)
Nonbonded interaction energy (kcalmolÀ1
)
)
van der Waals interaction
energy (kcalmolÀ1
Coulombicinteraction
energy (kcalmolÀ1
)
)
1
À85.2
À145.9
À132.1
À143.6
À14.2
À12.2
À12.6
À14.8
À33.7
À84.3
À71.8
À68.7
À37.3
À49.4
À47.7
À60.1
4
5a
5b
10
15
interaction whereas in the noncross-linking molecules
nonbonded interactions play an important role in stabi-
lization of their respective complexes.
9
11 11a
9a
N
HO
8
H
H134C
1
5
N
6a
O
7
2
6
4
3
13
O
3. Conclusions
12
1
The DNA binding ability for these noncross-linking
PBD dimers is noticeable inspite of the introduction of
amine functionality in one of the PBD rings in compar-
ison to DC-81. Modeling studies suggest that apart from
the covalent linkage formed between the imine PBD
subunit and G8, there are a number of favorable van
der Waals and coulombic interactions that are formed
between the DNA and the mixed imine–amine PBD
dimer. The complex formed by compound 5b (with a
five carbon chain linker) is energetically more stable
than the complex formed by 5a as the extra alkane
spacer units offer the molecule to make extra favorable
van der Waals and coulombic interactions with DNA.
However, 5b exhibits lower DTm value in comparison
to 4 because of the absence of carbonyl functionality
in the complex. Of all the molecules imine–amide PBD
dimer 4 forms the most stable complex. Nevertheless,
the imine–amine PBD dimers also exhibit interesting
profile of DNA binding ability in contrast to PBD
monomer (DC-81). This investigation exhibits the role
played by noncovalent interaction of PBD secondary
amine subunit. In addition these compounds show
promising in vitro cytotoxicity in different cancer cell
lines.
25
10'
H
10
O
11a'
H
21
8' O
1O5
17
9'
6'
19
9
11 11a
N
9a N
9a'
6a'
8
7
H
11'
20
16
18
1
1'
5
5'
O22
13O
6a
N
N
4
2
2'
7'
6
4'
3
3'
CH3
CH3
O
O
23
12
24
14
4
10'
10
9a N
H
19
1O5
11a'
9'
H 11'
17
9
11 11a
N
9a'
6a'
8' O
8
H
18
16
13 O
1'
2'
1
5'
5
O20
N
N
6a
7
2
7'
6'
6
4
3
4'
3'
CH3
CH3
O
O
21
22
12
14
5a
10'
10
9a N
H
21
8' O
1O5
11a'
17
9'
19
9
H11'
11 11a
N
9a'
6a'
8
H
20
16
18
1
1'
2'
5
5'
O22
13O
N
N
6a
7
2
7'
6
4
3
6'
4'
3'
CH3
CH3
O
O
23
12
24
14
5b
4. Experimental section
4.1. Synthetic chemistry
Figure 4. Numbering of PBD ring systems.
50kcalmolÀ1 less as compared to DC-81. Among the di-
mericPBDs the imine–amide PBD dimer 4 is associated
with the lowest nonbonded energy term and is stabilized
by the hydrogen bonding interaction between the carb-
onyl functionality and the amino group of G12. This
hydrogen bonding is precluded in 5a and 5b because
of the absence of the required carbonyl functionality
(see Table 5). Between 5a and 5b, the latter is associated
with better nonbonded as well as bonded energy compo-
nents and these together render higher stability for 5b by
about 11kcalmolÀ1 as compared to 5a.
Reaction progress was monitored by thin-layer chroma-
tography (TLC) using GF254 silica gel with fluorescent
indicator on glass plates. Visualization was achieved
with UV light and iodine vapor unless otherwise stated.
Chromatography was performed using Acme silica gel
(100–200mesh). The majority of reaction solvents were
purified by distillation under nitrogen from the indicated
drying agent and used fresh: dichloromethane (calcium
hydride), tetrahydrofuran (sodium benzophenone
ketyl), methanol (magnesium methoxide), acetonitrile
(calcium hydride).
The modeling of the complex of DNA with the cross-
linking PBD dimer 3 has not been considered in the
present study. In this the stability of the complex is pre-
dominantly due to the presence of additional covalent
1H NMR spectra were recorded on Varian Gemini 200/
300MHz spectrometer using tetramethyl silane (TMS)
as an internal standard. Chemical shifts are reported
in parts per million (ppm) down field from tetramethyl-

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
5433
Table 5. List of hydrophobic contacts and hydrogen bonded interactions (Fig. 4)
DC-81
4
5a
5b
Hydrophobic interactions
G8.C20Á Á ÁC1
A9.C20Á Á ÁC1
G10.C20Á Á ÁC6
C8.C20Á Á ÁC9
T9.C20Á Á ÁC6a
C10.C20Á Á ÁC2
G8.C20Á Á ÁC1
A9.C20Á Á ÁC1
G10.C20Á Á ÁC6
A11.C20Á Á ÁC19
G12.C20Á Á ÁC90
G13.C20Á Á ÁC10
C6.C20Á Á ÁC60a
T7.C20Á Á ÁC90
C8.C20Á Á ÁC9
T9.C20Á Á ÁC9
C10.C20Á Á ÁC2
G8.C20Á Á ÁC1
A9.C20Á Á ÁC1
G10.C20Á Á ÁC9
A11.C20Á Á ÁC90
G12.C20Á Á ÁC60a
G13.C20Á Á ÁC10
C6.C20Á Á ÁC10
T7.C20Á Á ÁC60a
C8.C20Á Á ÁC9
T9.C20Á Á ÁC6a
C10.C20Á Á ÁC2
G8.C20Á Á ÁC1
A9.C20Á Á ÁC1
G10.C20Á Á ÁC9
A11.C20Á Á ÁC18
G12.C20Á Á ÁC90
G13.C20Á Á ÁC10
C6.C20Á Á ÁC10
T7.C20Á Á ÁC90
C8.C20Á Á ÁC9
T9.C20Á Á ÁC6a
C10.C20Á Á ÁC2
Hydrogen bonded interactionsa
C8.O2Á Á ÁN10
C8.O2Á Á ÁN10
G12.N2Á Á ÁO25
C8.O2Á Á ÁN10
C8.O2Á Á ÁN10
a Imine N of PBD in all the complexes (1, 4, 5a, and 5b) is at a favorable distance (ꢀ3.3) to imino group of A9 of DNA however the angle is not
within hydrogen bonding limits (N–HÁ Á ÁN ꢀ 77ꢀ).
silane. Spin multiplicities are described as s (singlet), brs
(broad singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). Coupling constants are reported in Hertz
(Hz). Low resolution mass spectra were recorded on
VG-7070H Micromass mass spectrometer at 200ꢀC,
70eV with trap current of 200lA and 4kV acceleration
voltage. HRMS were recorded on VG Autospec N mass
spectrometer at 200ꢀC, 70eV with a trap current of
200lA and 7kV acceleration voltage.
3H), 5.20 (s, 2H), 7.05 (s, 1H), 7.20–7.45 (m, 5H), 7.50
(s, 1H); MS (EI) m/z 317 [M]+Å.
4.1.3. 4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (9).
Lithium hydroxide monohydrate (2N, 1.22mL) was
added to a solution of methyl 4-benzyloxy-5-methoxy-
2-nitrobenzoate 8 (317mg, 1mmol) in THF–H2O–
MeOH (4:1:1) and the mixture was stirred at room
temperature for 12h. After most of the THF and meth-
anol were evaporated, the aqueous phase was acidified
with 12N HCl to pH7 and re-extracted with CH2Cl2
4.1.1. Methyl 4-benzyloxy-3-methoxybenzoate (7). To a
solution of vanillin methyl ester 6 (182mg, 1mmol) in
acetone (30mL) was added, anhydrous K2CO3
(553mg, 4mmol) and benzyl bromide (256mg,
1.5mmol), the mixture was refluxed in an oil bath for
24h. The reaction was monitored by TLC using EtO-
Ac–hexane (2:8) and K2CO3 was removed by filtration
and the solvent was evaporated under the vacuum and
was purified by column chromatography (10% EtOAc–
hexane) to afford compound 7 as white solid (250mg,
92%); mp 117–118ꢀC. 1H NMR (CDCl3) d 3.94 (s,
3H), 3.98 (s, 3H), 5.20 (s, 2H), 6.88 (d, 1H,
J = 8.2Hz), 7.20–7.50 (m, 5H), 7.50–7.60 (m, 2H); MS
(EI) m/z 272 [M]+Å.
to give a 4-benzyloxy-5-methoxy-2-nitrobenzoicacid
9
as a pale yellow solid (251mg, 83%); mp 180–183ꢀC.
1H NMR (CDCl3) d 3.98 (s, 3H), 5.20 (s, 2H), 7.20 (s,
1H), 7.35–7.55 (m, 6H); MS (EI) m/z 303 [M]+Å.
4.1.4. Methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxylate (10). To a stirred sus-
pension of compound 9 (303mg, 1mmol) and thionyl
chloride (476mg, 4.0mmol) in dry benzene (15mL)
was added DMF (four to five drops) and the stirring
was continued for 6h. The benzene was evaporated in
vacuum and the resultant oil dissolved in dry THF
(20mL) and added dropwise over a period of 30min
to a stirred suspension of L-proline methyl ester hydro-
chloride (247mg 1.5mmol), triethylamine (303mg,
3mmol), and ice water (20mL) cooled in an ice bath.
After the completion of addition, the reaction mixture
was brought to ambient temperature and stirred for an
additional hour. The THF was evaporated in vacuum
and the aqueous layer was washed with ethyl acetate
(10mL). The aqueous phase was then adjusted to pH3
using 6N HCl and extracted with ethyl acetate and
washed with brine, dried over Na2 SO4, evaporated in
vacuum, and was purified by column chromatography
(30% EtOAc–hexane) to afford compound 10 as yellow
4.1.2. Methyl 4-benzyloxy-5-methoxy-2-nitrobenzoate
(8). A freshly prepared mixture of stannichcloride
(301mg, 1.156mmol) and fuming nitricaicd (98mg,
1.56mmol) in dichloromethane was added dropwise
over 5min with stirring to a solution of methyl-4-benzyl-
oxy-3-methoxybenzoate 7 (272mg, 1mmol) in dichloro-
methane (30mL) at À78ꢀC (dry ice/acetone). The
mixture was maintained at À78ꢀC for a further 5min,
quenched with water (20mL), and then allowed to
return to room temperature. The organiclayer was
separated and the aqueous layer was extracted with
dichloromethane (2 · 20mL). The combined organic
phase dried (Na2SO4), evaporated in vacuum, and it
was purified by column chromatography (20% EtOAc–
hexane) to give 8 as a yellow solid (247mg, 78%); mp
1
oil (352mg, 85%). H NMR (CDCl3) d 1.85–2.12 (m,
3H), 2.20–2.45 (m, 1H), 3.10–3.25 (m, 1H), 3.26–3.40
(m, 1H), 3.80 (s, 3H), 3.92 (s, 3H), 4.68–4.75 (m, 1H),
5.20 (s, 2H), 6.82 (s, 1H), 7.30–7.48 (m, 5H), 7.72 (s,
1H); MS (EI) m/z 414 [M]+Å.
1
128–132ꢀC. H NMR (CDCl3) d 3.89 (s, 3H), 3.95 (s,

5434
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
4.1.5. (2S)-N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-2-
(hydroxymethyl)pyrrolidine (11). A solution of ester 10
(414mg, 1mmol) in THF (20mL) was cooled to 0ꢀC
and treated portion-wise with LiBH4 (33mg, 1.5mmol).
The stirred reaction mixture was allowed to warm to
room temperature over 2.5h under an N2 atmosphere,
after which TLC revealed the complete consumption
of starting material. The mixture was cooled to 0ꢀC
and carefully treated with water (18mL) and then 2N
HCl (5mL). After concentration in vacuum, the mixture
was adjusted to pH7 with 10N NaOH saturated with
solid NaCl and then extracted with EtOAc (50mL).
The combined organic phase was washed with brine
(20mL), dried (Na2SO4), filtered, and evaporated in vac-
uum to furnish the pure alcohol 11 as oil (328mg, 85%).
1H NMR (CDCl3) d 1.65–2.20 (m, 4H), 2.80–3.20 (brs,
OH, exchangeable) 3.18–3.28 (m, 2H), 3.65–3.90 (m,
2H), 4.0 (s, 3H), 4.20–4.40 (m, 1H), 5.20 (s, 2H), 7.0
(s, 1H), 7.28–7.50 (m, 5H), 7.75 (s, 1H); MS (EI) m/z
355 [M À CH2OH]+Å.
(2S)-N-[4-(3-bromo-propoxy)-5-methoxy-2-nitrobenzo-
yl]pyrrolidine-2-carbaldehyde diethyl thioacetal 13a
(521mg, 1mmol) in dry acetone (30mL) was added
anhydrous K2CO3 (553mg, 4mmol) and the (11aS)-8-
hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyr-
rolo-[2,1-c][1,4]benzodiazepine-5-one 2 (248mg, 1mmol).
The reaction mixture was refluxed in an oil bath for 48h.
The reaction was monitored by TLC using EtOAc–hex-
ane (9:1) as a solvent system. K2CO3 was removed by fil-
tration and the solvent was removed under vacuum. The
crude product was purified by column chromatography
(90% EtOAc–hexane) to afford an yellow oil 14a
1
(482mg, 70%). H NMR (CDCl3) d 1.35–1.45 (m, 6H),
1.70–2.45 (m, 10H), 2.72–2.90 (m, 4H), 3.20–3.28 (m,
4H), 3.50–3.58 (m, 1H), 3.62–3.75 (m, 1H), 3.80–3.90
(m, 4H), 3.92–3.98 (m, 3H), 4.20 (t, 2H, J = 6.0Hz),
4.35 (t, 2H, J = 6.1Hz), 4.65–4.75 (m, 1H), 4.85 (d,
1H, J = 4.28Hz), 6.08 (s, 1H), 6.80 (s, 1H), 7.58 (s,
1H), 7.72 (s, 1H); MS (FAB) 689 [M + H]+Å.
4.1.9. (2S)-N-{4-[5-(7-Methoxy-(11aS)-1,2,3,10,11,11a-
hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one-8-
yloxy)pentyloxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-
2-carbaldehyde diethyl thioacetal (14b). The compound
14b was prepared according to the method described
for 14a by employing the (11aS)-8-hydroxy-7-methoxy-
1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzo-
diazepine-5-one 2 and 13b (549mg, 1mmol) to afford
4.1.6. (2S)-N-[4-Benzyloxy-5-methoxy-2-nitrobenzoyl]-
pyrrolidine-2-carbaldehyde (12). A solution of DMSO
(0.60mL, 8.4mmol) in CH2Cl2 (10mL) was added drop-
wise to a cooled solution of oxalyl chloride (0.46mL,
5.33mmol) at À60ꢀC (dry ice/acetone) under an N2
atmosphere. After the mixture was stirred at À70ꢀC
for 45min and then the alcohol 11 (1.15g, 3mmol) dis-
solved in 25mL of dry CH2Cl2 was added dropwise at
À60ꢀC. The reaction mixture was allowed to stir for
1.5h at À70ꢀC. Then a solution of TEA (1.67mL,
12mmol) in 15mL of dry CH2Cl2 was added dropwise
and the mixture was allowed to warm room tempera-
ture. The reaction mixture was diluted with water
(50mL), extracted with 1N HCl (25mL), saturated
aqueous NaHCO3 (25mL), and then brine (25mL).
1
14b as yellow oil (516mg, 72%). H NMR (CDCl3) d
1.30–1.40 (m, 6H), 1.65–2.35 (m, 14H), 2.65–2.75 (m,
4H), 3.18–3.32 (m, 3H), 3.45–3.75 (m, 2H), 3.80–3.85
(m, 4H), 3.85–4.0 (m, 5H), 4.65–4.72 (m, 1H), 4.85 (d,
1H, J = 4.38Hz), 6.0 (s, 1H), 6.78 (s, 1H), 7.52 (s, 1H),
7.65 (s, 1H); MS (FAB) 717 [M + H]+Å.
4.1.10. (2S)-N-{4-[3-(7-Methoxy-(11aS)-1,2,3,10,11,11a-
hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one-8-
yloxy)propoxy]-5-methoxy-2-aminobenzoyl}pyrrolidine-
2-carbaldehyde diethyl thioacetal (15a). The compounds
14a (688mg 1mmol) dissolved in methanol (20mL)
and added SnCl2Æ2H2O (1.125g, 5mmol) was refluxed
for 1.5h or until the TLC indicated that reaction was
complete. The methanol was evaporated by vacuum
and the aqueous layer was then carefully adjusted to
pH8 with 10% NaHCO3 solution and then extracted
with ethyl acetate (2 · 30mL). The combined organic
phase was dried over Na2SO4 and evaporated under
vacuum to afford the amino diethyl thioacetal 15a as a
yellow oil, which due to potential stability problems,29
was briefly characterized by 1H NMR and then used di-
rectly in the next step (514mg, 78%). 1H NMR (CDCl3)
d 1.20–1.42 (m, 6H), 1.60–2.42 (m, 10H), 2.60–2.85 (m,
4H), 3.18–3.28 (m, 2H), 3.40–3.75 (m, 5H), 3.78 (s,
3H), 3.85 (s, 3H), 4.05–4.25 (m, 4H), 4.60–4.70 (m,
2H), 6.05 (s, 1H), 6.25 (s, 1H), 6.80 (s, 1H), 7.52 (s, 1H).
The organicsolution was dried over Na SO4 and evap-
2
orated under vacuum. The residue was purified by col-
umn chromatography (30% EtoAc–hexane) to afford
compound 12 as yellow oil (345mg, 90%). H NMR
1
(CDCl3) d 1.88–2.40 (m, 5H), 3.18–3.38 (m, 2H), 4.02
(s, 3H), 4.70 (m, 1H), 5.22 (s, 2H), 6.86 (s, 1H), 7.25–
7.50 (m, 5H), 7.75 (s, 1H), 9.25 (s, 1H), 9.80 (s, 1H);
MS (EI) m/z 355 [M À CHO]+Å.
4.1.7. (11aS)-8-Hydroxy-7-methoxy-1,2,3,10,11,11a-hexa-
hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(2).
The compound 12 (384mg, 1.0mmol) was dissolved in
methanol (10mL) and 10%Pd–C (200mg) was added.
The mixture was hydrogenated at room temperature
under atmosphericpressure for 10h. The catalyst was
removed by filtration through Celite, then the solvent
was evaporated under vacuum and purified by column
chromatography (80% EtOAc–hexane) to afford 2 as plu-
ffy solid (154mg, 62%); mp 278–280ꢀC. 1H NMR
(CDCl3) d 1.70–2.35 (m, 4H), 3.19–3.29 (m, 1H), 3.45–
3.55 (m, 1H), 3.55–3.75 (m, 2H), 3.75–3.85 (m, 1H), 3.85
(s, 3H), 6.08 (s, 1H), 7.50 (s, 1H); MS (EI) m/z 248 [M]+Å.
4.1.11. (2S)-N-{4-[5-(7-Methoxy-(11aS)-1,2,3,10,11,11a-
hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one-8-
yloxy)pentyloxy]-5-methoxy-2-aminobenzoyl}pyrrolidine-
2-carbaldehyde diethyl thioacetal (15b). The compound
15b was prepared according to the method described
for the compound 15a employing the compound 14a
(716mg, 1mmol) to afford the amino diethyl thioacetal
4.1.8. (2S)-N-{4-[3-(7-Methoxy-(11aS)-1,2,3,10,11,11a-
hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one-8-
yloxy)propoxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-
carbaldehyde diethyl thioacetal (14a). To a solution of

A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
5435
15b as a yellow liquid (550mg, 80%). 1H NMR (CDCl3)
d 1.20–1.40 (m, 6H), 1.50–2.38 (m, 14H), 2.60–2.80 (m,
4H), 3.10–3.25 (m, 2H), 3.40–3.68 (m, 5H), 3.68–4.0
(m, 10H), 4.58–4.65 (m, 2H), 4.65–5.10 (brs, 2H), 6.0
(s, 1H), 6.20 (s, 1H), 6.78 (s, 1H), 7.48 (s, 1H).
method.28 Working solutions in aqueous buffer
(10mM NaH2PO4/Na2HPO4, 1mM Na2EDTA,
pH7.00 + 0.01) containing CT-DNA (100lm in phos-
phate) and the PBD (20lm) were prepared by addition
of concentrated PBD solutions in MeOH to obtain a
fixed [PBD]/[DNA] molar ratio of 1:5. The DNA–PBD
solutions were incubated at 37ꢀC for 0, 18h prior to
analysis. Samples were monitored at 260nm using a
Beckman DU-7400 spectrophotometer fitted with high
performance temperature controller, and heating was
applied at 1ꢀCminÀ1 in the 40–90ꢀC range. DNA
helix!coil transition temperatures (Tm) were obtained
from the maxima in the d(A260)/dT derivative plots.
Results are given as the mean standard deviation from
three determinations and are corrected for the effects of
MeOH co-solvent using a linear correction term.30
Drug-induced alterations in DNA melting behavior
are given by: DTm = Tm (DNA + PBD) À Tm (DNA
alone), where the Tm value for the PBD-free CT-DNA
is 69.2 0.01ꢀC. The fixed [PBD]/[DNA] ratio used
did not result in binding saturation of the host DNA
duplex for any compound examined.
4.1.12. 7-Methoxy-8-{3-[7-methoxy-(11aS)-1,2,3,10,11,
11a-hexahydro-5H-pyrrolo[2,1-c]-[1,4]benzodiazepine-5-
one-8-yloxy]propoxy}-(11aS)-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5a). A solution of
15a (658mg, 1mmol), HgCl2 (613.6mg, 2.26mmol) and
CaCO3 (246mg, 2.46mmol) in MeCN–water (4:1) was
stirred slowly at room temperature until TLC indicates
complete loss of starting material (12h). The reaction
mixture was diluted with EtOAc(30mL) and filtered
through a Celite bed. The clear yellow organic superna-
tant was extracted with saturated 5% NaHCO3 (20mL),
brine (20mL) and the combined organic phase is dried
(Na2SO4). The organiclayer was evaporated in vacuum
and purified by column chromatography (90% MeOH–
EtOAc) to give compound 5a as pale yellow oil
(267mg, 50%). This material was repeatedly evaporated
1
from CHCl3 in vacuum to generate the imine form. H
4.3. Molecular modeling studies
NMR (CDCl3) d 1.65–2.45 (m, 10H), 3.15–3.25 (m,
2H), 3.48–3.75 (m, 4H), 3.78–3.88 (m, 4H), 3.90 (s,
3H), 4.25–4.35 (m, 5H), 6.18 (s, 1H), 6.82 (s, 1H), 7.48
(s, 1H), 7.52 (s, 1H), 7.65 (d, 1H, J = 4.8Hz); MS
(FAB) 535 [M + H]+Å; HRMS [M + H]+Å calcd for
Calculations were performed using INSIGHT-II suite of
software (MSI, Inc.) running on Silicon Graphics
OCTANE system. The strategy and protocol used
for molecular modeling is similar to that adopted in
our previous study.19
C29H35N4O6 m/z 535.255660, obsd (FAB) m/z
25
D
535.257085; ½aŠ þ 530:3 (c 0.5, CHCl3); reverse phase
HPLC (C8 stationary phase, 85% MeOH/H2O mobile
phase, 254nm) tR = 4.21min,% peak area = 99.0%;
calcd for C29H34N4O6: C, 65.15; H, 6.41; N, 10.48;
found: C, 65.40; H, 6.31; N, 10.22.
(a) Modeling of DNA duplex and PBD dimer structures.
The 15-mer DNA sequence GGGGAGAGAGAGGGG
a symmetric sequence about the central triplet AGA,
which is the most preferred site for PBD binding was
considered. BIOPOLYMER module was used to build
the B-DNA duplex structure. PBD dimers 1, 4, 5a,
and 5b were separately constructed using the BUILDER
module of INSIGHT-II. Initially PBDs were ꢀsketchedꢁ
in 2D (two dimensions) and then converted into 3D
(three dimensions) using 2d–3d converter tool of the
BUILDER module. Care was taken to see that there is
C11(S)-geometry for all PBD dimers constructed, as this
stereochemistry is known to lead to energetically
favored adduct with that of guanine of B-DNA duplex
structure.5
4.1.13. 7-Methoxy-8-{5-[7-methoxy-(11aS)-1,2,3,10,11,
11a-hexahydro-5H-pyrrolo[2,1-c]-[1,4]benzodiazepine-5-
one-8-yloxy]pentyloxy}-(11aS)-1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5b). The com-
pound 5b was prepared according to the method de-
scribed for the compound 5a employing 15b (686mg,
1mmol) to afford 5b as a pale yellow oil (287mg,
1
51%). H NMR (CDCl3) d 1.20–2.45 (m, 14H), 3.10–
3.20 (m, 2H), 3.20–3.28 (m, 1H), 3.40–3.78 (m, 5H),
3.80 (s, 3H), 3.98 (s, 3H), 4.10–4.22 (m, 4H), 6.0 (s,
1H), 7.02 (s, 1H), 7.50 (s, 1H), 7.55 (s, 1H), 7.65 (d,
1H, J = 4.4Hz); 13C NMR (CDCl3) d 22.4, 22.8, 24.1,
28.6, 29.5, 30.8, 46.6, 48.3, 52.9, 53.6, 56.1, 57.7, 68.4,
68.7, 102, 110.5, 111.6, 115.3, 120.1, 140.6, 141.2,
142.1, 147.8, 150.8, 152, 163.3, 164.6, 166.3; MS
(FAB) 563 [M + H]+Å; HRMS [M + H]+Å calcd for
(b) Docking studies. PBDs were manually docked into
the minor groove of B-DNA duplex such that the
N10–C11 imine functionality and the exocyclic C2-
amino group of guanine (G8) are nearly at bonding dis-
tance and a covalent bond was then formed using the
create-bond tool. After the bond was created, the PBDs
in the minor groove were manually oriented such that
the PBDs are oriented toward the 30 end of covalently
linked B-DNA duplex strand. Dihedral angles about
C–C bonds were manually adjusted so that PBD dimers
form an isohelical fit within the minor groove of the B-
DNA duplex structure. CVFF force field was used to fix
the charges and the potentials required for energy calcu-
lations. The complexes formed were subjected to energy
minimization (EM) using conjugate gradient method till
they were fully converged that is, till the energy gradient
C31H39N4O6 m/z 563.286960, obsd (FAB) m/z
25
D
563.288484; ½aŠ þ 472:6 (c 0.5, CHCl3); reverse phase
HPLC (C8 stationary phase, 85% MeOH/H2O mobile
phase, 254nm) tR = 4.22min,% peak area = 97.3%;
calcd for C31H38N4O6: C, 66.17; H, 6.81; N, 9.96;
found: C, 66.39; H, 6.56; N, 9.65.
4.2. Thermal denaturation studies
Compounds were subjected to thermal denaturation
studies with duplex-form CT-DNA using by reported

5436
A. Kamal et al. / Bioorg. Med. Chem. 12 (2004) 5427–5436
8. Boyd, F. L.; Stewart, D.; Remers, W. A.; Barkley, M. D.;
Hurley, L. H. Biochemistry 1990, 29, 2387.
9. Thurston, D. E.; Morris, S. J.; Hartley, J. A. Chem.
Commun. 1996, 563.
was nearly equal to 0.001kJmolnm. Constraints were
applied to fix the B-DNA duplex structure during EM.
(c) Molecular dynamics. Molecular dynamic studies were
carried using the following protocol: heating phase
(equilibration) = 30ps and sampling phase = 100ps.
During simulations constraints were applied to fix the
B-DNA duplex structure. Intermittent structures of the
complex formed at every 10ps of simulation were col-
lected and subjected to EM. The minima of all the snap
shots were examined in order to select the lowest energy
conformation as a representative of DNA–PBD com-
plex for further studies.
10. Wilson, S. C.; Howard, P. W.; Forrow, S. M.; Hartley, J.
A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 1999, 42, 4028.
11. Reddy, B. S. P.; Damayanthi, Y.; Reddy, B. S. N.; Lown,
W. J. Anti-Cancer Drug Des. 2000, 15, 225.
12. Baraldi, P. G.; Balboni, G.; Cacciari, B.; Guiotto, A.;
Manfredini, S.; Romagnoli, R.; Spalluto, G.; Thurston, D.
E.; Howard, P. W.; Bianchi, N.; Rutigiiano, C.; Mischiati,
C.; Gambari, R. J. Med. Chem. 1999, 42, 5131.
13. Zhou, Q.; Duan, W.; Simmons, D.; Shayo, Y.; Raymond,
M. A.; Dorr, R. T.; Hurley, L. H. J. Am. Chem. Soc. 2001,
123, 4865.
14. Thurston, D. E.; Bose, D. S.; Thompson, A. S.; Howard,
P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C.; Neidle, S.;
Hartley, J. A.; Hurley, L. H. J. Org. Chem. 1996, 61, 8141.
15. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N.
A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 2001, 44, 737.
(d) Energy of interaction. The energy of the PBD–DNA
complex (Ecomplex) and the energies of DNA (EDNA) and
PBD (EPBD) individually after separating from the com-
plex were calculated. Energy of interaction (Eint) be-
tween DNA and PBD complex were calculated using
the following formula:
16. Gregson, S. J.; Howard, P. W.; Gullick, D. R.; Hama-
guchi, A.; Corcoran, K. E.; Brooks, N. A.; Hartley, J. A.;
Jenkins, T. C.; Patel, S.; Guille, M. J.; Thurston, D. E.
J. Med. Chem. 2004, 47, 1161.
17. Gregson, S. J.; Howard, P. W.; Corcoran, K. E.; Jenkins,
T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med. Chem.
Lett. 2001, 11, 2859.
18. Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.; Reddy,
G. S. K. Curr. Med. Chem.––Anti-Cancer Agents 2002, 2,
215.
19. Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.;
Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.;
Nagarajaram, H. A. J. Med. Chem. 2002, 45, 4679.
20. Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.;
Ramulu, P. Bioorg. Med. Chem. Lett. 2002, 12, 1917.
21. Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2002, 12, 1933.
Eint ¼ Ecomplex À ðEDNA þ EPBD
Þ
where Eint = energy of interaction of the complex,
Ecomplex = total energy of the complex, EDNA and
EPBD are the individual total energies of the DNA
and the PBD molecules calculated after they are
separated from each other.
Acknowledgements
We thank the National Cancer Institute, Maryland for
in vitro anticancer assay in human cancer cell lines.
The authors G.R., O.S., P.R., and N.L. are thankful
to CSIR, New Delhi for the award of research fellow-
ships.
22. Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg.
Med. Chem. Lett. 2003, 13, 3417.
References and notes
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