Dithiocarbamate/piperazine bridged pyrrolobenzodiazepines as DNA-minor groove binders: Synthesis, DNA-binding affinity and cytotoxic activity

Article abstract of DOI:10.1016/j.bioorg.2015.01.002

A new series of C8-linked dithiocarbamate/piperazine bridged pyrrolo[2,1-c][1,4]benzodiazepine conjugates (5a-c, 6a,b) have been synthesized and evaluated for their cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have

Full text of DOI:10.1016/j.bioorg.2015.01.002

Accepted Manuscript
Dithiocarbamate/Piperazine Bridged Pyrrolobenzodiazepines as DNA-minor
Groove Binders: Synthesis, DNA-Binding Affinity and Cytotoxic Activity
Ahmed Kamal, Kokkonda Sreekanth, Nagula Shankaraiah, Manda Sathish,
Shalini Nekkanti, Vunnam Srinivasulu
PII:
S0045-2068(15)00003-6
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.01.002
YBIOO 1782
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
10 November 2014
Please cite this article as: A. Kamal, K. Sreekanth, N. Shankaraiah, M. Sathish, S. Nekkanti, V. Srinivasulu,
Dithiocarbamate/Piperazine Bridged Pyrrolobenzodiazepines as DNA-minor Groove Binders: Synthesis, DNA-
Binding Affinity and Cytotoxic Activity, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.
2015.01.002
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Bioorganic Chemistry
journal homepage: www.elsevier.com
Dithiocarbamate/Piperazine Bridged Pyrrolobenzodiazepines as DNA-minor
Groove Binders: Synthesis, DNA-Binding Affinity and Cytotoxic Activity
Ahmed Kamal,*,a,b Kokkonda Sreekanth,a Nagula Shankaraiah,b Manda Sathish,a Shalini Nekkanti,b
Vunnam Srinivasulua
aMedicinalChemistry &Pharmacology ,CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad-500 037, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A
new
series
of
C8-linked
dithiocarbamate/piperazine
bridged
pyrrolo[2,1-
c][1,4]benzodiazepine conjugates (5a–c, 6a,b) have been synthesized and evaluated for their
cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have been
screened for their cytotoxicity against a panel of 60 human cancer cell lines. Compound 5a has
shown promising cytotoxic activity on selected cancer cell lines that display melanoma,
leukemia, CNS, ovarian, breast and renal cancer phenotypes. The consequence of further
replacement of the 3-cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate in 5b and 5c with 4-
methylpiperazine-1-carbodithioate yielded new conjugates 6a and 6b respectively. In addition,
the compounds 5c and 6a,b have been evaluated for their in vitro cytotoxicity on some of the
selected human cancer cell lines and these conjugates have exhibited significant cytotoxic
activity. Further, the DNA-binding ability of these new conjugates has been evaluated by using
thermal denaturation (ΔTm) studies. The correlation between structure and DNA-binding ability
has been investigated by molecular modeling studies which predicted that 6b exhibits superior
DNA-binding ability and these are in agreement with the experimental DNA-binding studies.
Keywords:
Pyrrolobenzodiazepine
piperazine
dithiocarbamates
DNA-binding affinity
cytotoxicity
2009 Elsevier Ltd. All rights reserved.
1. Introduction
products.23,24 Previously, we have designed and synthesized C8-
linked N-methyl piperazine PBD monomers25 and their dimers12
The minor groove binders are one of the most interesting and
widely studied classes of DNA ligands characterized by their
sequence specificity. DNA sequence specificity or selectivity has
become recognized as an important characteristic of many
cytotoxic agents1,2 such as duocarmycins,3 distamycin, netropsin4
and pyrrolo[1,4]benzodiazepines.5 Several of such molecules are
currently in clinical usage for the treatment of human
that displayed significant DNA-binding ability and anticancer
activity.26-30 These findings provided further impetus to explore the
linking of certain non-covalent interacting groups such as
dithiocarbamates to a PBD moiety, which has led to the design
and synthesis of a variety of PBD hybrids. Considerable interest
has been focused on dithiocarbamates which have been found to
possess a broad spectrum of biological activities like fungicidal,
antibacterial and anticancer activity.31 Recently, brassnin (3, Fig.
1), a dithiocarbamate isolated from cabbage, was reported as
cancer immunosuppressant and its structural modification has lead
to the design and synthesis of sulforamate (4, Fig. 1).31-34
malignancies.
Pyrrolo[2,1-c][1,4]benzodiazepines
(PBDs),
represent a group of exceptionally potent naturally occurring
antitumor antibiotics, derived from Streptomyces species;
examples of which include, DC-81 (1, Fig. 1), anthramycin,
tomaymycin and sibiromycin.6,7 Their interactions with DNA are
unique since they bind within the minor groove of DNA forming a
covalent aminal bond between the C11-position of the central B-
ring and N2-amino group of a guanine base.6,7 There is either an
imine or carbinolamine methyl ether moiety at the N10–C11
position.8–11 The latter is an electrophilic center responsible for
alkylating DNA. In this context, we have been engaged during the
last few years in the structural modifications and development of
new synthetic strategies for the PBD based ring system.12–21
On the other hand, piperazine motifs are amongst the key
scaffolds in today’s drug discovery owing to their potential
biological properties including anticancer activities.22 Moreover,
the piperazine scaffold occurs frequently in complex natural
Figure 1. Representative structures of PBD hybrids (1, 5a–c, and 6a,b),
dithiocarbamates (990207; 2, brassinin; 3 and sulforamate; 4).
1

Recently, bi-functional DNA-interacting agents comprising of (10a–c) as the starting material (Scheme 2). Reaction of (2S)-N-
two types of antitumor agents joined by a linker have attracted [4-(bromoalkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-
considerable attention as a new class of antitumor agents. In this carboxaldehyde diethylthioacetals (10a–c) with 3-cyano-3,3-
strategy, the coupling of a DNA-groove binder with a DNA- diphenylpropyl 1-piperazinecarbodithioate (9) gave C8-linked 3-
intercalator or the coupling of two DNA-groove-binders provides cyano-3,3-diphenylpropyl
1-piperazinecarbodithioate-
a basis for modulating the sequence-selective binding behavior nitrothioacetals (11a–c). The nitro group of these conjugates has
and/or tailoring the PBD-hybrid ligands for mixed-sequence been efficiently reduced by employing SnCl2.2H2O to afford the
recognition. Based on the potent antitumor activity exhibited by corresponding aminothioacetals 12a–c. Deprotection of the
the sulphonate35 and phosphonate substituted PBD27 hybrids, it thioacetal group with HgCl2-CaCO3 yielded the desired 3-cyano-
was considered of interest to design and synthesize new PBD 3,3-diphenylpropyl
1-piperazinecarbodithioate-pyrrolo[2,1-
conjugates by linking dithiocarbamates and piperazine moieties at c][1,4]benzodiazepine (PBD) conjugates (5a–c, Scheme 2).
the C8-position of the PBD with varying alkane spacers. The
Similarly, nitrothioacetal intermediates 13a,b have been
present paper is mostly focused on the DNA-binding affinity and
synthesized by reacting 10a–c with N-methylpiperazine in the
anticancer activity of the newly synthesized PBD conjugates with
presence of CS2 and anhydrous potassium phosphate. Further, the
an objective to improve the DNA sequence specificity.
reduction of 13a,b with SnCl2.2H2O has provided
aminothioacetals 14a, b. Deprotective cyclization of these
NC
Boc
N
NH
CS2
i
+
+
Br
aminothioacetals by using HgCl2-CaCO3 resulted in the formation
of the target imine conjugates 6a,b as shown in Scheme 2.
7
2.2. DNA binding studies
S
NC
The DNA-binding affinity of these PBD conjugates 5a–c and
6a,b has been investigated by thermal denaturation study of calf
thymus (CT) DNA. Binding of molecules into the DNA double
helix leads to increase in the helix melting temperature (ΔTm), the
temperature at which the double helix denatures into single
stranded DNA. It has been carried out with DNA/ligand molar
ratios of 5:1. The increase in the helix melting temperature of
DNA (ΔTm) for each conjugate has been examined at 0 h and 18 h
of incubation at 37 oC and data is presented in Table 1. From this
data, it was observed that all these conjugates have shown higher
ΔTm with respect to naturally occurring DC-81 (1). Conjugates
5a–c and 6a were found to exhibit moderate DNA binding
affinity, elevating the helix melting temperature of CT-DNA by
4.0, 2.1, 2.1 and 2.2 oC, respectively, after incubation at 37 oC for
18 h. N-Methyl piperazine-PBD conjugate with dithiocarbamate
6b has been found to be efficient in stabilizing the double stranded
N
S
Boc
N
8
ii
S
NC
N
S
HN
9
Scheme 1. . (i) K3PO4, acetone, r.t., 2 h; (ii) CF3COOH, CHCl3, rt, 8 h.
2. Results and discussion
2.1. Chemistry
The synthetic routes for the preparation of the target conjugates
5a–c and 6a, b were described in Scheme 2. The precursors 3-
cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate (9)32 and
(2S)-N-[4-(bromoalkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-
2-carboxaldehyde diethyl thioacetal (10a–c) have been prepared
by literature methods. The treatment of 3-cyano-3,3-diphenyl
propylbromide (7) with equimolar amount of N-boc piperazine in
the presence of CS2 lead to the formation of conjugate 8, which
was deprotected with TFA to give the intermediate 3-cyano-3,3-
diphenylpropyl 1-piperazinecarbodithioate (9) as shown in
Scheme 1.
o
CT-DNA (ΔTm = 10.9 C). These results demonstrate that such
PBD conjugates with piperazine and dithiocarbamate side chain
have significant DNA binding affinity as compared to naturally
occurring DC-81.
The SAR has been explained based on DNA-binding studies;
alkyl spacer can enhance the hydrophobic interactions and might
also achieve a superior isohelical fit within the minor groove of
DNA. The conjugate 6b has shown the best DNA-binding affinity
probably due to the flexible five carbon chain linker, and absence
of bulky end groups, which enables it to rotate and fit in the minor
Synthesis of the compounds 5a–c and 6a, b has been achieved by
employing
(2S)-N-[4-(bromoalkoxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxaldehyde
diethylthioacetals
S
S
CN
NO
Br
O
2 CH(SEt)2
S
NO2 CH(SEt)
( )n
Me
N
N
O
NO2 CH(SEt)
2
O
S
N
N
2
( )n
i
ii
( )n
N
MeO
N
MeO
N
MeO
O
O
O
13a−b
10a−c
11a−c
iii
iii
S
S
CN
NH2
S
NH
O
Me
N
N
O
S
N
N
CH(SEt)2
2 CH(SEt)2
( )n
( )n
N
N
MeO
MeO
O
14a−b
O
12a−c
iv
iv
6a; n = 4
5a; n = 3
5b; n = 4
5c; n = 5
6b;
n = 5
Scheme 2. (i) K2CO3, dry DMF, 48 h; (ii) N-methyl piperazine, CS2, K3PO4, acetone, rt. 2 h; (iii) SnCl2.2H2O, MeOH, reflux, 2 h; (iv) HgCl2, CaCO3, CH3CN-H2O, (4:1),
12 h, 55−58%.
2

groove without unfavorable close contacts. Hence, longer the
alkyl spacer, higher the DNA-binding potential has been observed.
Table 2. In vitro anticancer data for selected conjugates 5a and
5ba
Table 1. Thermal denaturation data for PBD hybrids with
piperazine and dithiocarbamate side chains using calf thymus
(CT) DNA
Response
parameter GI50
(μM) for
Response
parameter GI50
(μM) for
b
b
Panel cell
lines
Panel cell
lines
Induced ΔTm (oC)b values after incubation at 37
oC for
conjugate
conjugate
Conjugate
5a
5b
5a
5b
0 h
2.0
2.0
2.0
2.1
6.0
0.3
18 h
4.0
2.1
Leukemia
Ovarian
cancer
5a
5b
5c
6a
6b
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
-
0.53
0.32
3.29
0.35
0.49
0.25
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal
0.47
0.83
2.19
1.14
1.71
2.35
0.42
1.20
1.82
2.47
1.59
2.96
2.1
2.2
10.9
0.7
3.06
1.22
0.63
0.79
0.99
DC-81 (1)
aFor a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100
µM and ligand concentration = 20 µM in aqueous sodium phosphate buffer [10
mM sodium phosphate + 1 mM EDTA, pH [7.00 0.01].
Non-small cell lung cancer
cancer
bFor CT-DNA alone at pH 7.00 0.01, Tm = 69.1 oC 0.01 (mean value from
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
1.28
2.06
0.40
0.54
1.52
0.81
2.34
1.03
1.59
1.61
1.88
0.71
1.07
1.26
1.68
1.40
1.25
0.19
786-0
A498
ACHN
CAKI-1
SN12C
TK-10
0.23
1.10
0.81
0.35
1.04
1.83
0.55
1.28
1.19
1.16
5.97
0.41
1.16
1.26
0.59
10 separate determinations), all ΔTm values are 0.1 - 0.2 oC.
2.3. MTT assay
Amongst these PBD conjugates, compounds 5a and 5b have
been selected by the National Cancer Institute (NCI) in a panel of
disease-oriented human cancer cell line assay to investigate their
cytotoxic potential. According to the screening data, conjugate 5a
was found to be potentially cytotoxic against the nine cell panels
with GI50 values of <0.99 μM, as shown in Table 2. Relatively
higher sensitivity to the conjugates described here was found for
cell lines of melanoma (LOX IMVI, M14), leukemia (MOLT-4,
RPMI-8226, SR), CNS (SF-268, U251), ovarian (IGROV1,
OVCAR-3), breast (MCF-7) and renal cancer (786-0, ACHN,
CAKI-1, UO-31).
UO-31
Breast
cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
1.02
1.39
0.36
1.56
1.02
2.52
1.10
5.66
3.41
-
MCF7
NCI/ADR-
RES
MDA-MB-
231/ATCC
HS-578T
MDA-MB-
435
0.54
3.77
1.23
1.68
1.64
1.23
4.36
-
0.43
3.05
1.53
0.72
1.04
1.42
0.57
0.49
3.90
Moreover, the PBD conjugates 5c and 6a,b have also been
evaluated for their in vitro cytotoxicity in some selected human
cancer cell lines like MCF7 (breast), A2780 (ovarian), Colo205
(colon), PC3 (prostate), SiHa (cervix), A-549, Hop62 (lung) and
KB (leukemia), by employing the sulforhodamine B (SRB) assay.
The results displayed in Table 3 show that 5c and 6a,b were also
significantly cytotoxic, with GI50 values (the molar concentration
of the drug that inhibits 50% net cell growth inhibition) ranging
>100
3.78
-
KM12
SW-620
BT-549
T-47D
MDA-MB-
468
from 0.10 to
1.7 μM.
CNS cancer
Prostate
cancer
SF-268
SF-295
SF-539
NB-19
SNB-75
U251
0.53
1.11
1.46
3.89
1.36
0.41
1.23
-
1.69
1.67
1.64
1.11
PC-3
DU-145
1.45
0.82
0.69
1.47
Melanoma
LOX IMVI
MALME-
3M
0.86
1.28
0.90
1.01
1.80
1.46
1.98
1.20
2.58
3.04
3.04
2.35
3.96
2.82
4.17
3.55
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
aData obtained from the NCI’s in vitro disease-oriented human tumor cell
screen [34–36]. bThe responses parameter: GI50 is interpolated values
representing the molar concentrations at which percentage growth is +50.
Figure 2. Docked pose of 6b in the minor groove of DNA 5′-
GGGGCGAGAGAGGGG-3′.
3

Table 3. Cytotoxicity (GI50 µM) and (LC50 µM)a of conjugates 5c, 6a,b and adriamycin (ADR) against eight human cancer cell lines
Cell lines
5c
6a
GI50
6b
GI50
ADR
GI50
LC50
3.66
LC50
2.4
LC50
2.2
GI50
LC50
0.17
MCF7
2.56
0.15
0.13
<10E-7
A2780
n.d
n.d
0.16
0.14
>100
>100
0.14
0.12
28
0.002
14.7
<10E-7
>100
Colo 205
2.52
6.08
>100
PC-3
SiHa
2.01
n.d
>100
n.d
0.15
1.7
>100
>100
>100
0.12
0.15
0.14
26
0.23
1.9
13
<10E-7
>100
>100
>100
A-549
3.69
>100
0.16
>100
Hop-62
KB
2.46
n.d
>100
n.d
0.18
0.16
>100
>100
0.10
0.14
>100
30
<10E-7
0.16
<10E-7
>100
aResponse parameter; n.d: Not determined.
2.4. Molecular Modelling
In order to further substantiate the biological activity of these
conjugates, molecular modelling studies were performed. Seven
different B-DNA duplexes with varying sequences have been
constructed and investigated for the interaction with the ligands,
among which five DNA duplexes containing triplet AGA (Pu-G-
Pu), the preferred binding site for PBD, were found to be the
most favored sequences for docking. Among these five
sequences, docking results for the 15-mer sequence 5′-
GGGGCGAGAGAGGGG-3′ containing the central AGA has
shown excellent correlation with DNA-binding studies and has
been employed for further studies. Molecular docking
simulations carried out using Glide36 with default settings show
that these molecules prefer the minor groove over the major
groove and intercalation as well as the binding is based on non-
bonded
docked conformation of compound 6b was stable till 5 ns MD
simulation and the docked poses of all the remaining compounds
were reasonably stable up to 3 ns MD simulation. The snapshots
of this MD run confirmed the strong binding ability of the
compound 6b to DNA minor-groove as shown in Figure 3.
The energy of interaction (Eint) between the DNA and the
PBD conjugates in a complex was calculated as a measure of
stability of that complex as shown in Table 4. The complex
formed by 6b is energetically more stable than the complex
formed by 6a because of longer linker unit in 6b and more stable
than 5a–c due to absence of bulky end group which enables
better non-bonded interactions due to perfect isohelical fit in the
minor groove. It was very clear from the high negative
interaction energies that all the docked complexes are favorable
and stable.
interactions like hydrogen-bonding, Vander Waals contacts,
hydrophobic and electrostatic effects in addition to the covalent
linkage formed between the imine of PBD moiety and exocyclic
C2-amino group of the guanine. One of the representative
conjugates 6b has shown the best score probably due to flexible
five carbon chain linker, and absence of bulky end groups, which
enables it to rotate and fit in the minor groove without
unfavorable close contacts (Fig. 2). The best scored poses of
DNA-ligand complexes from the docking studies have been used
for molecular dynamic simulations. The results of molecular
docking studies are highly consistent with the cytotoxicity as
well as thermal denaturation data.
Table 4. Energy of interaction (Eint) calculated for DNA-PBD
conjugate complexes by generalized Born method after 5 ns MD
simulation.
-1
Complex
Eint (kcal mol
)
DNA-5a
DNA-5b
DNA-5c
DNA-6a
DNA-6b
-111.23
-101.86
-91.01
-105.30
-114.56
3. Conclusion
In conclusion, we have designed and synthesized a new series of
dithiocarbamate bridged piperazine-pyrrolobenzodiazepine
conjugates. These molecules have exhibited moderate to
significant DNA-binding ability. Introduction of N-
methylpiperazinedithiocarbamate with five-membered alkane
spacer to the PBD increased the DNA-binding activity
o
considerably in one of the PBD derivative 6b (ΔTm = 10.9 C).
Compound 5a has displayed cytotoxic potency against many cell
lines and exhibits a wide spectrum of activity against 33 cell lines
in nine cancer phenotypes with GI50 values of < 0.99 μM.
Further, detailed molecular modeling studies involving molecular
docking, molecular dynamics and MM-PBSA calculations
support the biological data. The promising results obtained for
these new PBD-dithiocarbomate derivatives presented in this
paper, make them possible candidates for the treatment of cancer,
and encourage us to advance in the synthesis and evaluation of
new PBD derivatives.
Figure 3. Snapshots of DNA-6b complex at 0 ns (A), 1 ns (B), 2 ns (C)
and 4 ns (D) time scale.
Molecular dynamics (MD) simulations were performed to
provide some insight into the dynamic nature of the complexes
and assess the effect of temperature and solvent. The simulation
time scale was 5 ns and 500 snapshots were taken, the final
binding interaction energies were calculated as the average of all
the 500 snapshots. It was clear from the RMSDs that the

The detailed molecular mechanism for these PBD conjugates is
being further investigated.
(S)-3-Cyano-3,3-diphenylpropyl
4-(3-(4-(2-
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-
nitrophenoxy)propyl)piperazine-1-carbodithioate (11a). To a
solution of 10a (521 mg, 1 mmol) in dry DMF (10 mL) was
added anhydrous K2CO3 (552 mg, 4 mmol) and compound 9 (381
mg, 1 mmol). The reaction mixture was stirred at room
temperature for 48 h. The K2CO3 was removed by suction
filtration and the solvent was evaporated under vacuum. The
crude product thus obtained was purified by column
chromatography using 60% ethyl acetate-hexane to afford title
compound 11a (657 mg, 80%). Light yellow solid; mp: 124−126
4. Experimental
4.1. General
The majority of the solvents were purified by distillation
under nitrogen from the indicated drying agent and used fresh:
dichloromethane (calcium hydride), tetrahydrofuran (sodium
benzophenone ketyl), acetone (potassium permanganate), and
acetonitrile (phosphorous pentoxide). Reaction progress was
monitored by thin-layer chromatography (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
(100–200 mesh). 1H and 13C NMR spectra were recorded on
INOVA (400 MHz) or Gemini Varian-VXR-unity (200 MHz) or
Bruker UXNMR/XWIN-NMR (300 MHz) spectrometer using
tetramethyl silane (TMS) as an internal standard. Chemical shifts
are reported in parts per million (ppm) downfield from
tetramethyl silane. Spin multiplicities are described as s (singlet),
br s (broad singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). Coupling constants are reported in Hertz (Hz). Low
1
ºC; H NMR (300 MHz, CDCl3): δppm: 7.71 (s, 1H), 7.29−7.57
(m, 10H), 6.83 (s, 1H), 4.88 (d, J = 3.39 Hz, 1H), 4.65−4.76 (m,
1H), 4.31 (t, J = 6.79 Hz, 2H), 4.18 (t, J = 6.04 Hz, 2H), 3.94 (s,
3H), 3.33−3.43 (m, 2H), 3.17−3.32 (m, 2H), 2.65−2.88 (m, 4H),
2.48−2.63 (m, 8H), 2.21−2.37 (m, 2H), 2.02−2.18 (m, 2H),
1.61−1.86 (m, 4H), 1.16−1.47 (m, 6H); MS (ESI): m/z 822 [M]+.
(S)-3-Cyano-3,3-diphenylpropyl
4-(4-(4-(2-
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-
nitrophenoxy)butyl)piperazine-1-carbodithioate (11b). The
compound 11b was prepared following the method described for
the compound 11a, employing 10b (535 mg, 1 mmol) and 9 (381
mg, 1 mmol), and the crude product was purified by column
chromatography (60% ethyl acetate-hexane) to afford the
compound 11b (668 mg, 80%). Light yellow solid; mp: 132−134
resolution mass spectra were recorded on
a VG-7070H
Micromass mass spectrometer at 200 oC, 70 eV with trap current
of 200 lA, and 4 kV acceleration voltage. FABMS spectra were
recorded on LSIMS-VG-AUTOSPEC-Micromass. Melting
points were recorded on Electrothermal 9100 and are
uncorrected. All computational studies were done with a Red Hat
Enterprise Linux version 5.0 using Maestro software version 9.5
(Schrödinger, LLC, New York, NY, 2013).
1
ºC; H NMR (300 MHz, CDCl3): δppm: 7.67 (s, 1H), 7.25−7.50
(m, 10H), 6.83 (s, 1H), 4.88 (d, J = 3.77 Hz, 1H), 4.61−4.76 (m,
1H), 4.31 (t, J = 6.79 Hz, 2H), 4.13 (t, J = 6.79 Hz, 2H), 3.95 (s,
3H), 3.33−3.43 (m, 2H), 3.17−3.32 (m, 2H), 2.67−2.87 (m, 4H),
2.41−2.60 (m, 8H), 2.21−2.37 (m, 2H), 2.04−2.19 (m, 2H),
1.88−2.03 (m, 2H), 1.53−1.86 (m, 4H), 1.21−1.40 (m, 6H); MS
(ESI): m/z 836 [M]+.
4.2. Chemistry
tert-Butyl
4-(((3-cyano-3,3-
(S)-3-Cyano-3,3-diphenylpropyl
4-(5-(4-(2-
diphenylpropyl)thio)carbonothioyl)piperazine-1-carboxylate
(8). To a stirred solution of N-boc piperazine (186 mg, 1 mmol)
and anhydrous potassium phosphate (425 mg, 2 mmol) in acetone
(20 mL) was added carbon disulfide 10 drops (~ 2.5 mmol) drop-
wise. After stirring for 30 min, 3-cyano-3,3-diphenylpropyl
bromide (7; 300 mg, 1 mmol) was added. The stirring was further
continued about 2 h, the resulted solid was filtered off and the
filtrate was concentrated. The residue was dissolved in ethyl
acetate (20 mL), and washed with water. The organic layer was
dried over anhydrous Na2SO4, evaporated the solvent and the
residue was purified by column chromatography using 50% ethyl
acetate/hexane to afford compound 8 (384 mg, yield 80%).
Cream colour solid; mp: 144−146 °C; 1H NMR (300 MHz,
CDCl3): δ ppm: 7.45 (d, J = 7.55 Hz, 4H), 7.36 (t, J = 7.55 Hz,
4H), 7.29 (d, J = 6.79 Hz, 2H), 4.22 (bs, 2H), 3.94 (bs, 2H), 3.53
(t, 4H), 3.32−3.41 (m, 2H), 2.74−2.83 (m, 2H), 1.46 (s, 9H); MS
(ESI): m/z 504 [M+Na]+.
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-
nitrophenoxy)pentyl)piperazine-1-carbodithioate (11c). The
compound 11c was prepared following the method described for
the compound 11a, employing 10c (549 mg, 1 mmol) and 9 (381
mg, 1 mmol), and the crude product was purified by column
chromatography (60% ethyl acetate-hexane) to afford the
compound 11c (680 mg, 80%). Light yellow solid; mp: 128−130
1
ºC; H NMR (200 MHz, CDCl3): δppm: 7.66 (s, 1H), 7.23−7.50
(m, 10H), 6.82 (s, 1H), 4.88 (d, J = 3.77 Hz, 1H), 4.67−4.77 (m,
1H), 4.31 (t, J = 6.79 Hz, 2H), 4.09 (t, J = 5.85 Hz, 2H), 3.94 (s,
3H), 3.33−3.44 (m, 2H), 3.17−3.32 (m, 2H), 2.65−2.90 (m, 4H),
2.48−2.62 (m, 8H), 2.38−2.47 (m, 2H), 2.22−2.37 (m, 2H),
2.00−2.19 (m, 2H), 1.86−1.98 (m, 2H), 1.48−1.77 (m, 4H),
1.22−1.41 (m, 6H); MS (ESI): m/z 850 [M]+.
(S)-3-Cyano-3,3-diphenylpropyl
4-(3-(5-amino-4-(2-
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-
methoxyphenoxy)propyl)piperazine-1-carbodithioate (12a).
The stirred mixture of 11a (822 mg, 1 mmol) and SnCl2.2H2O
(1.125 g, 5 mmol) in methanol (20 mL) was refluxed for 5 h or
until the TLC indicated that reaction was completed. The
methanol was evaporated by vacuum and the aqueous layer was
then carefully adjusted to pH 8 with 10% NaHCO3 solution and
then extracted with ethyl acetate and chloroform (2 x 30 ml and 2
x 30 mL). The combined organic phase was dried over Na2SO4
and evaporated under vacuum to afford the crude amino diethyl
thioacetal 12a (617 mg, 78%) as yellow oil, which was used
directly for the next step.
3-Cyano-3,3-diphenylpropyl piperazine-1-carbodithioate
(9). A solution of 8 (481 mg, 1 mmol) in dichloromethame (15
o
mL) was added trifluoroacetic acid (1.14 gm, 10 mmol) at 0 C
and the mixture was stirred at room temperature for 8 h. The
solvent was evaporated and the solution was carefully adjusted to
pH 8 with saturated NaHCO3 solution and then extracted with
chloroform (2 x 15 mL). The combined organic phase was
washed with brine (15 mL), dried over anhydrous Na2SO4 and
evaporated under vacuum to afford the compound 9 (285 mg,
75%). Thick liquid. 1H NMR (300 MHz, CDCl3): δ ppm: 7.46 (d,
J = 8.0 Hz, 4H), 7.38 (dd, J = 5.50 Hz, 1.9 Hz, 4H), 7.30 (t, 2H),
4.29 (bs, 2H), 3.87 (bs, 2H), 3.39 (m, 2H), 2.93 (bs, 4H), 2.83
(m, 2H), 1.72 (bs, 1H); MS (ESI): m/z 382.4 [M +H]+.
(S)-3-Cyano-3,3-diphenylpropyl
4-(4-(5-amino-4-(2-
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-
5

methoxyphenoxy)butyl)piperazine-1-carbodithioate
(12b).
1H NMR (400 MHz, CDCl3) δppm: 7.67 (d, J = 4.71 Hz, 1H),
7.29−7.54 (m, 10H), 6.79 (s, 1H), 3.96−4.18 (m, 4H), 3.95 (s,
3H), 3.49−3.88 (m, 4H), 3.25−3.45 (m, 2H), 2.73−2.91 (m, 2H),
2.22−2.63 (m, 8H), 1.38−2.17 (m, 10H); 13C NMR (100 MHz,
CDCl3) δ ppm: 196.1, 164.9, 163.3, 152.3, 148.9, 143.7, 140.1,
129.4, 128.1, 126.2, 117.4, 116.1, 109.9, 107.8, 69.9, 56.8, 56.1,
54.2, 53.1, 52.4, 49.9, 39.7, 38.4, 29.7, 28.6, 24.7, 23.7; HRMS
(ESI): m/z calcd for C39H45N5O3S2 696.3003, found 696.3011
[M+H]+.
The compound 12b was prepared following the method described
for the compound 12a, employing the 11b (836 mg, 1 mmol) and
SnCl2.2H2O (1.125 g, 5 mmol) to afford the amino diethyl
thioacetal 12b as a yellow oil (628 mg, 78%), which was used
directly for the next step without further purification.
(S)-3-Cyano-3,3-diphenylpropyl
4-(5-(5-amino-4-(2-
(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-
methoxyphenoxy)pentyl)piperazine-1-carbodithioate (12c).
The compound 12c was prepared following the method described
for the compound 12a, employing the 11c (850 mg, 1 mmol) and
SnCl2.2H2O (1.125 g, 5 mmol) to afford the amino diethyl
thioacetal 12c as a yellow oil (639 mg, 78%), which was used
directly for the next step without further purification.
(S)-4-(4-(2-(Bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-
2-methoxy-5-nitrophenoxy)butyl
4-methylpiperazine-1-
carbodithioate (13a). To a solution of 1-methylpiperazine (100
mg, 1 mmol) in dry acetone (10 mL) was added carbon disulfide
10 drops (~2.5 mmol) and anhydrous K3PO4 (425 mg, 2 mmol).
o
The mixture was stirred <10 C for 1 h. Then 10b (535 mg, 1
(S)-3-Cyano-3,3-diphenylpropyl 4-(3-((7-methoxy-5-oxo-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
8-yl)oxy)propyl)piperazine-1-carbodithioate (5a). A solution
of 12a (792 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and
CaCO3 (246 mg, 2.46 mmol) in acetonitrile:water (4:1) was
stirred slowly at room temperature until TLC indicates complete
loss of starting material. The reaction mixture was diluted with
ethyl acetate (30 mL) filtered through a celite pad. The clear
organic supernatant was extracted with saturated 5% NaHCO3
(20 ml), brine (20 ml) and the combined organic phase is dried
(Na2SO4). The organic layer was evaporated in vacuum and
purified by column chromatography (95% CH2Cl2-MeOH) to
give compound 5a (366 mg, 55% yield) as cream yellow solid.
This material was repeatedly evaporated from CHCl3 in vacuum
mmol) was added. The reaction mixture was stirred at room
temperature for 12 h. TLC using ethylacetate as a solvent system
monitored the reaction. The potassium phosphate was removed
by suction filtration and the solvent was removed under vacuum.
The crude product was purified by column chromatography using
40% ethyl acetate-hexane to afford pure compound 13a (504 mg,
1
80%). Light yellow solid; mp: 80−82 ºC; H NMR (200 MHz,
CDCl3) δppm: 7.64 (s, 1H), 6.77 (s, 1H), 4.83 (d, J = 4.15 Hz,
1H), 4.61−4.76 (m, 1H), 4.14 (t, J = 5.81, 6.12 Hz, 2H), 3.95 (s,
3H), 3.39 (t, J = 6.64, 5.83 Hz, 2H), 3.17−3.30 (m, 2H),
2.51−2.90 (m, J = 4.98 Hz, 14H), 2.33 (s, 3H), 1.87−2.12 (m,
6H), 1.23−1.42 (q, 6H); MS (ESI): m/z 631 [M]+.
(S)-5-(4-(2-(Bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-
to generate the imine form. mp: 114–116 oC; [α]D = +0.26 (c =
2-methoxy-5-nitrophenoxy)pentyl
4-methylpiperazine-1-
25
1
0.9 in CHCl3); H NMR (400 MHz, CDCl3): δppm: 7.64 (d, J =
carbodithioate (13b). To a solution of 1-methylpiperazine (100
mg, 1 mmol) in dry acetone (10 mL) was added carbon disulfide
10 drops (~2.5 mmol) and anhydrous K3PO4 (425 mg, 2 mmol).
The mixture was stirred <10 °C for 1 h. Then 10c (549 mg, 1
mmol). The reaction mixture was stirred at room temperature for
12 h. TLC using ethylacetate as a solvent system monitored the
reaction. The potassium phosphate was removed by suction
filtration and the solvent was removed under vacuum. The crude
product was purified by column chromatography using 40% ethyl
acetate-hexane to afford pure compound of 13b (528 mg, 82%).
Light yellow solid; mp: 86−88 ºC; 1H NMR (200 MHz, CDCl3) δ
ppm: 7.67 (s, 1H), 6.83 (s, 1H), 4.88 (d, 1H), 4.65−4.78 (m, 1H),
4.11 (t, J = 7.03 Hz, 2H), 3.96 (s, 3H), 3.36 (t, J = 7.03 Hz, 2H),
3.21−3.30 (m, 2H), 2.75−2.88 (m, 4H), 2.52 (t, J = 5.47 Hz, 4H),
2.35 (s, 3H), 2.17−2.30 (m, 2H), 2.12 (t, J = 7.81 Hz, 2H),
2.00−2.07 (m, 2H), 1.94 (t, J = 7.81 Hz, 2H), 1.75−1.88 (m, 2H),
1.67 (t, J = 3.90 Hz, 2H), 1.57−1.65 (m, 2H), 1.16−1.44 (m, 6H);
MS (ESI): m/z 645 [M]+.
3.82 Hz, 1H), 7.28−7.55 (m, 10H), 6.80 (s, 1H), 6.36 (s, 1H),
3.96−4.23 (m, 4H), 3.95 (s, 3H), 3.44−3.86 (m, 4H), 3.25−3.43
(m, 2H), 2.73−2.91 (m, 2H), 2.20−2.62 (m, 7H), 1.38−2.15 (m,
6H) ppm; 13C NMR (100 MHz, CDCl3) δ ppm: 197.1, 164.8,
162.9, 152.1, 149.7, 143.7, 140.7, 139.7, 128.9, 127.4, 119.2,
118.5, 117.8, 112.8, 79.4, 59.3, 56.9, 56.6, 54.7, 53.6, 51.1, 44.1,
38.7, 29.3, 28.9, 28.1, 24.1; HRMS (ESI): m/z calcd for
C37H41N5O3S2 668.2691, found 668.2699 [M+H]+.
(S)-3-Cyano-3,3-diphenylpropyl 4-(4-((7-methoxy-5-oxo-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
8-yl)oxy)butyl)piperazine-1-carbodithioate
(5b).
The
compound 5b was prepared following the method described for
the preparation of the compound 5a, employing 12b (806 mg, 1
mmol) and HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246 mg,
2.46 mmol) to afford the compound 5b as a pale yellow solid
(388 mg, 57%). mp: 100–102 oC; [α]D = +5.4 (c = 1 in CHCl3);
25
1H NMR (400 MHz, CDCl3): δppm: 7.67 (d, J = 4.71 Hz, 1H),
7.29−7.57 (m, 10H), 6.81 (s, 1H), 6.34 (s, 1H), 3.97−4.44 (m,
4H), 3.94 (s, 3H), 3.45−3.80 (m, 4H), 3.30−3.46 (m, 2H),
2.74−2.91 (m, 2H), 2.23−2.65 (m, 8H), 1.35−2.20 (m, 7H); 13C
NMR (100 MHz, CDCl3) δ ppm: 196.9, 164.6, 163.1, 152.4,
149.6, 143.5, 141.1, 139.6, 129.2, 127.4, 119.1, 118.5, 117.6,
112.8, 79.2, 59.1, 57.1, 56.6, 54.6, 53.6, 51.4, 44.2, 38.7, 29.4,
28.9, 28.1, 24.8, 24.2; HRMS (ESI): m/z calcd for C38H43N5O3S2
682.2847, found 682.2853 [M+H]+.
(S)-4-(5-Amino-4-(2-(bis(ethylthio)methyl)pyrrolidine-1-
carbonyl)-2-methoxyphenoxy)butyl
4-methylpiperazine-1-
carbodithioate (14a). The compound 13a (631 mg, 1 mmol)
dissolved in methanol (20 mL) and added SnCl2.2H2O (1.125 g, 5
mmol) was refluxed for 5 h. The methanol was evaporated by
vacuum and the aqueous layer was then carefully adjusted to pH
8 with 10% NaHCO3 solution and then extracted with ethyl
acetate and chloroform (2 x 30 ml and 2 x 30 mL). The combined
organic phase was dried over Na2SO4 and evaporated under
vacuum to afford the crude amino diethyl thioacetal 14a (468
mg, 78%) as yellow oil, which was used directly in the next step
without further purification.
(S)-3-Cyano-3,3-diphenylpropyl 4-(5-((7-methoxy-5-oxo-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
8-yl)oxy)pentyl)piperazine-1-carbodithioate
(5c).
The
compound 5c was prepared following the method described for
the preparation of the compound 5a, employing 12c (820 mg, 1
mmol), to afford the compound 5c as a pale yellow solid (382
(S)-5-(5-Amino-4-(2-(bis(ethylthio)methyl)pyrrolidine-1-
carbonyl)-2-methoxyphenoxy)pentyl 4-methylpiperazine-1-
carbodithioate (14b). The compound 14b was prepared
o
25
mg, 55%). mp: 110–112 C; [α]D = +0.44 (c = 0.7 in CHCl3);
6

following the method described for the compound 14a,
employing the compound 13b (645 mg, 1 mmol) to afford the
amino diethyl thioacetal 14b as a yellow oil (479 mg, 78%),
which was used directly in the next step.
Results are given as means
standard deviation from three
determinations and are corrected for the effects of DMSO co-
solvent using a linear correction term. Drug-induced alterations
in DNA melting behaviour are given by ∆Tm = Tm (DNA + PBD)
Tm (DNA alone), where the Tm value for the PBD-free CT-
(S)-4-((7-Methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-
o
DNA is 69.2 0.01 C. The fixed [PBD]/[DNA] ratio used did
benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butyl
4-
not result in binding saturation of the host DNA duplex for any
compound examined.
methylpiperazine-1-carbodithioate (6a). A solution of 14a
(601 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246
mg, 2.46 mmol) in acetonitrile:water (4:1) was stirred slowly at
room temperature for overnight. The reaction mixture was diluted
with ethyl acetate (30 mL) filtered through a celite pad. The clear
organic supernatant was extracted with saturated 5% NaHCO3
(20 mL), brine (20 mL) and the combined organic phase was
dried over (Na2SO4). The organic layer was evaporated under
vacuum and purified by column chromatography using 4%
MeOH-CHCl3 to give compound 6a (276 mg, 58%) as light
yellow solid. This material was repeatedly evaporated from
4.4. MTT assay
In-routine compounds 5c, 6a and 6b have been evaluated for
their in vitro cytotoxicity in selected human cancer cell lines of
MCF7 (breast), A2780 (ovarian), Colo205 (colon), PC3
(prostate), SiHa (cervix), A-549, Hop62 (lung) and KB
(Leukemia) origin. A protocol of 48 h continuous drug exposure
has been used and a sulforhodamine B (SRB) protein assay has
been used to estimate cell viability or growth.38,39
o
1
CHCl3 in vacuum to generate the imine form. mp: 94–96 C; H
NMR (400 MHz, CDCl3) δppm: 7.61 ppm (d, J = 4.68 Hz, 1H),
7.45 (s, 1H), 6.73(s, 1H), 3.80−4.07 (m, 4H), 3.79 (s, 3H),
3.61−3.74 (m, 2H), 3.3 (t, J = 7.03 Hz, 4H), 2.44 (t, J = 5.46 Hz,
2H), 2.27 (s, 3H), 1.77−2.05 (m, 4H), 1.51−1.54 (m, 1H), 1.36
(d, J = 5.46 Hz, 1H), 1.06−1.25 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ ppm: 196.9, 164.5, 163.5, 152.3, 148.9, 140.2, 117.1,
108.1, 107.2, 79.5, 57.1, 54.7, 52.8, 51.7, 47.2, 36.8, 31.1, 28.1,
26.7, 24.6; HRMS (ESI): m/z calcd for C23H32N4O3S2 477.1952,
found 477.1961 [M+H]+.
The cell lines were grown in RPMI 1640 medium containing
10% fetal bovine serum and 2 mM L-glutamine and were
inoculated into 96-well microtiter plates in 90 µL at plating
densities depending on the doubling time of individual cell lines.
The microtiter plates were incubated at 37 oC, 5% CO2, 95% air,
and 100% relative humidity for 24 h prior to addition of
experimental drugs. Aliquots of 10 µL of the drug dilutions were
added to the appropriate microtiter wells already containing 90
µL of cells, resulting in the required final drug concentrations.
Plates were incubated further for 48 h and assay was terminated
by the addition of 50 lL of cold trichloro acetic acid (TCA) (final
concentration, 10% TCA) and incubated for 60 min at 4 oC.
(S)-5-((7-Methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl
4-
methylpiperazine-1-carbodithioate (6b). A solution of 14b
(615 mg, 1 mmol), HgCl2 (613 mg, 2.26 mmol) and CaCO3 (246
mg, 2.46 mmol) in acetonitrile:water (4:1) was stirred slowly at
room temperature for overnight. The reaction mixture was diluted
with ethyl acetate (30 mL) filtered through a celite pad. The clear
organic supernatant was extracted with saturated 5% NaHCO3
(20 mL), brine (20 mL) and the combined organic phase was
dried (Na2SO4). The organic layer was evaporated under vacuum
and purified by column chromatography using 4% MeOH-CHCl3
to give compound 6b (284 mg, 58%) as light yellow solid. This
material was repeatedly evaporated from CHCl3 in vacuum to
generate the imine form. mp: 90–92 C; H NMR (400 MHz,
CDCl3) δppm: 7.62 (d, J = 3.90 Hz, 1H), 7.22 (s, 1H), 6.75 (s,
1H), 3.92−4.11 (m, 4H), 3.90 (s, 3H), 3.41−3.85 (m, 3H), 3.28 (t,
J = 7.03 Hz, 2H), 2.45 (m, 8H), 2.29 (s, 3H), 1.41−2.18 (m, 8H);
13C NMR (100 MHz, CDCl3) δppm: 197.1, 164.7, 163.6, 152.4,
149.0, 140.4, 117.2, 108.3, 107.1, 79.0, 56.9, 54.6, 52.7, 50.9,
50.2, 46.9, 36.6, 31.4, 30.08, 29.7, 26.6, 23.9; HRMS (ESI): m/z
calcd for C24H34N4O3S2 491.2111, found 491.2119 [M+H]+.
The plates were washed five times with tap water and air-
dried. Sulforhodamine B (SRB) solution (50 µL) at 0.4% (w/v) in
1% acetic acid was added to each of the wells, and plates were
incubated for 20 min at room temperature. The residual dye was
removed by washing five times with 1% acetic acid. The plates
were air-dried. Bound stain was subsequently eluted with 10 mM
trizma base, and the absorbance was read on an ELISA plate
reader at a wavelength of 540 nm with 690 nm reference
wavelength. Percent growth was calculated on a plate–byplate
basis for test wells relative to control wells.
o
1
Percentage growth was expressed as the (ratio of average
absorbance of the test well to the average absorbance of the
control wells) x 100.
4.5. Molecular modelling Studies
The hybrids were built and prepared using Ligprep 2.7 and
geometrically minimized with Macromodel 10.1 followed by
conformational analysis in Maestro 9.5. Truncated Newton
Conjugate Gradient (TNCG) minimization method was used with
500 iterations and convergence threshold of 0.05 kJ/mol. All the
DNA duplexes were prepared using protein preparation wizard.
The Glide XP 6.5 algorithm was employed for docking. The
lowest energy pose for each compound was further subjected to
molecular dynamics simulations using Desmond 3.5 with OPLS-
AA force field in explicit solvent with the TIP3P water model.
Before MD simulations, the system was pre-equilibrated using
Constant-Volume (NVT) MD simulation for the first 100 ps
during which temperature of the system was raised from 0 to 300
K and for further simulation the temperature was maintained at
300 K. Subsequently, the system was equilibrated in NPT which
is composed of minimization and short MD simulation (12 and
24 ps) to relax the model system. After that, long equilibration
MD simulation was performed for first 2 ns and long production
4.3. DNA binding studies
Compounds were subjected to thermal denaturation studies
with duplex-form CT-DNA using reported method. Working
solutions in aqueous buffer (10 mM NaH2PO4/Na2HPO4, 1 mM
Na2EDTA, pH 7.00 + 0.01) containing CT-DNA (100 µM in
phosphate) and the PBD (20 µM) were prepared by addition of
concentrated PBD solutions in DMSO to obtain a fixed
[PBD]/[DNA] molar ratio of 1:5. The DNA–PBD solutions were
incubated at 37 oC for 0 and 18 h prior to analysis. Samples were
monitored at 260 nm using a Beckman–Coulter DU 800
spectrophotometer fitted with high performance temperature
o
1
controller, and heating was applied at 1 C min in the range of
40–90 oC. DNA helix→coil transition temperatures I were
obtained from the maxima in the d(A260)/dT derivative plots.
7

24. A. Kamal, Rajender, M. K. Reddy, G. Balakishan, T. B. Shaik, M.
Chourasia, G. N. Sastry, Bioorg. Med. Chem. 17 (2009) 1557.
25. A. Kamal, P. S. Reddy, D. R. Reddy, E. Laxman, Bioorg. Med.
Chem. 14 (2006) 385.
MD simulation for 5 ns. Data were collected every 10 ps during
the MD runs. The complexes present in trajectory file after
production phase of MD simulations, were clustered according to
the RMSD of backbone. The interaction energies in these clusters
were calculated using the using MM/ GBSA present in Prime 3.3.
26. L. Reddy, B. Odhav, K. D. Bhoola, Pharmacology
Therapeutics 99 (2003) 1.
&
27. J. W. Blunt, B. R. Copp, M. H. Munro, P. T. Northcote, M. R.
Prinsep, Nat. Prod. Rep. 28 (2011) 196.
28. A. Kamal, N. Laxman, G. Ramesh, O. Srinivas, P. Ramulu,
Bioorg. Med. Chem. Lett. 12 (2002) 1917.
Acknowledgments
The authors K.S, M.S, V.S and S.N are thankful to the
National Cancer Institute (NCI), USA for providing the data on
the sixty cell panel of human cancer cell lines. The authors are
also grateful to CSIR, New Delhi, for the award of Research
Fellowships
29. A. Kamal, R. Ramu, V. Tekumalla, G. B. Khanna, M. S.
Barkume, A. S. Juvekar, S. M. Zingde, Bioorg. Med. Chem. 16
(2008) 7218.
30. A. Kamal, P. P. Kumar, B. N. Seshadri, O. Srinivas, M. S. Kumar,
S. Sen, N. Kurian, A. S. Juvekar, S. M. Zingde, Bioorg. Med.
Chem. 16 (2008) 3895.
31. A. Kamal, M. N. Khan, K. S. Reddy, S. K. Ahmed, M. S. Kumar,
A. S. Juvekar, S. Sen, S. M. Zingde, Bioorg. Med. Chem. Lett. 17
(2007) 5345.
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8

Graphical Abstract
S
N
O
Me
N
N
S
H
)
( )
n
N
MeO
6b; n = 5
In vitro 0.10 0.14 M (GI
O
−
μ
50
T
= 10.9 oC at 37 oC in 18 h
Δ
m
9

Research Highlights
ꢀ C8-linked dithiocarbamate/piperazine-PBD
conjugates (5a–c, 6a,b) were synthesized.
ꢀ The conjugates 5a and 5b have been
screened for their cytotoxicity by NCI.
ꢀ The compounds 5c and 6a,b have been also
exhibited significant in vitro cytotoxicity.
ꢀ Molecular modeling studies predict that 6b
exhibits superior than other compounds.
10