DNA binding potential and cytotoxicity of newly designed pyrrolobenzodiazepine dimers linked through a piperazine side-armed-alkane spacer

Article abstract of DOI:10.1016/j.bmc.2005.08.020

New pyrrolobenzodiazepine (PBD) dimers have been developed that are composed of two DC-81 subunits tethered to their C8 positions through piperazine moiety side-armed with alkaneoxy linkers (composed of 2-5 carbons). DNA thermal denaturation studies show

Full text of DOI:10.1016/j.bmc.2005.08.020

Bioorganic & Medicinal Chemistry 14 (2006) 385–394
DNA binding potential and cytotoxicity of newly designed
pyrrolobenzodiazepine dimers linked through a piperazine
side-armed-alkane spacerq
Ahmed Kamal,* P. S. Murali Mohan Reddy, D. Rajasekhar Reddy and E. Laxman
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 16 June 2005; revised 9 August 2005; accepted 9 August 2005
Available online 26 September 2005
Abstract—New pyrrolobenzodiazepine (PBD) dimers have been developed that are composed of two DC-81 subunits tethered to
their C8 positions through piperazine moiety side-armed with alkaneoxy linkers (composed of 2–5 carbons). DNA thermal dena-
turation studies show that after 18 h of incubation with calf thymus DNA at a 1:5 ligand/DNA ratio, one of them, 6a, increases
the DTm value by 24.0 ꢀC. Thus, incorporation of a piperazine moiety instead of an inert alkanedioxy linker alone significantly
enhances the DNA binding ability, and the analogous dimer 4 that lacks a piperazine moiety in the linker spacer elevates melting
by only 15.1 ꢀC under identical experimental conditions. This illustrates the effect of introducing a piperazine ring in the middle of
such an alkanedioxy linker which produces several hydrophobic interactions and could also achieve a superior isohelical fit within
the DNA minor groove. Interestingly, these dimers 6a–d are significantly more cytotoxic than 4 in a number of human cancer cell
lines, in particular, compound 6c is highly potent for almost all the nine human cancer cell lines.
ꢁ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ed to their property of sequence selective covalent bind-
ing with the 2-NH2 group of guanine in the minor
groove of duplex DNA via an acid labile aminal bond
to the electrophilic imine at the N10-C11 position.6,7
The N10-C11 carbinolamine bond exists in the equiva-
lent imine of carbinolamine methyl ether form depend-
ing on the precise structure of the compound and
method of isolation.8 PBD monomers span three DNA
base pairs with the preference for Pu-G-Pu triplets.
The PBDs have been shown to interfere with the interac-
tion of endonuclease enzymes with DNA9 and to block
transcription by inhibiting RNA polymerase in a se-
quence-specific manner.10 Thurston and co-workers
reported the first C8/C80-linked PBD dimer (DSB-120)
(4) in which two DC-81 subunits are joined through
their aromatic A-ring at phenol positions by an inert
propyldioxy linkage.11,12 These C8-diether-linked PBD
dimers exhibit enhanced DNA binding affinity com-
pared to the monomer DC-81 and span six DNA base
pairs actively recognizing a 50-GATC sequence, that is,
doubling of the DNA binding size.13 However, these
lack in vivo antitumor activity probably because of the
low bioavailability and also due to excessive electrophi-
licity at the N10-C11 imine moiety.14 Subsequently, a
PBD dimer (SJG-136) (5) containing C2/C20-exomethyl-
ene functionalities was designed to reduce the electro-
philicity of the molecule by decreasing the deactivation
There have been considerable attempts with the objec-
tive of targeting specific sequences in DNA by synthetic
ligands with the idea of designing both drugs and molec-
ular probes for DNA polymorphism compounds that
bind in the minor groove of DNA.2–4 These classes of
DNA ligands possess several biological activities partic-
ularly antiviral and antitumor activity.5 One of the class
of compounds is able to induce permanent DNA dam-
age through direct or indirect irreversible interaction
with DNA nucleotides comprised of antibiotic com-
pounds of natural, synthetic, and semi-synthetic origin.
One group of naturally occurring alkylating antitumor
antibiotics are pyrrolobenzodiazepines (PBDs). These
antitumor antibiotics are a class of sequence selective
DNA binding agents derived from Streptomyces spe-
cies,6 well-known members of which include DC-81
(1), tomaymycin (2), and anthramycin (3) (Fig. 1). The
cytotoxicity and antitumor activity of these are attribut-
Keywords: Pyrrolobenzodiazepine; DNA-binding ability; Cytotoxicity;
Piperazine.
q
See Ref. 1.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail addresses: ahmedkamal@iict.res.in; ahmedkamal@
iictnet.org
0968-0896/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.020

386
A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
OH
H
10
N
OCH3
H
9
11
N
N
H3C
HO
HO
H
H
8
1
11a
5
2
N
N
4
N
H3CO
H3CO
7
CONH2
6
3
O
O
O
3
1
2
N
N
O
O
H
H
N
N
OCH3 H3CO
O
N
O
4
N
O
O
H
H
N
N
OCH3 H3CO
5
O
O
O
N
(H2C)n
O
N
N (CH2)n
N
H
H
N
OCH3
N
H3CO
6a n = 2
6b n = 3
6c n = 4
6d n = 5
O
O
Figure 1. Structures of the PBD monomer DC-81 (1), tomaymycin (2), anthramycin (3), DSB-120 (4), SJG-136 (5), piperazine-linked PBD dimers
(6a–d).
of cellular nucleophiles.15 This compound exhibits sig-
nificant in vivo potential and is under clinical evaluation
(Fig. 1). Additionally, in a recent report it has been ob-
served that the length of the linker modulates DNA
cross-linking property and cytotoxic potency. Recently,
a large number of structurally modified PBDs have been
synthesized and evaluated for their biological activity
particularly for their DNA binding potential and antitu-
mor property. Interestingly, a number of these com-
pounds have been selected for preclinical studies but
unfortunately most of them did not proceed beyond that
stage mainly because of problems related to poor bio-
availability and low water solubility.16
no effort has been made to prepare and investigate
PBD dimers linked through a piperazine spacer. It
was considered worthwhile to prepare and evaluate
PBD dimers linked through a piperazine moiety
side-armed with symmetrical alkane spacers at the
C8-position that contain two DC-81 subunits. More-
over, the main objective to incorporate a piperazine
moiety is to improve the bioavailability and DNA
binding ability of such PBD dimers. Therefore, we
herein report the synthesis and biological evaluation
of novel PBD dimers linked through a piperazine
moiety.
During the last decade, many piperazine derivatives have
been synthesized as useful chemotherapeutic agents for
various diseases. Bis-1,4-dialkyl-piperazines have been
extensively investigated and have been reported as anti-
bacterial17–19 and antineoplastic agents.17,20,21 Michejda
and co-workers22 reported symmetrical bifunctional
agents as a promising antitumor class of compounds with
remarkable selectivity against colon cancers that possess a
piperazine moiety in its linker spacer. Recently, soluble
cationic trans-diamine dichloroplatinum(II) complexes
with piperazine ligands have been prepared that exhibit
significant cytotoxic activity against cisplatin resistant
ovarian cancer cells.23 Moreover, these platinum-pipera-
zine complexes are taken up by cancer cells much more
rapidly and bind to cellular DNA and to calf thymus
DNA much faster than cisplatin.
2. Results and discussion
2.1. Chemistry
Synthesis of PBD dimers linked through piperazine moi-
ety has been carried out by employing the commercially
available vanillin. Oxidation of vanillin followed by
esterification employing literature procedure24 provides
the starting material vanillin methyl ester (7). The
monoalkylation of 7 has been achieved by using three
molar equivalents of the dibromoalkanes. Nitration of
8a–d followed by ester hydrolysis and coupling of
(2S)-pyrrolidine-carboxaldehyde diethyl thioacetal25 af-
fords 11a–d. Dimerization of 11a–d has been carried
out by employing piperazine in the presence of potassi-
um carbonate in refluxing acetone to provide the dimers
(12a–d) linked through a piperazine moiety. These nitro
dimers have been reduced with tin chloride to give the
amino dimers (13a–d). Finally, deprotection of the
During the course of preparation of structurally mod-
ified PBDs, it has been observed that in the literature

A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
387
thioacetal group employing the literature procedure12,26
affords the desired target molecules 6a–d (Scheme 1).
the two DC-81 PBD subunits. Further, it is interesting
to observe that as the linker chain increases from two
to four carbons there is a decrease in the DNA binding
ability, while on further increase to five carbon chain
the DNA stabilization is moderately enhanced, as in
the case of 6d. These results indicate that incorporation
of a piperazine moiety in the alkane spacer linker
enhances the DNA binding potential significantly par-
ticularly in the case of 6a (Table 1). Melting tempera-
ture (DTm) curves of CT-DNA alone and for a
representative ligand complex with CT-DNA for com-
pound 6a have been illustrated in Figure 2. In compar-
ison to DC-81 and the previously synthesized PBD
dimer DSB-120, these PBD dimers linked through a
piperazine moiety in its alkane chain spacer are likely
to possess better hydrophobic interactions with the
DNA to make them more stable ligands. In this pro-
cess, the linker length could play an important role
in the increase or decrease in the binding ability based
on the favorability of such interactions. Furthermore,
2.1.1. Thermal denaturation studies. The DNA binding
ability of the novel piperazine-linked PBD dimers
(6a–d) was investigated by thermal denaturation studies
using calf thymus (CT) DNA. The studies for these
compounds (6a–d) were carried out by DNA/ligand
molar ratios of 5:1. The increase in the helix melting
temperature (DTm) for each compound was examined
after 0, 18, and 36 h incubation at 37 ꢀC. Data for
compounds 1 and 4 are included in Table 1 for com-
parison. The compound 6a elevates the helix melting
temperature of the CT-DNA by 24.0 ꢀC after incuba-
tion at 37 ꢀC for 18 h. The naturally occurring DC-81
(1) gives a DTm of 0.7 ꢀC, whereas synthetic DC-81 di-
mer 4 (DSB-120) gives a DTm of 15.1 ꢀC under identi-
cal experimental conditions. This result illustrates the
significant effect of introducing the piperazine moiety
side-armed with symmetrical alkane spacers between
HO
O
NO2
OCH3
O
Br(H2C)n
Br(H2C)n
ii
i
OCH3
OCH3
H3CO
H3CO
H3CO
O
O
O
9a-d
8a-d
7
iii
CH(SEt)2
O
NO2
OH
O
NO2
N
Br(H2C)n
Br(H2C)n
iv
H3CO
H3CO
O
O
11a-d
10a-d
v
(EtS)2HC
O2N
O
O
(H2C)n
N
N (CH2)n O
CH(SEt)2
NO2
N
N
OCH3
12a-d
H3CO
O
vi
(EtS)2HC
H2N
O
(H2C)n
O
N
N (CH2)n O
CH(SEt)2
NH2
N
N
OCH3
13a-d
H3CO
O
vii
N
O
(H2C)n
O
N
N
(CH2)n O
N
H
H
N
OCH3
N
H3CO
6a-d
n = 2, 3, 4, 5
O
Scheme 1. Reagents and conditions: (i) Br(CH2)nBr, K2CO3, CH3COCH3, reflux, 48 h. (ii) SnCl4/HNO3, CH2Cl2, ꢀ25 ꢀC, 5 min. (iii) 2 N LiOH,
THF, MeOH, H2O (3:1:1), rt, 12 h. (iv) SOCl2 then DMF, THF, H2O, 2(S)-pyrrolidine-carboxaldehyde diethylthioacetal, Et3N, 3 h. (v) Piperazine,
K2CO3, CH3COCH3, reflux, 48 h. (vi) SnCl2Æ2H2O, MeOH, reflux, 1.5 h. (vii) Bi(OTf)3ÆxH2O or HgCl2, CaCO3, CH3CN–H2O (4:1), 3–8 h.

388
A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
Table 1. Thermal denaturation data for piperazine-linked PBD dimers with calf thymus (CT) DNA
[PBD]:[DNA] molar ratiob
DTm (ꢀC) after incubation at 37 ꢀC for
a
PBD dimers
0 h
18 h
36 h
6a
6b
1:5
1:5
1:5
1:5
1:5
1:5
21.8
14.6
10.4
18.4
10.2
0.3
24.0
16.1
14.5
18.4
15.1
0.7
25.0
16.2
15.4
18.4
15.4
0.7
6c
6d
DSB-120 (4)
DC-81 (1)
a For CT-DNA alone at pH 7.00 0.01, Tm = 69.6 ꢀC 0.01 (mean value from 10 separate determinations), all DTm values are 0.1–0.2 ꢀC.
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 lM and ligand concentration = 20 lM in aqueous sodium phosphate
buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
non-small cell lung, colon, CNS, melanoma, ovarian,
a
3,0000
renal, prostate, and breast cancers). The concentration
causing 50% cell growth inhibition (GI50), total cell
growth inhibition (TGI, 0% growth), and 50% cell
death (LC50, ꢀ50% growth) compared with the con-
Abs
trol was calculated (Table 2). The mean graph mid-
point values of log10 TGI, and log10 LC50 as well as
log10 GI50 for 6a–d are listed in Table 3. However,
0,0000
3,0000
the cytotoxicity (IC50) of compounds 1 and 4 has been
reported as 0.33 and 0.0005 lM. In general, all the
compounds are active but compounds 6b and 6c show
significant cytotoxic activity against some human can-
cer cell lines.
40
50
60.0
70.0
80.0
90.0
Temperature (˚C)
b
It is observed from the data given in Table 3 that the
length of the linker plays an important role for the cyto-
toxic activity. Interestingly, as the alkane chain increases
from two to four, the cytotoxic activity markedly increas-
es. However, the increase of the carbon spacer to further
longer chain (five carbon) in the case of 6d moderately
reduces the cytotoxicity nevertheless comparable to 6b.
The cytotoxicity data cannot be correlated to the DNA
thermal denaturation values. As demonstrated by the
mean graph pattern for compound 6c (Fig. 3), it is seen
that it exhibits not only significant activity but more sen-
Abs
0,0000
50
60.0
70.0
80.0
90.0
100.0
Temperature (˚C)
Figure 2. Thermal melting curves for CT-DNA and its ligand complex
[CT-DNA without ligand (a), CT-DNA + compound 6a (b)].
Table 1 shows that the Tm for global melting of this
duplex DNA increases upon incubation in most of
these dimers. This is expected due to initial non-cova-
lent recognition and binding of the molecule to its
favored DNA sites and then followed by slower
covalent reversible fixation of the bound ligand.
Table 3. Log10 GI50 Log10 TGI and Log10 LC50 mean graph midpoints
(MG_MID) of in vitro cytotoxicity data for the compounds 6a–d
against human tumor cell lines
Compound
Log10 GI50
Log10 TGI
Log10 LC50
6a
6b
6c
6d
ꢀ4.69
ꢀ5.19
ꢀ7.70
ꢀ5.14
ꢀ4.16
ꢀ4.22
ꢀ5.95
ꢀ4.26
ꢀ4.03
ꢀ4.01
ꢀ4.47
ꢀ4.04
2.1.2. Cytotoxicity. These compounds 6a–d were evalu-
ated for in vitro activity against sixty human tumor
cell lines, derived from nine cancer types (leukemia,
Table 2. Log GI50 (concentration in mol/L causing 50% growth inhibition) values for piperazine-linked PBD dimersa
Cancer
Compound 6a
Compound 6b
Compound 6c
Compound 6d
Leukemia
Non-small-cell lung
Colon
ꢀ5.70
ꢀ4.57
ꢀ4.58
ꢀ4.80
ꢀ4.70
ꢀ4.35
ꢀ4.60
ꢀ4.40
ꢀ4.53
ꢀ6.69
ꢀ6.60
ꢀ5.78
ꢀ5.71
ꢀ5.47
ꢀ5.36
ꢀ5.70
ꢀ5.57
ꢀ6.07
ꢀ7.81
ꢀ7.68
ꢀ7.66
ꢀ7.97
ꢀ7.78
ꢀ7.54
ꢀ7.59
ꢀ7.31
ꢀ7.58
ꢀ5.48
ꢀ5.47
ꢀ5.59
ꢀ5.45
ꢀ5.62
ꢀ5.32
ꢀ6.88
ꢀ5.39
ꢀ5.49
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
a Each cancer type represents the average of six to eight different cancer cell lines.

Figure 3. Log10 GI50, Log10 TGI, and Log10 LC50 data from the NCI 60 cell line screen for PBD dimer 6c.

390
A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
1
sitivity for certain cancer cell lines and has been selected
for in vivo evaluation.
8a as a white solid (236 mg, 82%). Mp: 110–113 ꢀC. H
NMR (CDCl3): d 3.64 (t, 2H, J = 6.2 Hz), 3.90 (s, 3H),
3.95 (s, 3H), 4.19 (t, 2H, J = 6.3 Hz), 6.88 (d, 1H,
J = 6.1 Hz), 7.50 (s, 1H), 7.65 (d, 1H, J = 8.4 Hz). MS
(EI) m/z 289 [M]+Å.
3. Conclusion
Introduction of a piperazine moiety in the alkane spacer
of the PBD dimers increased the DNA binding activity
considerably in one of the PBD dimers 6a. More impor-
tantly, these newly designed piperazine incorporated
PBD dimers possess interesting and promising in vitro
growth inhibition in the 60 cancer cell line assay. There-
fore, inclusion of a piperazine moiety in the linker spacer
not only enhances the potency of such PBD dimers but
also their solubility profile. The in vitro cytotoxicity re-
sults obtained from this investigation will also be helpful
in the design of improved interstrand DNA cross-link-
ing molecules.
4.3. Methyl-4-(3-bromopropoxy)-3-methoxybenzoate (8b)
The compound 8b was prepared following the method
described for the compound 8a, employing vanillin
methyl ester 7 (182 mg, 1 mmol) and 1,3-dibromopro-
pane (605 mg, 3 mmol), and the crude product was puri-
fied by column chromatography (10% EtOAc–hexane)
to afford the compound 8b as a white solid (260 mg,
1
86%). Mp: 117–118 ꢀC. H NMR (CDCl3): d 2.37–2.42
(m, 2H), 3.62 (t, 2H, J = 6.2 Hz), 3.92 (s, 3H), 3.96 (s,
3H), 4.20 (t, 2H, J = 6.1 Hz), 6.85 (d, 1H, J = 8.3 Hz),
7.50 (s, 1 H), 7.65 (d, 1H, J = 8.5 Hz). MS (EI) m/z
304 [M+1]+Å.
4. Experimental
4.4. Methyl-4-(4-bromobutoxy)-3-methoxybenzoate (8c)
4.1. Synthetic chemistry
The compound 8c was prepared following the method
described for the compound 8a, employing vanillin
methyl ester 7 (182 mg, 1 mmol) and 1,4-dibromobutane
(648 mg, 3 mmol), and the crude product was purified by
column chromatography (10% EtOAc–hexane) to afford
the compound 8c as a white solid (260 mg, 82%). Mp:
122–124 ꢀC. H NMR (CDCl3): d 1.98–2.20 (m, 4H),
3.50 (t, 2H, J = 6.4 Hz), 3.93 (s, 3H), 3.96 (s, 3H), 4.15
(t, 2H, J = 6.3 Hz), 6.82 (d, 1H, J = 8.2 Hz), 7.50 (s,
1H), 7.60 (d, 1H, J = 8.4 Hz). MS (EI) m/z 317 [M]+Å.
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. Chro-
matography was performed using Acme silica gel (100–
200 mesh). 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), acetone
(potassium permanganate), and acetonitrile (phospho-
rous pentoxide).
1
4.5. Methyl-4-(5-bromopentyloxy)-3-methoxybenzoate
(8d)
1H NMR spectra were recorded on a Varian Gemini
200 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 resolution mass spectra were recorded
on a VG-7070H Micromass mass spectrometer at
200 ꢀC, 70 eV with trap current of 200 lA, and 4 kV
acceleration voltage. FABMS spectra were recorded
The compound 8d was prepared following the method de-
scribed for the compound 8a, employing vanillin methyl
ester 7 (182 mg, 1 mmol) and 1,5-dibromopentane
(689mg, 3 mmol), and the crude product was purified
by column chromatography (10% EtOAc–hexane) to af-
ford the compound 8d as a white solid (275 mg, 83%).
Mp: 125–127 ꢀC. H NMR (CDCl3): d 1.62–2.05 (m,
6H), 3.45 (t, 2H,J = 6.4 Hz), 3.93 (s, 3H), 3.96 (s, 3H),
4.15 (t, 2H,J = 6.3 Hz), 6.82 (d, 1H, J = 8.2 Hz), 7.55 (s,
1H), 7.60 (d, 1H, J = 8.8 Hz). MS (EI) m/z 331 [M]+Å.
1
on
LSIMS-VG-AUTOSPEC-Micromass.
Melting
4.6. Methyl-4-(2-bromoethoxy)-5-methoxy-2-nitrobenzo-
ate (9a)
points were recorded on Electrothermal 9100 and are
uncorrected.
A freshly prepared mixture of stannic chloride (260 mg,
1.156 mmol) and fuming nitric acid (98 mg, 1.56 mmol)
in dichloromethane was added dropwise for over 5 min
with stirring to a solution of methyl-4-(2-bromoeth-
oxy)-3-methoxybenzoate (8a) (289 mg, 1 mmol) in
dichloromethane (30 mL) at ꢀ25 ꢀC (dry ice/carbon tet-
rachloride). The mixture was maintained at ꢀ25 ꢀC for a
further 5 min, quenched with water (20 mL), and then
allowed to return to room temperature. The organic
layer was separated and the aqueous layer was extracted
with dichloromethane (2 · 20 mL). The combined
organic phase was dried over anhydrous Na2SO4, evap-
orated under vacuum, and purified by column chroma-
4.2. Methyl-4-(2-bromoethoxy)-3-methoxybenzoate (8a)
To a solution of vanillin methyl ester 7 (182 mg,
1 mmol) in acetone (30 mL) were added anhydrous
potassium carbonate (553 mg, 4 mmol) and 1,2-dib-
romoethane (561 mg, 3 mmol) and the mixture was re-
fluxed for 48 h. The progress of the reaction was
monitored by TLC. After completion of the reaction,
potassium carbonate was removed by filtration and the
solvent was evaporated under vacuum to get the crude
product. This was further purified by column chroma-
tography (10% EtOAc–hexane) to afford the compound

A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
391
tography (20% EtOAc–hexane) to afford the compound
9a as an oily liquid (300 mg, 90%). 1H NMR (CDCl3): d
3.65 (t, 2H, J = 6.2 Hz), 3.90 (s, 3H), 3.95 (s, 3H), 4.19
(t, 2H, J = 6.3 Hz), 7.05 (s, 1H), 7.42 (s, 1H). MS (EI)
m/z 334 [M]+Å.
2H, J = 6.2 Hz), 7.20 (s, 1H), 7.40 (s, 1H). MS (EI)
m/z 334 [M]+Å.
4.12. 4-(4-Bromobutoxy)-5-methoxy-2-nitrobenzoic acid
(10c)
4.7. Methyl-4-(3-bromopropoxy)-5-methoxy-2-nitro-
benzoate (9b)
The compound 10c was prepared by the same method
described earlier for 10a employing 9c (362 mg,
1 mmol), which gave 10c as a white solid (324 mg,
93%). Mp 176–178 ꢀC. H NMR (CDCl3): d 2.0–2.15
(m, 4H), 3.50 (t, 2H, J = 6.3 Hz), 3.96 (s, 3H), 4.15 (t,
2H, J = 6.1 Hz), 7.18 (s, 1H), 7.38 (s, 1H). MS (EI) m/
z 348 [M]+Å.
1
Compound 8b (303 mg, 1 mmol) was nitrated by the
similar method described earlier for 9a to afford the
compound 9b as a pale yellow oily liquid (313 mg,
1
90%). H NMR (CDCl3): d 2.30–2.45 (m, 2H), 3.62 (t,
2H, J = 6.3 Hz), 3.89 (s, 3H), 3.96 (s, 3H), 4.12 (t, 2H,
J = 6.1 Hz), 7.10 (s, 1H), 7.22 (s, 1H). MS (EI) m/z
347 [Mꢀ1]+Å.
4.13. 4-(5-Bromopentyloxy)-5-methoxy-2-nitrobenzoic
acid (10d)
4.8. Methyl-4-(4-bromobutoxy)-5-methoxy-2-nitrobenzo-
ate (9c)
The compound 10d was prepared by the same method
described earlier for 10a employing 9d (376 mg, 1 mmol),
which gave 10d as a white solid (330 mg, 91%). 1H NMR
(CDCl3): d 1.9–2.1 (m, 6H), 3.45 (t, 2H, J = 6.4 Hz), 3.95
(s, 3H), 4.1–4.15 (t, 2H, J = 6.2 Hz), 7.18 (s, 1H), 7.38 (s,
1H) MS (EI) m/z 362 [M]+Å.
Compound 8c (317 mg, 1 mmol) was nitrated by the
similar method described earlier for 9a to afford the
compound 9c as a pale yellow oily liquid (218 mg,
1
88%). H NMR (CDCl3): d 1.95–2.10 (m, 4H), 3.45 (t,
2H, J = 6.2 Hz), 3.88 (s, 3H), 3.95 (s, 3H), 4.10 (t, 2H,
J = 6.10 Hz), 7.05 (s, 1H), 7.40 (s, 1H). MS (EI) m/z
362 [M]+Å.
4.14. (2S)-N-[4-(2-Bromoethoxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal
(11a)
4.9. Methyl-4-(5-bromopentyloxy)-5-methoxy-2-nitro-
benzoate (9d)
DMF was added to a stirred suspension of compound
10a (320 mg, 1 mmol) and thionyl chloride (476 mg,
4 mmol) in dry benzene (15 mL) and the stirring was
continued for 6 h. The benzene was evaporated under
vacuum and the resultant oil was dissolved in dry
THF (20 mL) and added dropwise over a period of
30 min to a stirred suspension of (2S)-pyrrolidine-2-car-
boxaldehyde diethyl thioacetal (205 mg, 1 mmol), trieth-
ylamine (303 mg, 3 mmol), and ice water (2 mL) in dry
THF cooled in an ice bath. After the addition was com-
pleted, the reaction mixture was brought to ambient
temperature and stirred for an additional hour. The
THF was evaporated under vacuum and the aqueous
layer was washed with ethyl acetate (10 mL). The aque-
ous phase was then adjusted to pH 3 using 6 N HCl and
extracted with ethyl acetate and washed with brine,
dried over anhydrous Na2SO4, and evaporated under
vacuum. The crude product was purified by column
chromatography (30% EtOAc–hexane) to afford the
compound 11a as an oily liquid (456 mg, 90%). 1H
NMR (CDCl3): d 1.22–1.40 (m, 6H), 1.82–2.50 (m,
4H), 2.60–2.90 (m, 4H), 3.19–3.28 (m, 2H), 3.65 (t,
2H, J = 6.2 Hz), 3.95 (s, 3H), 4.35 (t, 2H, J = 6.3 Hz),
4.59–4.71 (m, 1H), 4.82 (d, 1H, J = 4.2 Hz), 6.78 (s,
1H), 7.70 (s, 1H). MS (FAB) 507 [M]+Å.
Compound 8d (331 mg, 1 mmol) was nitrated by the
similar method described earlier for 9a to afford the
methyl 4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzo-
1
ate 9d as a light yellow oily liquid (345 mg, 91%). H
NMR (CDCl3): d 1.60–2.00 (m, 6H), 3.40 (t, 2H), 3.90
(s, 3H), 3.95 (s, 3H), 4.20 (t, 2H), 7.05 (s, 1H), 7.40 (s,
1H). MS (EI) m/z 376 [M]+Å.
4.10. 4-(2-Bromoethoxy)-5-methoxy-2-nitrobenzoic acid
(10a)
A solution of 2 N lithium hydroxide monohydrate
(1.221 mL) was added to a solution of methyl 4-(2-bromo-
ethoxy)-5-methoxy-2-nitrobenzoate (9a) (334 mg,
1 mmol) in THF–H2O–MeOH (3:1:1) and the mixture
was stirred at room temperature for 12 h. After most
of the THF and methanol had been evaporated, the
aqueous phase was acidified with 12 N HCl to pH 7
and extracted with ethyl acetate to give the
compound 10a as a white solid (288 mg, 90%). Mp
120–125 ꢀC. 1H NMR (CDCl3):
d 3.64 (t, 2H,
J = 6.3 Hz), 3.98 (s, 3H), 4.20 (t, 2H, J = 6.2 Hz),
7.20 (s, 1H), 7.42 (s, 1H). MS (EI) m/z 320 [M]+Å.
4.15. (2S)-N-[4-(3-Bromopropoxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal
(11b)
4.11. 4-(3-Bromopropoxy)-5-methoxy-2-nitrobenzoic acid
(10b)
The compound 10b was prepared by the same method
described earlier for 10a employing 9b (348 mg,
1 mmol), which gave 10b as a white solid (297 mg,
89%). Mp 118–120 ꢀC. H NMR (CDCl3): d 2.28–2.48
(m, 2H), 3.6 (t, 2H, J = 6.4 Hz), 3.98 (s, 3H), 4.25 (t,
The compound 11b was prepared following the method
described for the compound 11a by employing the com-
pound 10b (334 mg, 1 mmol) and (2S)-pyrrolidine-2-car-
boxaldehyde diethyl thioacetal, which gave the product
1
1
as an oily liquid (464 mg, 89%). H NMR (CDCl3): d

392
A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
1.30–1.42 (m, 6H), 1.85–2.50 (m, 6H), 2.70–2.85 (m,
4H), 3.20–3.35 (m, 2H), 3.65 (t, 2H, J = 6.20 Hz), 3.95
(s, 3H), 4.29 (t, 2H, J = 6.3 Hz), 4.63-4.75 (m, 1H),
4.85 (d, 1H, J = 4.2 Hz), 6.79 (s, 1H), 7.70 (s, 1H). MS
(FAB) 521 [M]+Å.
azine and 11b (521 mg, 1 mmol), and the crude
product was purified by column chromatography
(80% EtOAc–hexane) to afford the compound 12b as
a white solid (860 mg, 85%). 1H NMR (CDCl3): d
1.28–1.41 (m, 12H), 1.90–2.32 (m, 12H), 2.42–2.88
(m, 20H), 3.18–3.32 (m, 4H), 3.96 (s, 6H), 4.18 (t,
4H, J = 6.4 Hz), 4.62–4.75 (m, 2H), 4.82 (d, 2H,
J = 4.30 Hz), 6.75 (s, 2H), 7.67 (s, 2H). MS (FAB)
967 [M+1]+Å.
4.16. (2S)-N-[4-(4-Bromobutoxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal
(11c)
Thecompound11cwaspreparedfollowingthemethodde-
scribed for the compound 11a by employing the com-
pound 10c (348 mg, 1 mmol) and (2S)-pyrrolidine-2-
carboxaldehyde diethyl thioacetal, which gave 11c as an
4.20. 1,10-[[1,4-Di(butane-1,4-diyl)hexahydropiper-
azine]dioxy]-bis[(2-nitro-5-methoxy-1,4-phenylene)car-
bonyl]]-bis[pyrrolidine-2-carboxaldehyde diethyl
thioacetal] (12c)
1
oily liquid (476 mg, 89%). H NMR (CDCl3): d 1.30–
1.43 (m, 6H), 1.85–2.38 (m, 8H), 2.69–2.88 (m, 4H),
3.20–3.32 (m, 2H), 3.51 (t, 2H, J = 6.4 Hz), 3.97 (s, 3H),
4.16 (t, 2H, J = 6.4 Hz), 4.63–4.76 (m, 1H), 4.88 (d, 1H,
J = 6.4 Hz),6.79(s,1H),7.67(s,1H).MS(FAB)535[M]+Å.
The compound 12c was prepared according to the
method described for the compound 12a, employing
piperazine and 11c (535 mg, 1 mmol), and the crude
product was purified by column chromatography
(80% EtOAc–hexane) to afford the compound 12c as
a white solid (875 mg, 88%). 1H NMR (CDCl3): d
1.30–1.43 (m, 12H), 1.70–2.38 (m, 16H), 2.51–2.66
(m, 20H), 3.20–3.32 (m, 4H), 3.97 (s, 6H), 4.18 (t,
4H, J = 6.2 Hz), 4.62–4.76 (m, 2H), 4.87 (d, 2H,
J = 4.20 Hz), 6.79 (s, 2H), 7.63 (s, 2H). MS (FAB)
995 [M]+Å.
4.17. (2S)-N-[4-(5-Bromopentyloxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal
(11d)
The compound 11d was prepared following the method
described for the compound 11a by employing the com-
pound 10d (362 mg, 1 mmol) and (2S)-pyrrolidine-2-car-
boxaldehyde diethyl thioacetal, which gave 11d as an
4.21. 1,10-[[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]di-
oxy]-bis[(2-nitro-5-methoxy-1,4-phenylene)carbonyl]]-
bis[pyrrolidine-2-carboxaldehyde diethyl thioacetal] (12d)
1
oily liquid (505 mg, 92%). H NMR (CDCl3): d 1.25–
1.43 (m, 6H), 1.60–2.30 (m, 10H), 2.63–2.82 (m, 4H),
3.18–3.30 (m, 2H), 3.42 (t, 2H, J = 6.20 Hz), 3.96 (s,
3H), 4.10 (t, 2H, J = 6.10 Hz), 4.60–4.70 (m, 1H), 4.85
(d, 1H, J = 4.20 Hz), 6.78 (s, 1H), 7.62 (s, 1H). MS
(FAB) 549 [M]+Å.
The compound 12d was prepared following the meth-
od described for the compound 12a, employing piper-
azine and 11d (549 mg, 1 mmol), and the crude
product was purified by column chromatography
(80% EtOAc–hexane) to afford the compound 12d
as a white solid (908 mg, 90%). 1H NMR (CDCl3):
d 1.20–1.40 (m, 12H), 1.50–2.35 (m, 20H), 2.45–2.82
(m, 20H), 3.15–3.30 (m, 4H), 3.96 (s, 6H), 4.06 (t,
4H, J = 6.20 Hz), 4.60–4.75 (m, 2H), 4.80 (d, 2H,
J = 4.20 Hz), 6.75 (s, 2H), 7.60 (s, 2H). MS (FAB)
1023 [M]+Å.
4.18. 1,10-[[1,4-Di(ethane-1,2-diyl)hexahydropiperazine]
dioxy]-bis[(2-nitro-5-methoxy-1,4-phenylene)carbonyl]]-
bis[pyrrolidine-2-carboxaldehyde diethyl thioacetal] (12a)
To a solution of (2S)-N-[4-(2-bromoethoxy)-5-methoxy-
2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde
diethyl
thioacetal (11a) (507 mg, 1 mmol) in dry acetone
(30 mL) were added anhydrous K2CO3 (552 mg,
4 mmol) and the piperazine (43 mg, 0.5 mmol). The
reaction mixture was refluxed for 48 h. The reaction
was monitored by TLC using ethyl acetate/hexane
(8:2) as a solvent system. The potassium carbonate
was removed by suction filtration and the solvent was
evaporated under vacuum. The crude product was puri-
fied by column chromatography (80% EtOAc–hexane)
to afford the compound 12a as a white solid (845 mg,
4.22. 1,10-[[1,4-Di(ethane-1,2-diyl)hexahydropiperazine]di-
oxy]-bis[(2-amino-5-methoxy-1,4-phenylene)carbonyl]]-
bis[pyrrolidine-2-carboxaldehyde diethyl thioacetal] (13a)
The compound 12a (939 mg, 1 mmol) dissolved in
methanol (20 mL) and SnCl2Æ2H2O (2.256 g, 10 mmol)
added was refluxed for 1.5 h. The reaction mixture
was cooled and the methanol was evaporated under
vacuum, and the residue was carefully adjusted to
pH 8 with saturated NaHCO3 solution and then
extracted with ethyl acetate (2 · 30 mL). The com-
bined organic phase was washed with brine
(15 mL), dried over anhydrous Na2SO4, and evapo-
rated under vacuum to afford the crude amino dieth-
yl thioacetal 13a as a yellow oil (747 mg, 85%). 1H
NMR (CDCl3): d 1.20–1.40 (m, 12H), 1.60–2.38 (m,
8H), 2.58–2.68 (m, 16H), 2.82 (t, 4H, J = 6.3 Hz),
3.57–3.70 (m, 4H), 3.75 (s, 6H), 4.09 (t, 4H,
J = 6.2 Hz), 4.56–4.70 (m, 4H), 6.19 (s, 2H), 6.78 (s,
2H). MS (FAB) 879 [M+H]+Å.
1
90%). H NMR (CDCl3): d 1.29–1.41 (m, 12H), 1.70–
2.39 (m, 8H), 2.61–2.95 (m, 20H), 3.19–3.30 (m, 4H),
3.92 (s, 6H), 4.20 (t, 4H, J = 6.2 Hz), 4.61–4.72 (m,
2H), 4.82 (d, 2H, J = 4.30 Hz), 6.78 (s, 2H), 7.72 (s,
2H). MS (FAB) 939 [M]+Å.
4.19. 1,10-[[1,4-Di(propane-1,3-diyl)hexahydropiperazine]
dioxy]-bis[(2-nitro-5-methoxy-1,4-phenylene)carbonyl]]-
bis[pyrrolidine-2-carboxaldehyde diethyl thioacetal] (12b)
The compound 12b was prepared following the meth-
od described for the compound 12a, employing piper-

A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
393
4.23. 1,10-[[1,4-Di(propane-1,3-diyl)hexahydropiper-
azine]dioxy]-bis[(2-amino-5-methoxy-1,4-phenylene)car-
bonyl]]-bis[pyrrolidine-2-carboxaldehyde diethyl thioacetal]
(13b)
1.95–2.40 (m, 8H), 2.59–2.97 (m, 12H), 3.50–3.87 (m,
6H), 3.95 (s, 6H), 4.15–4.28 (m, 4H), 6.78 (s, 2H), 7.50
(s, 2H), 7.66 (d, 2H, J = 4.4 Hz). 13C NMR
(CDCl3 + DMSO-d6): d 22.4, 24.0, 29.2, 39.0, 39.3,
39.6, 39.9, 40.1, 40.4, 40.7, 46.8, 53.5, 56.8, 66.8, 110.7,
111.7, 120.2, 140.9, 147.3, 150.5, 163.7. MS (FAB) 631
[M+1]+Å. Anal. Calcd for C34H42N6O6: C, 64.75; H,
6.71; N, 13.32. Found: C, 64.69; H, 6.53; N, 13.22.
The compound 13b was prepared following the
method described for the compound 13a employing
the compound 12b (967 mg, 1 mmol) to afford the
amino diethyl thioacetal 13b as
a
yellow oil
(802 mg, 83%). 1H NMR (CDCl3): d 1.19–1.40 (m,
12H), 1.90–2.35 (m, 12H), 2.45–2.88 (m, 20H),
3.56–3.65 (m, 4H), 3.95 (s, 6H), 4.00 (t, 5H,
J = 6.1 Hz), 4.55–4.88 (m, 4H), 6.20 (s, 2H), 6.75
(s, 2H). MS (FAB) 907 [M]+Å.
4.27. 1,10-[[1,4-Di(propane-1,3-diyl)hexahydropiperazine]di-
oxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyr-
rolo[2,1-c][1,4]benzodiazepin-5-one] (6b)
The compound 6b was prepared following the method
described for the compound 6a employing 13b
(907 mg, 1 mmol), to afford the compound 6b as a pale
4.24. 1,10-[[1,4-Di(butane-1,4-diyl)hexahydropiperazine]dioxy]-
bis[(2-amino-5-methoxy-1,4-phenylene)carbonyl]]-bis[pyr-
rolidine-2-carboxaldehyde diethyl thioacetal] (13c)
25
yellow solid (415 mg, 63%). ½aꢁ +329.0 (c 1.01 CHCl3);
D
1H NMR (CDCl3): d 1.61–2.18 (m, 12H), 2.23–2.75 (m,
12H), 3.40–3.70 (m, 6H), 3.92 (s, 6H), 4.00–4.21 (m,
4H), 6.75 (s, 2H), 7.48 (s, 2H), 7.60 (d, 2H,
J = 4.4 Hz). MS (FAB) 659 [M+1]+Å. Anal. Calcd for
C36H46N6O6: C, 65.71; H, 7.05; N, 12.77. Found: C,
65.67; H, 6.98; N, 12.70.
The compound 13c was prepared following the method
described for the compound 13a employing the com-
pound 12c (995 mg, 1 mmol) to afford the amino diethyl
1
thioacetal 13c as a yellow oil (794 mg, 85%). H NMR
(CDCl3): d 1.20–1.40 (m, 12H), 1.90–2.38 (m, 16H),
2.45–2.75 (m, 20H), 3.57–3.65 (m, 4H), 3.95 (s, 6H),
4.05 (t, 4H, J = 6.2 Hz), 4.56–4.68 (m, 4H), 6.20 (s,
2H), 6.76 (s, 2H). MS (FAB) 935 [M]+Å.
4.28. 1,10-[[1,4-Di(butane-1,4-diyl)hexahydropiperazine]di-
oxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one] (6c)
4.25. 1,10-[[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]di-
oxy]-bis[(2-amino-5-methoxy-1,4-phenylene)carbonyl]]-bis[pyrroli-
dine-2-carboxaldehyde diethyl thioacetal] (13d)
The compound 6c was prepared according to the meth-
od described for the compound 6a employing 13c
(935 mg, 1 mmol), to afford the compound 6c as a pale
26
yellow solid (419 mg, 61%). ½aꢁ +315.05 (c 1.0 CHCl3);
D
The compound 13d was prepared following the method
described for the compound 13a employing the com-
pound 12d (1023 mg, 1 mmol) to afford the amino dieth-
yl thioacetal 13d as a yellow oil (787 mg, 83%). 1H NMR
(CDCl3): d 1.20–1.40 (m, 12H), 1.90–2.38 (m, 20H),
2.45–2.82 (m, 20H), 3.57–3.65 (m, 4H), 3.96 (s, 6H),
4.05 (t, 4H, J = 6.2 Hz), 4.55–4.68 (m, 4H), 6.30 (s,
2H), 6.78 (s, 2H). MS (FAB) 963 [M]+Å.
1H NMR (CDCl3): d 1.61–2.18 (m, 8H), 2.23–2.75 (m,
20H), 3.40–3.70 (m, 6H), 3.95 (s, 6H), 4.10–4.23 (m,
4H), 6.75 (s, 2H), 7.48 (s, 2H), 7.60 (d, 1H,
J = 4.3 Hz). MS (FAB) 687 [M+1]+Å. Anal. Calcd for
C38H50N6O6: C, 66.53; H, 7.34; N, 12.25. Found: C,
66.45; H, 7.34; N, 12.17.
4.29. 1,10-[[1,4-Di(pentane-1,5-diyl)hexahydropiperazine]di-
oxy]bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrol-
o[2,1-c][1,4]benzodiazepin-5-one] (6d)
4.26. 1,10-[[1,4-Di(ethane-1,2-diyl)hexahydropiperazine]di-
oxy]-bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one] (6a)
The compound 6d was prepared following the
method described for the compound 6a employing 13d
(963 mg, 1 mmol), to afford the compound 6d as a pale
A solution of amino thioacetal 13a (878 mg, 1 mmol),
HgCl2 (1.19 g, 4.4 mmol), and CaCO3 (480 mg,
4.8 mmol) in acetonitrile–water (4:1) was stirred slowly
at room temperature overnight until TLC indicated
the complete disappearance of starting material. The
reaction mixture was diluted with ethyl acetate
(30 mL) and filtered through a Celite. The clear yellow
organic supernatant was extracted with ethyl acetate
(2 · 20 mL). The organic layer was washed with
saturated NaHCO3 solution (20 mL) and brine
(20 mL), and the combined organic phase was dried over
anhydrous Na2SO4. The organic layer was evaporated
under vacuum and the crude product was purified by
column chromatography (95% CH2Cl2–MeOH) to af-
ford the compound 6a as a pale yellow solid (410 mg,
65%). This material was repeatedly evaporated from
CHCl3 in vacuum to generate the imine for-
26
yellow solid (450 mg, 63%). ½aꢁ +343.5 (c 1.0 CHCl3);
D
1H NMR (CDCl3): d 1.55–2.38 (m, 20H), 2.60–3.15
(m, 12H), 3.45–3.80 (m, 6H), 3.96 (s, 6H), 4.01–4.20
(m, 4H), 6.81 (s, 2H), 7.50 (s, 2H), 7.78 (d, 1H,
J = 4.6 Hz). 13C NMR (CDCl3 + DMSO-d6): d 22.7,
23.4, 27.6, 28.8, 38.5, 38.8, 39.1, 39.3, 39.6, 39.9, 40.2,
45.9, 50.3, 53.0, 56.5, 109.8, 110.8, 115.0, 119.5, 139.8,
146.9, 149.9, 163.7. MS (FAB) 715 [M+1]+Å. Anal. Calcd
for C40H54N6O6: C, 67.20; H, 7.61; N, 11.76. Found: C,
67.21; H, 7.58; N, 11.71.
4.30. Thermal denaturation studies
The DNA binding affinity of the novel piperazine linked
PBD dimers (6a–d) has been evaluated through thermal
denaturation studies with duplex-form calf thymus
DNA (CT-DNA) using modified reported proce-
26
D
1
m.½aꢁ ¼ þ353:5 (c 1.0 CHCl3); H NMR (CDCl3): d

394
A. Kamal et al. / Bioorg. Med. Chem. 14 (2006) 385–394
dure.27,28 Working solutions in aqueous buffer (10 mM
NaH2PO4/Na2HPO4, 1 mM Na2EDTA, pH
6. Thurston, D. E. Molecular Aspects of Anticancer Drug-
DNA Interactions; The Macmillan Press Ltd.: London,
UK, 1993, pp 54–88.
7. Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley, L.
H. J. Biol. Chem. 1982, 257, 6207–6216.
8. (a) Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433–
465; (b) Kopka, M. L.; Goodless, D. S.; Baiklov, I.;
Grzeskowaik, K.; Cascio, D.; Dickerson, R. E. Biochem-
istry 1994, 33, 13593–13610.
9. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.;
Thurston, D. E. Nucleic Acids Res. 1993, 21, 3671–
3675.
10. Puvvada, M. S.; Forrow, S. A.; Hartley, J. A.; Stephenson,
P.; Gibson, I.; Jenkins, T. C.; Thurston, D. E. Biochem-
istry 1997, 36, 2478–2484.
11. Smellie, M.; Kelland, L. R.; Thurston, D. E.; Souhami, R.
L.; Hartley, J. A. Br. J. Cancer 1994, 70, 48–53.
12. Thurston, D. E.; Bose, D. S.; Thompson, A. S.; Howard,
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Hartley, J. A.; Hurley, L. H. J. Org. Chem. 1996, 61, 8141–
8147.
7.00 0.01) counting CT-DNA (100 lM in phosphate)
and the PBD (20 lM) were prepared by addition of con-
centrated PBD solutions in DMSO to obtain a fixed
[PBD]/[DNA] molar ratio of 1:5. The DNA–PBD solu-
tions are incubated at 37 ꢀC for 0, 18, and 36 h prior to
analysis. Samples are monitored at 260 nm using a Beck-
man DU-7400 spectrophotometer fitted with high per-
formance temperature controller and were heated at
1 ꢀC /min in the range of 40–95 ꢀC. DNA helix coil tran-
sition temperatures (Tm) were obtained from the maxi-
ma in the d(A260)/dT derivative plots. Drug-induced
alterations in DNA melting temperatures are given by:
DTm = Tm (DNA + PBD) ꢀ Tm (DNA alone), where
the Tm value for the PBD-free CT-DNA is 69.6 0.01.
4.31. In vitro evaluation of cytotoxic activity
The compounds 6a–d were evaluated for in vitro activity
against sixty human tumor cell lines, derived from nine
cancer types (leukemia, non-small cell lung, colon,
CNS, melanoma, ovarian, renal, prostate, and breast can-
cers). For each compound, dose–response curves against
each cell line were measured at a minimum of five concen-
trations at 10-fold dilutions. A protocol of 48 h continu-
ous drug exposure was used, and a sulforhodamine B
(SRB) protein assay has been used to estimate cell viabil-
ity or growth.
13. Hartley, J. A.; Berardini, M. D.; Souhami, R. L. Anal.
Biochem. 1991, 193, 131–134.
14. Walton, M. I.; Goddard, P.; Kelland, L. R.; Thurston, D.
E.; Harrap, K. R. Cancer Chemother. Pharmacol. 1996, 38,
431–438.
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, 737–748.
16. Kamal,A.;Rao,M.V.;Laxman,N.;Ramesh,G.;Reddy,G.
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