Remarkable DNA binding affinity and potential anticancer activity of pyrrolo[2,1-c][1,4]benzodiazepine-naphthalimide conjugates linked through piperazine side-armed alkane spacers

Article abstract of DOI:10.1016/j.bmc.2008.06.034

A series of pyrrolobenzodiazepine-naphthalimide conjugates tethered through a piperazine ring system have been designed, synthesized, and evaluated for their anticancer activity. These new conjugates exhibit very high DNA binding affinity and cytotoxic ac

Full text of DOI:10.1016/j.bmc.2008.06.034

Bioorganic & Medicinal Chemistry 16 (2008) 7218–7224
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Remarkable DNA binding affinity and potential anticancer activity
of pyrrolo[2,1-c][1,4]benzodiazepine–naphthalimide conjugates linked
through piperazine side-armed alkane spacers
a
a
a
b
Ahmed Kamal a, , R. Ramu , Venkatesh Tekumalla , G. B. Ramesh Khanna , Madan S. Barkume ,
*
Aarti S. Juvekar b, Surekha M. Zingde b
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre Khargar, Navi Mumbai 410 210, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrrolobenzodiazepine–naphthalimide conjugates tethered through a piperazine ring system
have been designed, synthesized, and evaluated for their anticancer activity. These new conjugates exhi-
bit very high DNA binding affinity and cytotoxic activity against a number of cell lines.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 May 2008
Revised 19 June 2008
Accepted 20 June 2008
Available online 25 June 2008
Keywords:
Pyrrolobenzodiazepines
Naphthalimides
Piperazine ring
DNA binding affinity
Anticancer activity
1. Introduction
mitonafide (1) and amonafide (2), as shown in Figure 1, have been
tested in clinical trials. Mitonafide has been studied in phase I and
Efforts to increase the DNA recognizing ability of DNA interact-
ing agents has led to the approach of dimerizing such molecules. In
the past few years, a variety of dimeric DNA binders have been re-
ported and some of them exhibited potent cytotoxicity and higher
in vivo antitumor activity compared to the monomers.1 Further,
the bifunctional DNA-interactive agents in which the two function-
alities possess two different mechanisms of interaction with DNA
and are capable of recognizing heterogeneous DNA sequences, cou-
pled by a spacer, have also attracted considerable attention as a
new class of antitumor agents.2 In this strategy, coupling of a
groove binder with a DNA-affinic intercalator or coupling two
groove-binding moieties provides a basis for modulating the se-
quence-selective binding behavior or tailoring the hybrid ligands
for mixed-sequence recognition.
Naphthalimides are DNA-intercalating agents with high antitu-
mor activity,1a which bind to DNA by insertion between the base
pairs of the double helix. The main driving forces for their DNA
binding ability are charge transfer interactions and stacking be-
tween the base pairs. The development of naphthalimide class of
compounds as anticancer agents started in the year 1973. Both
phase II clinical trials where it exhibited good activity against solid
tumors.3 Unfortunately, mitonafide was found to possess central
nervous system toxicity. Two other compounds of the bis-naph-
thalimide series, LU 79553 (3) and DMP 840 (4), are reported to
be in clinical trials.4–6 The absence of nitro group in the chromo-
phore of LU 79553 may be beneficial, since the nitro group on
the monomeric compounds was expected to be the cause for the
central nervous system toxicity as observed in the clinical trials
of mitonafide.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of
antitumor agents which include natural products such as anthra-
mycin, tomaymycin, sibiromycin, and DC-81 (5).1c They exert their
biological activity by covalent binding to the minor groove of the
DNA to form an aminal linkage between the electrophilic carbinol-
amine present at the C11 position and the N2 of the guanine.7
Molecular modeling, solution NMR, fluorimetry, and DNA foot-
printing experiments reveal that the PBD monomers recognize
three base pairs of DNA with an alkylating preference for Pu-G-
Pu sequences. Recently, there has been increasing interest in the
design and synthesis of DNA interstrand crosslinking agents as
well as conjugates to enhance the sequence selectivity and in-
crease selectivity for tumor cells.6 One of the recently developed
PBD dimers, SJG-136 (6) with C2-exomethylene substitution has
* Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.034

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
7219
O
N
O
O
Me
N
Me
O
N
N
N
N
O
H
H
3
O
O
H
N
O
Me
H
N
R
N
N
O
H
Me
H
1 R = NO2
2 R = NH2
O
4
O2N
NO2
H
11
N
N
N
HO
H3CO
8
O
O
H
H
N
N
N
H3CO
OCH3
O
O
O
6
5
O
N
O
N
CH2
O
n
H
N
H3CO
O
7a-e
n = 3-5, 8, 10
Figure 1. Chemical structures of mitonafide (1), amonafide (2), LU 79553 (3), DMP 840 (4), DC-81 (5), SJG-136 (6), and PBD–naphthalimide hybrids (7a–e).
been selected for clinical trials.8 Cyclopropaindole–PBD,9,10 pyrrole
and imidazole polyamide–PBD,11–13 and cyclic amine-PBD14 conju-
gates have been designed and synthesized to explore their antican-
cer potential and DNA binding ability. The type of spacer employed
to link the two pharmacophores plays an important role in the de-
sign of such hybrid molecules. An appropriately optimized flexible
linker leads to improved cytotoxicity. In view of the interesting
biological activity exhibited by PBDs, there has been considerable
interest in the structural modification of PBDs in our labora-
tory.15–17 In this endeavor a series of novel PBD–naphthalimide hy-
brids18 (7a–e) have been prepared, which exhibited promising
anticancer activity against numerous cell lines. The National Can-
cer Institute (NCI), USA has investigated the cytotoxicity of a mem-
ber of this series, 7c, using hollow fiber assay. This compound
exhibited a good cell killing ability at a concentration of 37.5 mg/
kg/dose in selected cell lines. In view of its promising activity it
has been selected for detailed studies by the NCI. In continuation
to the efforts in this direction, we now report the synthesis and
biological evaluation of a new series of PBD–naphthalimide hy-
brids, coupled through a piperazine moiety with side-armed sym-
metrical and unsymmetrical alkane spacers.19
etal as the key step. The nitrothioacetals 13a–c were coupled
with 2-[ -(piperazine-1-yl)alkyl]benz[de]isoquinoline-1,3-diones
x
(11a–c) to give C8-linked naphthalimide nitrothioacetals 14a–e.
The nitrothioacetals 13b and 13c, having three and four carbon
spacer, were coupled with piperazine-linked naphthalimide inter-
mediate 11a, having two carbon spacers, providing C8-linked
naphthalimide–piperazine–nitrothioacetals 14b and 14c, consist-
ing of unsymmetrical alkane spacers. The nitrothioacetals, 13a–c
were coupled with the corresponding piperazine-linked naphthal-
imide intermediates 11a–c having identical alkane spacer to give
C8-linked naphthalimide nitrothioacetals 14a, 14d and 14e. The ni-
tro group of these compounds has been efficiently reduced employ-
ing SnCl2ꢀ2H2O to afford the corresponding aminothioacetals 15a–e.
Deprotection of the thioacetal groups with HgCl2 and CaCO3 affor-
ded the desired naphthalimide–PBD hybrids 16a–e (Scheme 2).
3. DNA interactions: thermal denaturation studies
The DNA binding affinity of these new PBD hybrids 16a–e has
been evaluated through thermal denaturation studies with du-
plex-form of calf thymus DNA (CT-DNA) by using modified reported
procedure.20,21 The thermal denaturation studies for these com-
pounds have been carried out at PBD/DNA molar ratios of 1:5.
2. Chemistry
The increase in melting temperature (DTm) for each compound
The synthesis of new PBD hybrids was carried out employing
piperazine-linked naphthalimides 11a–c. These intermediates
were obtained by alkylation of naphthalimide with dibromoalk-
anes followed by coupling with N-Boc piperazine and deprotection
of Boc with trifluoroacetic acid. The mono alkylation of 8 was
achieved by using excess dibromoalkanes (Scheme 1).
has been examined at 0 and 18 h of incubation at 37 °C (Table 1).
Data for DC-81 and DSB-120 are included in Table 1 for comparison.
The results show that these hybrids stabilize the thermal he-
lix ? coil transition (Tm) for CT-DNA duplex more efficiently. There
is a slight increase in melting temperature, when the incubation
time is increased from 0 to 18 h at 37 °C. The new PBD–naphthal-
Synthesis of the other precursor (2S)-N-[4-(x-bromoalkyloxy)-
imide hybrids have shown DTm values ranging from 12.9 to 26.5 °C
5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl-
thioacetals 13a–c was carried out in six steps according to the lit-
erature method,17 starting from the commercially available
after incubation for 18 h. In this series, compound 16d, which has a
three-carbon chain on either side of piperazine, has lower DNA
binding affinity compared to other compounds, and this compound
elevates the hybrid melting temperature by 12.9 °C after incuba-
tion for 18 h. Compound 16b, which has piperazine side armed
vanillin and coupling of 4-(
x-bromoalkyloxy)-5-methoxy-2-nitro-
benzoic acid and (2S)-pyrrolidine-2-carboxaldehyde diethylthioa-

7220
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
O
O
O
iii
NH
O
N CH2
N
N CH2
N
NBoc
NH
n
n
O
O
8
11a-c
10a-c
8
i
n = 2, 3, 4
9
ii
10
Scheme 1. Reagents and conditions: (i) dibromo alkanes, K2CO3, acetonitrile, reflux, 12 h; (ii) N-Boc piperazine, K2CO3, acetonitrile, reflux, 8 h; (iii) CF3COOH, CHCl3, rt, 8 h.
Table 1
HO
Thermal denaturation data for PBD–naphthalimide hybrids (16a–e) with calf thymus
(CT) DNA
H3CO
COOH
12
Compoundb
Induced
D
Tm (°C) after incubation at 37 °C
a
0 h
18 h
16a
16b
16c
16d
16e
DC-81
DSB-120
21.9
25.9
23.1
12.0
19.9
0.3
22.7
26.5
23.9
12.9
20.8
0.7
NO2
Br CH2
O
CH(SEt)2
m
N
H3CO
10.2
15.1
O
a
For CT-DNA alone at pH 7.00 0.01, Tm = 69.4 °C 0.01 (mean value from 10
Tm values are 0.1–0.2 °C.
For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100
and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM
sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
13a-c
separate determinations), all
D
i
b
l
M
O
N
O
l
NO2
CH2
N
n
N
N
CH2
O
CH(SEt)2
m
was observed, followed by an increase from three to four carbons.
This illustrates the significant effect of alkane spacers between
PBD–piperazine–naphthalimide moieties. The spacers in 16b and
16c appear to impart the molecules a better fit in the minor groove
of DNA, leading to enhanced binding affinity. In the same experi-
N
H3CO
14a-e
O
ii
O
N
O
ment, the naturally occurring DC-81 exhibits a
DTm of 0.7 °C and
its dimer (DSB-120) gives a Tm of 15.4 °C. This demonstrates that
D
NH2
CH2
N
n
CH2
O
CH(SEt)2
m
these new PBD hybrids have remarkable DNA binding affinity. It is
interesting to note that these hybrids with unsymmetrical side
N
H3CO
15a-e
chains (16b–c) have higher
DTm values than those with identical
O
side chains (16a and 16c and 16d).
iii
4. Cytotoxicity
O
N
O
N
CH2
N
n
N
Compounds 16a–c have been evaluated for their in vitro cyto-
toxicity in selected human cancer cell lines such as Hop62 (lung),
SiHa (cervix), MCF7 and ZR-75-1 (breast), Colo205 (colon), PC3
(prostate), and A2780 (ovarian), by employing sulforhodamine B
(SRB) assay. The results described in Table 2 show that all the
CH2
O
H
m
N
H3CO
O
16a-e
16a: n = 2, m = 2
16b: n = 2, m = 3
16c: n = 2, m = 4
16d: n = 3, m = 3
16e: n = 4, m = 4
Table 2
In vitro anticancer activity data for the representative PBD–naphthalimide hybrids
(16a–c)
a
Compound
IC50
(l
M)
Hop62b
SiHac
MCF7d
ZR-75-1d
PC3e
Colo205f
A2780g
Scheme 2. Reagents and conditions: (i) compounds 11a–c, K2CO3, acetonitrile,
reflux, 12 h; (ii) SnCl2–2H2O, methanol, reflux, 3.5 h; (iii) HgCl2, CaCO3, acetonitrile/
H2O, rt, 15 h.
16a
16b
16c
ADR
1.2
<1.0
<1.0
1
1.0
0.7
0.8
1.5
1.4
0.5
0.5
0.5
<1.0
<1.0
<1.0
0.6
0.8
0.5
0.5
0.5
1.6
0.5
0.5
2.0
0.5
0.5
0.6
0. 5
with two- and three-carbon chains, has higher DNA binding affin-
ity and elevates the helix melting temperature of CT-DNA by
26.5 °C after incubation at 37 °C for 18 h. The PBD hybrids 16c
and 16d, which have six carbon alkane spacer (m + n), between
PBD and naphthalimide, but differ in carbon spacers between
PBD–piperazine and piperazine–naphthalimide moieties, have
shown different DNA binding affinity. With the increase in the
chain length of the linker connecting the naphthalimide to pipera-
zine from two to three carbons, a decrease in melting temperature
a
Dose of the compound required to inhibit cell growth by 50% compared to
untreated cell controls. Values are derived from IC50 graphs. All experiments were
done in triplicate wells and each experiment was repeated thrice.
b
Lung cancer.
Cervix cancer.
Breast cancer.
Prostate cancer.
Colon cancer.
Ovarian cancer.
c
d
e
f
g

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
7221
new compounds are significantly cytotoxic, with the concentration
of the drug that produced 50% inhibition of cell growth (IC50) rang-
(10% EtOAc–hexane) to afford the compound 9b (280 mg, 88%).
1H NMR (CDCl3): d 2.10–2.40 (m, 2H, CH2), 3.40–3.58 (m, 2H,
CH2Br), 4.22–4.40 (m, 2H, NCH2), 7.75 (t, 2H, J = 7.3 Hz, ArH),
8.20 (d, 2H, J = 8.0 Hz, ArH), 8.60 (d, 2H, J = 7.4 Hz, ArH); MS (EI):
m/z 318 [M]+.
ing from 0.5 to 1.6
shown higher DNA binding affinity, displayed higher cytotoxicity
with IC50 values ranging between 0.5 and 1 M against all the cell
lines examined, when compared to 16a, that comprised symmetri-
cal alkane spacers exhibiting a correlation between Tm and cyto-
lM. Compounds 16b and 16c, which have
l
D
6.4. 2-(4-bromobutyl)benz[de]isoquinoline-1,3-dione (9c)
toxicity of the molecules. Compound 16a exhibited higher
cytotoxicity against A2780, PC3, and SiHa cell lines with IC50 values
ranging from 0.5 to 1.0 lM.
The compound 9c was prepared following the method described
for the preparation of the compound 9a, employing 8 (197 mg,
1 mmol) and 1,4-dibromobutane (648 mg, 3 mmol), and the crude
product was purified by column chromatography (10% EtOAc–hex-
ane) to afford the compound 9c (305 mg, 92%). 1H NMR (CDCl3): d
1.80–2.15 (m, 4H, 2 ꢁ CH2), 3.38–3.52 (m, 2H, CH2Br), 4.05–4.22
(m, 2H, NCH2), 7.75 (t, 2H, J = 7.2 Hz, ArH), 8.20 (d, 2H, J = 8.0 Hz,
ArH), 8.60 (d, 2H, J = 7.4 Hz, ArH); MS (EI): m/z 332 [M]+.
5. Conclusion
A series of new PBD conjugates, in which DNA covalent binding
PBD moiety conjugated with a DNA-intercalating naphthalimide
ring system, through piperazine moiety with carbon side chain
arms have been designed, synthesized, and evaluated for their
biological activity. These unique bifunctional compounds exhibit
remarkable DNA binding ability in comparison to some of the cross
linking agents. These compounds also exhibit potent in vitro anti-
tumor activity against some of the cancer cell lines examined.
6.5. 2-[20-[4-(tert-Butoxycarbonyl)piperazine-1-
yl]ethyl]benz[de]isoquinoline-1,3-dione (10a)
To
a solution of 2-(2-bromoethyl)benz[de]isoquinoline-1,3-
dione (9a) (304 mg, 1 mmol) in dry acetone (30 mL) were added
anhydrous K2CO3 (552 mg, 4 mmol) and the N-Boc piperazine
(186 mg, 1 mmol). The reaction mixture was refluxed for 8 h. The
reaction was monitored by TLC using ethyl acetate/hexane (4:1)
as a solvent system. The potassium carbonate was removed by suc-
tion filtration, the solvent was evaporated under reduced pressure,
and the crude product was purified by column chromatography
(80% EtOAc–hexane) to afford the compound 10a (348 mg, 85%).
1H NMR (CDCl3): d 1.42 (s, 9H, C(CH3)3), 2.52 (t, 4H, J = 4.9 Hz,
2 ꢁ NCH2), 2.68 (t, 2H, J = 6.3 Hz, NCH2), 3.35 (t, 4H, J = 4.6 Hz,
2 ꢁ NCH2), 4.29 (t, 2H, J = 6.1 Hz, NCH2), 7.73 (t, 2H, J = 7.4 Hz,
ArH), 8.12 (d, 2H, J = 8.4 Hz, ArH), 8.55 (d, 2H, J = 7.2 Hz, ArH);
MS (FAB): 410 [M+1]+.
6. Experimental
6.1. Chemistry
Reaction progress was monitored by thin-layer chromatogra-
phy (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). Most of the reaction solvents were
purified by distillation under nitrogen from the indicated drying
agent and used fresh: dichloromethane (calcium hydride), acetone
(potassium permanganate), and acetonitrile (phosphorous pentox-
ide). 1H NMR spectra were recorded on a Varian Gemini 200 MHz
spectrometer using tetramethyl silane (TMS) as an internal stan-
dard. Chemical shifts are reported in parts per million (ppm)
downfield from tetramethyl silane. Spin multiplicities are de-
scribed 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
6.6. 2-[30-[4-(tert-Butoxycarbonyl)piperazine-1-
yl]propyl]benz[de]isoquinoline-1,3-dione (10b)
The compound 10b was prepared following the method
described for the preparation of the compound 10a, employing
N-Boc piperazine and 9b (318 mg, 1 mmol), and the crude product
was purified by column chromatography (80% EtOAc–hexane) to
afford the compound 10b (364 mg, 86%). 1H NMR (CDCl3): d 1.40
(s, 9H, C(CH3)3), 1.82–2.0 (m, 2H, CH2), 2.40–2.60 (m, 6H,
3 ꢁ NCH2), 3.22–3.40 (m, 4H, 2 ꢁ NCH2), 4.10–4.25 (m, 2H,
NCH2), 7.75 (t, 2H, J = 7.3 Hz, ArH), 8.18 (d, 2H, J = 8.0 Hz, ArH),
8.55 (d, 2H, J = 7.5 Hz, ArH); MS (FAB): 424 [M+1]+.
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.
6.2. 2-(2-bromoethyl)benz[de]isoquinoline-1,3-dione (9a)
6.7. 2-[40-[4-(tert-Butoxycarbonyl)piperazine-1-
yl]butyl]benz[de]isoquinoline-1,3-dione (10c)
To a solution of compound 8 (197 mg, 1 mmol) in acetonitrile
(30 mL), anhydrous potassium carbonate (553 mg, 4 mmol) and
1,2-dibromoethane (561 mg, 3 mmol) were added and the mixture
was refluxed for 12 h. After completion of the reaction, potassium
carbonate was removed by filtration and the solvent was evapo-
rated under reduced pressure to get the crude product. This was
further purified by column chromatography (10% EtOAc–hexane)
to afford the compound 9a (273 mg, 90%). 1H NMR (CDCl3): d
3.62 (t, 2H, J = 7.1 Hz, CH2Br), 4.56 (t, 2H, J = 7.1 Hz, NCH2), 7.74
(t, 2H, J = 7.6 Hz, ArH), 8.18 (d, 2H, J = 8.2 Hz, ArH), 8.58 (d, 2H,
J = 7.2 Hz, ArH); MS (EI): m/z 304 [M]+.
The compound 10c was prepared following the method
described for the preparation of the compound 10a, employing
N-Boc piperazine and 9c (332 mg, 1 mmol), and the crude product
was purified by column chromatography (80% EtOAc–hexane) to
afford the compound 10c (372 mg, 85%). 1H NMR (CDCl3): d 1.40
(s, 9H, C(CH3)3), 1.60–1.80 (m, 4H, 2 ꢁ CH2), 2.50–2.72 (m, 6H,
3 ꢁ NCH2), 3.20–3.35 (m, 4H, 2 ꢁ NCH2), 4.10–4.24 (m, 2H,
NCH2), 7.75 (t, 2H, J = 7.3 Hz, ArH), 8.20 (d, 2H, J = 8.2 Hz, ArH),
8.60 (d, 2H, J = 7.6 Hz, ArH); MS (FAB): 437 [M]+.
6.3. 2-(3-bromopropyl)benz[de]isoquinoline-1,3-dione (9b)
6.8. 2-[20-(Piperazine-1-yl)ethyl]benz[de]isoquinoline-1,3-
The compound 9b was prepared following the method de-
scribed for the preparation of the compound 9a, employing 8
(197 mg, 1 mmol) and 1,3-dibromopropane (605 mg, 3 mmol),
and the crude product was purified by column chromatography
dione (11a)
To a solution of 10a (409 mg, 1 mmol) in chloroform (15 mL)
was added trifluoroacetic acid (1.14 g, 10 mmol) and the mixture

7222
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
was stirred at room temperature for 12 h. The solution was care-
fully adjusted to pH 8 with saturated NaHCO3 solution and then
extracted with chloroform (2ꢁ 15 mL). The combined organic
phase was washed with brine (15 mL), dried over anhydrous
Na2SO4, and evaporated under reduced pressure to afford the com-
pound 11a (238 mg, 77%). 1H NMR (CDCl3): d 2.45–2.65 (m, 6H,
3 ꢁ NCH2), 2.80–2.92 (m, 4H, 2 ꢁ NCH2), 4.20–4.35 (m, 2H,
NCH2), 7.75 (t, 2H, J = 7.3 Hz, ArH), 8.20 (d, 2H, J = 8.2 Hz, ArH),
8.60 (d, 2H, J = 7.6 Hz, ArH); MS (EI): m/z 310 [M+1]+.
6 ꢁ NCH2, 2 ꢁ SCH2), 3.15–3.30 (m, 2H, CH2), 3.92 (s, 3H, OCH3),
4.10–4.20 (m, 2H, OCH2), 4.26–4.36 (m, 2H, NCH2), 4.60–4.70 (m,
1H, CH), 4.82 (d, 1H, J = 4.3 Hz, CH(SEt)2), 6.77 (s, 1H, ArH), 7.65
(s, 1H, ArH), 7.75 (t, 2H, J = 7.4 Hz, ArH), 8.20 (d, 2H, J = 8.0 Hz,
ArH), 8.6 (d, 2H, J = 7.6 Hz, ArH); FABMS: 750 [M]+.
6.13. (2S)-N-{4-[4-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-butyl]-oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (14c)
6.9. 2-[30-(Piperazine-1-yl)propyl]benz[de]isoquinoline-1,3-
dione (11b)
The compound 14c was prepared following the method de-
scribed for the compound 14a, employing 13c (535 mg, 1 mmol)
and 11a (309 mg, 1 mmol), and the crude product was purified
by column chromatography (5% MeOH–EtOAc) to afford the com-
pound 14c (611 mg, 80%). 1H NMR (CDCl3): d 1.25–1.40 (m, 6H,
2 ꢁ SCH2CH3), 1.60–2.15 (m, 8H, 4 ꢁ CH2), 2.35–2.85 (m, 16H,
6 ꢁ NCH2, 2 ꢁ SCH2), 3.15–3.30 (m, 2H, CH2), 3.92 (s, 3H, OCH3),
4.10–4.15 (m, 2H, OCH2), 4.22–4.36 (m, 2H, NCH2), 4.60–4.72 (m,
1H, CH), 4.80 (d, 1H, J = 4.2 Hz, CH(SEt)2), 6.75 (s, 1H, ArH), 7.60
(s, 1H, ArH), 7.75 (t, 2H, J = 7.4 Hz, ArH), 8.20 (d, 2H, J = 8.2 Hz,
ArH), 8.56 (d, 2H, J = 7.6 Hz, ArH); MS (FAB): 765 [M+1]+.
The compound 11b was prepared following the method de-
scribed for the preparation of the compound 11a, employing the
compound 10b (424 mg, 1 mmol) to afford the compound 11b
(246 mg, 76%). 1H NMR (CDCl3): d 1.80–2.0 (m, 2H, CH2), 2.25–
2.40 (m, 6H, 3 ꢁ NCH2), 2.85–3.0 (m, 4H, 2 ꢁ NCH2), 4.15–4.30
(m, 2H, NCH2), 7.75 (t, 2H, J = 7.4 Hz, ArH), 8.20 (d, 2H, J = 8.0 Hz,
ArH), 8.60 (d, 2H, J = 7.5 Hz, ArH); MS (EI): m/z 324 [M+1]+.
6.10. 2-[40-(Piperazine-1-yl)butyl]benz[de]isoquinoline-1,3-
dione (11c)
6.14. (2S)-N-{4-[3-[4-[3-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)propyl]piperazin-1-yl]propyl]-oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (14d)
The compound 11c was prepared following the method de-
scribed for the preparation of the compound 11a, employing the
compound 10c (438 mg, 1 mmol) to afford the compound 11c
(263 mg, 78%). 1H NMR (CDCl3): d 1.50–1.80 (m, 4H, 2 ꢁ CH2),
2.22–2.45 (m, 6H, 3 ꢁ NCH2), 2.80–2.96 (m, 4H, 2 ꢁ NCH2), 4.05–
4.20 (m, 2H, NCH2), 7.75 (t, 2H, J = 7.4 Hz, ArH), 8.20 (d, 2H,
J = 8.0 Hz, ArH), 8.60 (d, 2H, J = 7.6 Hz, ArH); MS (EI): m/z 338
[M+1]+.
The compound 14d was prepared following the method de-
scribed for the compound 14a, employing 13b (521 mg, 1 mmol)
and 11b (323 mg, 1 mmol), and the crude product was purified
by column chromatography (5% MeOH–EtOAc) to afford the com-
pound 14d (626 mg, 82%). 1H NMR (CDCl3): d 1.25–1.42 (m, 6H,
2 ꢁ SCH2CH3), 1.70–2.40 (m, 8H, 4 ꢁ CH2), 2.60–3.30 (m, 18H,
6 ꢁ NCH2, 2 ꢁ SCH2, CH2), 3.92 (s, 3H, OCH3), 4.05–4.30 (m, 4H,
OCH2, NCH2), 4.70–4.80 (m, 1H, CH), 4.82 (d, 1H, J = 4.2 Hz,
CH(SEt)2), 6.77 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.75 (t, 2H,
J = 7.3 Hz, ArH), 8.18 (d, 2H, J = 8.0 Hz, ArH), 8.55 (d, 2H,
J = 7.5 Hz, ArH); MS (FAB): 764 [M]+.
6.11. (2S)-N-{4-[2-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-ethyl]-oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (14a)
To a solution of (2S)-N-[4-(2-bromoethoxy)-5-methoxy-2-nitro-
benzoyl)pyrrolidine-2-carboxaldehyde
diethylthioacetal
(13a)
(507 mg, 1 mmol) in dry acetonitrile (30 mL) were added anhy-
drous K2CO3 (552 mg, 4 mmol) and the compound 11a (309 mg,
1 mmol). The reaction mixture was refluxed for 12 h. The reaction
was monitored by TLC using methanol/ethylacetate (1:19) as a sol-
vent system. The potassium carbonate was removed by suction fil-
tration, the solvent was evaporated under reduced pressure, and
the crude product was purified by column chromatography (5%
MeOH–EtOAc) to afford the compound 14a (588 mg, 80%). 1H
NMR (CDCl3): d 1.22–1.40 (m, 6H, 2 ꢁ SCH2CH3), 1.70–2.35 (m,
4H, 2 ꢁ CH2), 2.55–2.95 (m, 16H, 6 ꢁ NCH2, 2 ꢁ SCH2), 3.15–3.32
(m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.15–4.35 (m, 4H, OCH2, NCH2),
4.57–4.72 (m, 1H, CH), 4.80 (d, 1H, J = 4.3 Hz, CH(SEt)2), 6.77 (s,
1H, ArH), 7.60–7.80 (m, 3H, ArH), 8.20 (t, 2H, J = 8.0 Hz, ArH),
8.55 (d, 2H, J = 7.6 Hz, ArH); FABMS: 736 [M]+.
6.15. (2S)-N-{4-[4-[4-[4-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)butyl]piperazin-1-yl]-butyl]-oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (14e)
The compound 17e was prepared following the method de-
scribed for the compound 14a, employing 13c (535 mg, 1 mmol)
and 11c (337 mg, 1 mmol), and the crude product was purified
by column chromatography (5% MeOH–EtOAc) to afford the com-
pound 14e (641 mg, 81%). 1H NMR (CDCl3): d 1.25–1.40 (m, 6H,
2 ꢁ SCH2CH3), 1.60–2.33 (m, 12H, 6 ꢁ CH2), 2.52–3.0 (m, 16H,
6 ꢁ NCH2, 2 ꢁ SCH2), 3.12–3.30 (m, 2H, CH2), 3.95 (s, 3H, OCH3),
4.02–4.25 (m, 4H, OCH2, NCH2), 4.60–4.72 (m, 1H, CH), 4.80 (d,
1H, J = 4.3 Hz, CH(SEt)2), 6.75 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.75
(t, 2H, J = 7.4 Hz, ArH), 8.18 (d, 2H, J = 8.2 Hz, ArH), 8.56 (d, 2H,
J = 7.6 Hz, ArH); MS (FAB): 793 [M+1]+.
6.12. (2S)-N-{4-[3-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-propyl]-oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(14b)
6.16. (2S)-N-{4-[2-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-ethyl]-oxy-5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (15a)
The compound 14b was prepared following the method de-
scribed for the compound 14a, employing 13b (521 mg, 1 mmol)
and 11a (309 mg, 1 mmol), and the crude product was purified
by column chromatography (5% MeOH–EtOAc) to afford the com-
pound 14b (615 mg, 82%). 1H NMR (CDCl3): d 1.30–1.42 (m, 6H,
2 ꢁ SCH2CH3), 1.70–2.30 (m, 6H, 3 ꢁ CH2), 2.40–2.82 (m, 16H,
The compound 14a (736 mg, 1 mmol) was dissolved in metha-
nol (20 mL) and SnCl2ꢀ2H2O (1.13 g, 5 mmol) was added and the
mixture was refluxed for 3.5 h. The reaction mixture was cooled
and the methanol was evaporated under vacuum, and the residue

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
7223
was carefully adjusted to pH 8 with saturated NaHCO3 solution and
then extracted with ethyl acetate (2ꢁ 30 mL). The combined or-
ganic phase was washed with brine (15 mL), dried over anhydrous
Na2SO4 and evaporated under reduced pressure to afford the crude
amino diethyl thioacetal 15a as yellow oil (536 mg, 76%), which
due to potential stability problems was directly used in the next
step without isolation.
(m, 4H, H-1, H-2), 2.45–2.92 (m, 12H, 6 ꢁ NCH2), 3.55–3.82 (m,
3H, H-3, H-11a), 3.92 (s, 3H, OCH3), 4.05–4.40 (m, 4H, OCH2,
NCH2), 6.77 (s, 1H, H-6), 7.45 (s, 1H, H-9), 7.62 (d, 1H, J = 4.4 Hz,
H-11), 7.76 (t, 2H, J = 7.7 Hz, ArH), 8.20 (d, 2H, J = 8.2 Hz, ArH),
8.60 (d, 2H, J = 7.3 Hz, ArH); FABMS: 582 [M]+. Anal. Calcd for
C33H35N5O5: C, 68.14; H, 6.07; N, 12.04. Found: C, 68.10; H, 6.11;
N, 12.01.
6.17. (2S)-N-{4-[3-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-propyl]-oxy-5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (15b)
6.22. 7-Methoxy-8-{3-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl] propyl}-oxy-(11aS)-1,2,3,11a
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiaze-pin-5-one (16b)
The compound 16b was prepared following the method de-
scribed for the preparation of the compound 16a, employing 15b
(720 mg, 1 mmol) to afford the compound 16b as a pale yellow so-
lid (304 mg, 51%). Mp 81–83 °C; ½aꢂ2D6 +282.5 (c 0.2, CHCl3); 1H
NMR (CDCl3): d 1.60–2.16 (m, 6H, H-1, H-2, side-chain CH2),
2.25–2.80 (m, 12H, 6 ꢁ NCH2), 3.50–3.82 (m, 3H, H-3, H-11a),
3.94 (s, 3H, OCH3), 4.05–4.18 (m, 2H, OCH2), 4.22–4.38 (m, 2H,
NCH2), 6.80 (s, 1H, H-6), 7.45 (s, 1H, H-9), 7.62 (d, 1H, J = 4.3 Hz,
H-11), 7.76 (t, 2H, J = 7.3 Hz, ArH), 8.20 (d, 2H, J = 8.0 Hz, ArH),
8.60 (d, 2H, J = 7.3 Hz, ArH); MS (FAB): 597 [M+1]+. Anal. Calcd
for C34H37N5O5: C, 68.55; H, 6.26; N, 11.76. Found: C, 68.58; H,
6.24; N, 11.80.
The compound 15b was prepared following the method de-
scribed for the compound 15a, employing the compound 14b
(750 mg, 1 mmol) to afford the amino diethyl thioacetal 15b as a
yellow oil (561 mg, 78%).
6.18. (2S)-N-{4-[4-[4-[2-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]-butyl]-oxy-5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (15c)
The compound 15c was prepared following the method de-
scribed for the compound 15a, employing the compound 14c
(764 mg, 1 mmol) to afford the amino diethyl thioacetal 15c as a
yellow oil (536 mg, 73%).
6.23. 7-Methoxy-8-{4-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-
5H-pyrrolo [2,1-c][1,4]benzodiaze-pin-5-one (16c)
6.19. (2S)-N-{4-[3-[4-[3-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)propyl]piperazin-1-yl]-propyl]-oxy-5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(15d)
The compound 16c was prepared following the method de-
scribed for the preparation of the compound 16a, employing 15c
(734 mg, 1 mmol) to afford the compound 16c as a pale yellow so-
lid (329 mg, 54%). Mp 72–74 °C; ½aꢂ2D6 +286.5 (c 0.5, CHCl3); 1H
NMR (CDCl3): d 1.56–2.15 (m, 8H, H-1, H-2, 2 ꢁ side-chain CH2),
2.25–2.80 (m, 12H, 6 ꢁ NCH2), 3.45–3.82 (m, 3H, H-3, H-11a),
3.92 (s, 3H, OCH3), 4.0–4.15 (m, 2H, OCH2), 4.22–4.37 (m, 2H,
NCH2), 6.72 (s, 1H, H-6), 7.42 (s, 1H, H-9), 7.60 (d, 1H, J = 4.3 Hz,
H-11), 7.72 (t, 2H, J = 7.4 Hz, ArH), 8.16 (d, 2H, J = 8.1 Hz, ArH),
8.56 (d, 2H, J = 7.4 Hz, ArH); MS (FAB): 611 [M+1]+. Anal. Calcd
for C35H39N5O5: C, 68.95; H, 6.45; N, 11.49. Found: C, 68.91; H,
6.50; N, 11.52.
The compound 15d was prepared following the method de-
scribed for the compound 15a, employing the compound 14d
(764 mg, 1 mmol) to afford the amino diethyl thioacetal 15d as a
yellow oil (557 mg, 76%).
6.20. (2S)-N-{4-[4-[4-[4-(1,3-Dioxo-benz[de]isoquinolin-2-
yl)butyl]piperazin-1-yl]-butyl]-oxy-5-methoxy-2-
aminobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (15e)
6.24. 7-Methoxy-8-{3-[4-[3-(1,3-dioxo-benz[de]isoquinolin-2-
yl)propyl]piperazin-1-yl]propyl}-oxy-(11aS)-1,2,3,11a tetrahy-
dro-5H-pyrrolo [2,1-c][1,4]benzodiaze-pin-5-one (16d)
The compound 15e was prepared following the method de-
scribed for the compound 15a, employing the compound 14e
(792 mg, 1 mmol) to afford the amino diethyl thioacetal 15e as a
yellow oil (618 mg, 78%).
The compound 16d was prepared following the method de-
scribed for the preparation of the compound 16a, employing 15d
(734 mg, 1 mmol) to afford the compound 16d as a pale yellow so-
lid (323 mg, 53%). Mp 65–67 °C; ½aꢂ2D6 +34.5 (c 0.5, CHCl3); 1H NMR
(CDCl3): d 1.75–2.18 (m, 8H, H-1, H-2, 2 ꢁ side-chain CH2), 2.22–
2.80 (m, 12H, 6 ꢁ NCH2), 3.45–4.30 (m, 10H, H-3, H-11a, OCH2,
NCH2, OCH3), 6.75 (s, 1H, H-6), 7.45 (s, 1H, H-9), 7.60 (d, 1H,
J = 4.2 Hz, H-11), 7.72 (t, 2H, J = 7.4 Hz, ArH), 8.20 (d, 2H,
J = 8.1 Hz, ArH), 8.58 (d, 2H, J = 7.3 Hz, ArH); MS (FAB): 610 [M]+.
Anal. Calcd for C35H39N5O5: C, 68.95; H, 6.45; N, 11.49. Found: C,
68.92; H, 6.42; N, 11.54.
6.21. 7-Methoxy-8-{2-[4-[2-(1,3-dioxo-benz[de]isoquinolin-2-
yl)ethyl]piperazin-1-yl]ethyl}-oxy-(11aS)-1,2,3,11a tetrahydro-
5H-pyrrolo [2,1-c][1,4]benzodiazep-in-5-one (16a)
A solution of amino thioacetal 15a (706 mg, 1 mmol), HgCl2
(597 mg, 2.2 mmol), and CaCO3 (240 mg, 2.4 mmol) in acetoni-
trile-water (4:1) was stirred slowly at room temperature for
15 h. The reaction mixture was diluted with ethyl acetate
(30 mL) and filtered through Celite. The clear yellow organic super-
natant was extracted with ethyl acetate (2ꢁ 20 mL). The organic
layer was washed with saturated NaHCO3 (20 mL) and brine
(20 mL), and the combined organic phase was dried over anhy-
drous Na2SO4. The organic layer was evaporated under reduced
pressure, and the crude product was purified by column chroma-
tography (15% CHCl3–MeOH) to afford the compound 16a as a pale
yellow solid (314 mg, 54%). This material was repeatedly evapo-
rated from CHCl3 in vacuum to generate the imine form. Mp 75–
77 °C; ½aꢂ2D6 +278.5 (c 0.5, CHCl3); 1H NMR (CDCl3): d 1.90–2.40
6.25. 7-Methoxy-8-{4-[4-[4-(1,3-dioxo-benz[de]isoquinolin-2-
yl)butyl]piperazin-1-yl]butyl}-oxy-(11aS)-1,2,3,11a tetrahydro-
5H-pyrrolo [2,1-c][1,4]benzodiaze-pin-5-one (16e)
The compound 16e was prepared following the method de-
scribed for the preparation of the compound 16a, employing 15e
(762 mg, 1 mmol) to afford the compound 16e as a pale yellow so-
lid (325 mg, 51%). Mp 68–70 °C; ½aꢂ2D6 +58.4 (c 0.5, CHCl3); 1H NMR

7224
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7218–7224
(CDCl3): d 1.50–2.10 (m, 12H, H-1, H-2, 4 ꢁ side-chain CH2), 2.25–
2.80 (m, 12H, 6 ꢁ NCH2), 3.45–3.86 (m, 3H, H-3, H-11a), 3.95 (s,
3H, OCH3), 4.05–4.25 (m, 4H, OCH2, NCH2), 6.75 (s, 1H, H-6), 7.45
(s, 1H, H-9), 7.62 (d, 1H, J = 4.2 Hz, , H-11), 7.75 (t, 2H, J = 7.3 Hz,
ArH), 8.20 (d, 2H, J = 8.1 Hz, ArH), 8.56 (d, 2H, J = 7.4 Hz, ArH);
MS (FAB): 638 [M]+. Anal. Calcd for C37H43N5O5: C, 69.68; H,
6.80; N, 10.98. Found: C, 69.71; H, 6.76; N, 11.03.
tance. Three of the authors R.R, V.T, and G.B.R.K are thankful to
CSIR, New Delhi, for the award of research fellowships.
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6.26. DNA thermal denaturation studies
The DNA binding affinity of these novel PBD hybrids (16a–e)
has been evaluated through thermal denaturation studies with du-
plex-form calf thymus DNA (CT-DNA) using modified reported pro-
cedure.20,21 Working solutions in aqueous buffer (10 mM NaH2PO4/
Na2HPO4, 1 mM Na2EDTA, pH 7.00 0.01) containing CT-DNA
(100 lM in phosphate) and the PBD (20 lM) 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 are incu-
bated at 37 °C for 0, 18, and 36 h prior to analysis. Samples are
monitored at 260 nm using a Beckman DU-7400 spectrophotome-
ter fitted with high performance temperature controller, and were
heated at 1 °C /min in the range of 40–95 °C. DNA helix coil transi-
tion temperatures (Tm) were obtained from the maxima in the
d(A260)/dT derivative plots. Drug-induced alterations in DNA melt-
ing temperatures are given by
alone), where the Tm value for the PBD-free CT-DNA is
69.4 °C 0.01.
D
Tm = Tm(DNA + PBD) ꢃ Tm (DNA
6.27. In vitro evaluation of cytotoxic activity
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The compounds 16a–c were evaluated for in vitro activity
against selected human tumor cell lines, derived from six cancer
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colon cancer, and ovarian cancer). For each compound, dose–re-
sponse curves against each cell line were measured. Sulforhoda-
mine B (SRB) protein assay22,23 has been used to estimate cell
viability or growth.
Acknowledgments
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The authors are grateful to the Department of Biotechnology
(BT/PR/7037/Med/14/933/2006), New Delhi, for the financial assis-