Synthesis and biological activity of fluoroquinolone-pyrrolo[2,1-c][1,4] benzodiazepine conjugates

Article abstract of DOI:10.1016/j.bmc.2005.01.010

Fluoroquinolones have been synthesized and linked to DC-81 at C8 position through different alkyl chain spacers. These PBD conjugates have exhibited good DNA binding affinity, and a representative compound shows promising in vitro anticancer activity.

Full text of DOI:10.1016/j.bmc.2005.01.010

Bioorganic & Medicinal Chemistry 13 (2005) 2021–2029
Synthesis and biological activity of fluoroquinolone-
pyrrolo[2,1-c][1,4]benzodiazepine conjugates
Ahmed Kamal,* V. Devaiah, K. Laxma Reddy and M. Shiva Kumar
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 7 December 2004; revised 7 January 2005; accepted 7 January 2005
Available online 29 January 2005
Abstract—Fluoroquinolones have been synthesized and linked to DC-81 at C8 position through different alkyl chain spacers. These
PBD conjugates have exhibited good DNA binding affinity, and a representative compound shows promising in vitro anticancer
activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
In literature, quinolone ring system has been extensively
exploited for the development of a broad spectrum of anti-
microbial drugs.6 The compounds based on quinolones
skeleton particularly fluoroquinolones are the only direct
inhibitors of the DNA synthesis by binding to the en-
zyme DNA complex, they stabilize DNA strand breaks
created by DNA gyrase and topoisomerase II and IV.7
Further, in the literature a number of antiviral and anti-
tumour agents have been developed in which fluorine sub-
stitution has played a key role in their biological activity.
Recently, some quinolones like 7-(2,6-dimethyl-4-pyr-
idyl) derivative WIN57294 (5a) have been identified as
potent antineoplastic agents.8 Similarly, 1,4-dihydro-4-
oxanaphthyridine-3-carboxylic acids, for example, AT-
3639 (5b) have displayed good in vitro as well as in vivo
activity against murine tumour cells in mouse P-388 leu-
kemia models.9 More recently, quinobenzoxazines such
as A-62176 (5c) demonstrated broad activity against
human and murine tumour cell lines10 (Fig. 2).
The imine or carbinolamine-containing pyrrlo[2,1-
c][1,4]benzodiazepines are a family of low molecular
weight antitumour antibiotics originally isolated from
various Streptomyces species1 and examples of which in-
clude, DC-81 (1), anthramycin (2), tomaymycin and sib-
iromycin. These antibiotics bind selectively in the minor
groove of DNA through a covalent aminal bond be-
tween the electrophilic C11 position of the PBD moiety
and the nucleophilic N2-amino group of a guanine
base,2 resulting in the possible biological activity. In
the literature, large number of PBD conjugates and its
dimers have been synthesized and evaluated for their
biological activity, particularly for their antitumour po-
tential.3 Thurston and co-workers have synthesized
some new PBD dimers, for example, SJG-136 (3a) and
DRG-16 (3b) that shows markedly superior in vitro
cytotoxic potency and interstrand DNA cross-linking
reactivity, and SJG-136 is presently under clinical evalu-
ation.4 Moreover, recently mixed imine–amide (4) and
imine–amine PBD dimers have been designed and syn-
thesized in this laboratory for investigating the contribu-
tion from the non-covalent interactions by one of the
subunit in these dimers. It has been observed that the
contribution by such a non-covalent component is very
significant as DNA binding affinity is remarkable in
such mixed-type of PBD dimers5 (Fig. 1).
The development of conjugates or hybrid molecules
between two type of cytotoxic moieties represent a new
approach in the discovery of new antitumour agents, as
they could posses not only high potency but also different
alkylation sites, both the aspects are useful for tumour
treatment. Our recent efforts in the structural modifica-
tion of the PBD-ring system have led to the synthesis
of a variety of PBD hybrids11 that exhibited not only
good DNA binding affinity but also promising in
vitro anticancer activity. Moreover, we have also
been involved in the development of new synthetic
strategies12 including solid-phase synthesis13 for this
ring system. It is observed from the literature that
Keywords: Pyrrolobenzodiazepines; Fluoroquinolones; DNA binding
ability; Cytotoxicity.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.010

2022
A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
OH
10
N
11a
H
N
OCH3
H
9
6
11
HO
H3C
H
8
1
2
NH2
N
N
7
H3CO
5
4
3
O
O
O
Anthramycin (2)
DC-81 (1)
N
N
H
O
O
H
n
N
N
O
OCH3 H3CO
O
SJG-136, n = 1 (3a)
DRG-16, n = 3 (3b)
H
N
O
H
N
O
(CH2)n O
H
N
H3CO
N
OCH3
O
O
imine-amide PBD dimers (4)
Figure 1. Structures of pyrrolobenzodiazepines and their dimers.
O
O
N
O
N
F
COOH
F
COOH
F
COOH
H3C
N
N
N
N
N
H2N
H2N
N
O
N
S
CH3
WIN57294 (5a)
AT-3639 (5b)
A-62176 (5c)
F
F
F
F
O
N
n
O
N
O
N
H
O
n
N
O
H
O
N
O
H3CO
N
OEt
6a-c, n = 1-3
Figure 2. Structures of 1,8-naphthyridines, quinobenzoxazines and fluoroquinolone-PBD conjugates.
H3CO
OH
O
1,4-dihydro-4-oxo-3-pyridine carboxylic acids (quino-
lone-3-carboxylic acids) moiety is usually essential for
the antitumour activity, however to prepare such a moiety
linked to the PBD ring system posed synthetic problems.
Numerous attempts to hydrolyze the ester functionality of
the quinolone ring either before or after the imine forma-
tion has not provided the desired product. Nevertheless,
attempts are in progress to prepare the free acid group
containing fluoroquinolone–PBD conjugates by employ-
ing enzymatic procedures. However, it was of interest to
prepare compounds, in which fluoroquinolone moiety
has been linked to the PBD-ring system even without
the free 3-carboxylic acid functionality at the fluoroquino-
lone ring particularly to understand the effect of the incor-
poration of fluoroquinolone moiety on the DNA binding
potential. We herein report the synthesis of PBDs that are
linked to the fluoroquinolone through different alkyl
chain spacers at C8 position. Interestingly, these fluoro-
quinolone–PBDs (6a–c) have shown good DNA binding
affinity and promising in vitro cytotoxicity.
2. Results and discussion
2.1. Synthesis
Synthesis of quinolone–PBD conjugates 6a–c has been
carried out by employing the (2S)-N-(4-benzyloxy-5-
methoxy-2-nitrobenzoyl)-pyrrolidine-2-carboxaldehyde-
diethyl thioacetal (11) as the starting material. This

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
2023
compound has been prepared by a literature method,14
which upon debenzylation gives (2S)-N-(4-hydroxy-5-
methoxy-2-nitrobenzoyl)-pyrrolidine-2-carboxaldehyde-
diethyl thioacetal (12). The main precursors 13a–c
have been obtained by coupling of compound 12 and
N-alkylbromo-6,7-difluoro-4-hydroxyquinolone-3-ethyl-
carboxylate (10a–c). These nitrothioacetals have been
reduced with SnCl2Æ2H2O to give 14a–c. The deprotec-
tion of these amino thioacetal by employing HgCl2/
CaCO3 afford the target imine compounds 6a–c (Scheme
2).15 The precursors 10a–c have been prepared by the
monoalkylation of compound (9) with dibromoalkanes.
The compound ethyl-6,7-difluoro-4-hydroxy-3-quino-
line carboxylate (9) has been prepared by the cyclization
of diethyl-2-(3,4-difluoroanilinomethylene)malonate (8),
which has been obtained by the condensation of 3,4-
difluoroaniline (7) with diethyl ethoxymethylenemalo-
nate (Scheme 1).
helix ! coil or melting stabilization (DTm) for the CT-
DNA duplex at pH 7.0, incubated for 18 h at 37 ꢁC,
where PBD/DNA molar ratio is 1:5. Interestingly, in this
study PBD conjugates have shown moderate melting
temperature values (1.6–3.9 ꢁC). Data for compounds
6a–c and DC-81 are included in Table 1. It is interesting,
to observe that both the PBD conjugates 6b and 6c the
DTm values are higher when the length of alkyl chain
spacer is four or five. In the same experiment the natu-
rally occurring DC-81 exhibits a DTm of 0.7 ꢁC.
2.4. RED100-restriction endonuclease digestion assay
Many studies have employed restriction endonuclease
inhibition to confirm the relative binding affinity of
DNA-interactive small molecule ligands.17–20 A quanti-
tative restriction enzyme digestion (RED100) assay has
been developed in which the inhibition of DNA cleavage
by BamH1 is used to probe the DNA binding capability
of PBD monomers.21 We have earlier investigated this
assay for preferences of base pair selectivity of the
imine–amide PBD dimers.22 Recently, this study has
been carried out to determine the DNA binding ability
of A-C8/C-C2-exo unsaturated alkoxyamido-linked
PBD dimers.23 Moreover, this technique has also been
used to study the covalent DNA interaction of PBD di-
mers and it is capable to distinguish between the mono-
meric and dimeric families.4 The BamH1 cleavage
sequence G#GATCC overlaps with several favoured
PBD binding sites suggesting ligand binding has the po-
tential to inhibit the BamH1 cleavage activity. The study
has been carried out to determine the ability of 6a–c,
which inhibits the DNA linearization by BamH1. The
results of this experiment for compounds 6a, 6b and 6c
are shown in Figure 3 suggest that the fluoroquino-
lone–PBDs inhibit BamH1. There are differences in the
inhibitory activity exhibited by PBDs evaluated in this
assay. It is observed that the ranking order is
6c > 6b > 6a for inhibition of BamH1 cleavage is in
agreement with the DNA binding affinity as determined
2.2. Cytotoxicity
As representative members, compounds 6b–c have been
evaluated for their in vitro cytotoxicity in selected hu-
man cancer cell lines of colon (HT-29, HCT-15), lung
(A-549, HOP-62), cervix (SiHa) origin by using SRB
method. Usually, when the concentration of the solution
is 10ꢀ6 mol/L, the inhibition of the solution is more than
50%, and then the compound is considered as an effec-
tive agent. According to this standard, it has been ob-
served from Table 2 that both 6b and 6c exhibit a
strong effect to HCT-15, A-549 cell lines. However, the
in vitro cytotoxicity (IC50) for naturally occurring DC-
8116 is 0.38 and 0.33 lM in L1210 and PC6 cell lines.
2.3. DNA interactions: thermal denaturation studies
The DNA binding ability for these C8-linked PBD con-
jugates has been examined by thermal denaturation
studies using calf thymus (CT) DNA. Melting studies
show that these compounds stabilize the thermal
EtO2C CO2Et
F
F
i
F
NH2
F
N
H
7
8
ii
O
N
O
OH
N
O
F
F
F
F
OEt
iii
OEt
Br
n
9
10a-c
n = 1-3
Scheme 1. Reagents and conditions: (i) diethylethoxymethylenemalonate, 110 ꢁC, 1.5 h, 98%; (ii) diphenyl ether, 250 ꢁC, 1 h, 81%; (iii)
dibromoalkanes, K2CO3, acetone, reflux, 48 h, 80–90%.

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A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
NO2
NO2
BnO
HO
CH(SEt)2
CH(SEt)2
i
N
N
H3CO
H3CO
O
O
11
12
ii
F
F
O
O
N
O
NO2
CH(SEt)2
n
N
H3CO
OEt
O
13a-c
n = 1-3
iii
F
F
O
O
N
O
NH2
n
CH(SEt)2
N
H3CO
OEt
O
14a-c
n = 1-3
iv
6a-c
Scheme 2. Reagents and conditions: (i) EtSH–BF3OEt2, CH2Cl2, 12 h, rt, 76% (ii) 10a–c, K2CO3, acetone, reflux, 48 h, 90–95%; (iii) SnCl2Æ2H2O,
MeOH, reflux, 6–8 h, 80–85%; (iv) HgCl2, CaCO3, CH3CN–H2O (4:1), rt, 12 h, 60–70%.
Table 1. Thermal denaturation data for fluoroquinolone–PBD conju-
gates with calf thymus (CT) DNA
PBD conjugates
[PBD]:[DNA]
molar ratioa
DTm (ꢁC)b after
incubation at
37 ꢁC for
0 h
18 h
6a
6b
1:5
1:5
1:5
1:5
1.4
1.6
3.4
0.3
1.6
2.0
3.9
0.7
Figure 3. RED100-restriction endonuclease digestion assay for fluor-
oquinolone–PBDs conjugates CT-DNA inhibitory activity of 6a, 6b
and 6c on the cleavage of plasmid pBR322 by restriction endonuclease
BamH1 (20 units in 2 lL) for 1 h at 37 ꢁC. The cut (C) and uncut (UC)
products were separated by agarose gel electrophoresis and visualized
by ethidium bromide staining under UV illumination. Lane 1: control
pBR322; lane 2: complete digest of pBR322 by BamH1.
6c
DC-81
a For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion = 100 lM and ligand concentration = 20 lM in aqueous sodium
phosphate buffer [10 mM sodium phosphate + 1 mM EDTA,
pH 7.00 0.01].
b For CT-DNA alone at pH 7.00 0.01, Tm = 69.2 ꢁC 0.01 (mean
value from 20 separate determinations), all DTm values are 0.05–
0.1 ꢁC.
by thermal denaturation. These results clearly demon-
strate that as the linker chain increases from three to five

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
Table 2. The percentage growth inhibition data for fluoroquinolone–PBD hybrids
2025
Cell lines
Lung
Colon
HT-29
10ꢀ5
Colon
HCT-15
10ꢀ5
Lung
HOP-62
10ꢀ5
Cervix
SiHa
10ꢀ5
A-549
10ꢀ5
Compd (mol/L)
10ꢀ4
10ꢀ6
10ꢀ4
10ꢀ6
10ꢀ4
10ꢀ6
10ꢀ4
10ꢀ6
10ꢀ4
10ꢀ6
6b
6c
77
79
40
65
9
25
NT
NT
96
47
52
59
78
79
43
69
49
55
21
NT
10
29
8
10
49
43
28
33
12
25
carbon spacer as in the case of 6c there is an enhance-
ment in the inhibitory activity (Table 2).
flow was introduced to the reaction mixture to remove
the ethanol formed. Then, hexane was added to dissolve
the product. The reaction mixture was warmed (100 ꢁC)
and allowed to cool to room temperature to give corre-
sponding malonate as a white crystalline compound 8
(5.04 g, 85%): mp 76–77 ꢁC; IR (KBr) 1739 cmꢀ1 (ester),
2.5. Conclusion
In conclusion, these new quinolone–PBD conjugates ex-
hibit DNA binding affinity and promising in vitro antit-
umour activity. Interestingly, upon increase of the size
of the alkane linker from three to five carbons the
DNA melting temperatures enhance considerably. The
restriction endonuclease studies also demonstrate this
aspect and suggest that these molecules selectively inter-
act with G-sequences in DNA and low affinity with AT-
rich sequences. Moreover the DNA binding potential of
this quinolone–PBD conjugates is more than the natu-
rally occurring PBD (DC-81). However, the in vitro
anticancer activity for the representative compounds
6b and 6c is moderate.
1688 cmꢀ1 1H NMR CDCl3: d (ppm) 1.20 (t, 3H,
;
J = 7.12 Hz), 1.24 (t, 3H, J = 7.12 Hz), 4.18 (q, 2H,
J = 7.12 Hz), 4.24 (q, 2H, J = 7.12 Hz), 6.79–6.88 (m,
1H), 6.92–6.88 (m, 1H), 7.07–7.25 (m, 1H), 8.27 (d,
1H, J = 12.83 Hz), 10.97 (d, NH, J = 12.83 Hz); MS
(EI) m/z; 299. Anal. Calcd for C14H15F2NO4: C, 56.19;
H, 5.05; N, 4.68. Found: C, 56.09; H, 4.95; N, 4.59.
3.2. Synthesis of ethyl 6,7-difluoro-4-hydroxy-3-quinoline-
carboxylate (9)
Diphenylether (14 mL) was heated in an oil bath at 250
ꢁC and the malonate 8 (3.56 g, 12.0 mmol) was slowly
added. The mixture was kept under reflux for 1 h. Dur-
ing this time vapours evolved and a white solid was
formed. The solid was filtered and washed with hexane
to remove the excess of diphenylether. The correspond-
ing ester 9 was obtained as a white solid.
3. Experimental
Reaction progress was monitored by thin-layer chroma-
tography (TLC) using GF254 silica gel with fluorescent
indicator on glass plates. Visualization was achieved
with UV light and iodine vapor unless otherwise stated.
Chromatography was performed using Acme silica gel
(100–200 and 60–120 mesh). The majority of reaction
solvents were purified by distillation under nitrogen
from the indicated drying agent and used fresh: dichlo-
romethane (calcium hydride), tetrahydrofuran (sodium
benzophenone ketyl), methanol (magnesium methox-
ide), acetonitrile (calcium hydride).
3.3. Synthesis of ethyl-1-(4-bromopropyl)-6,7-difluoro-4-
oxo-1,4-dihydro-3-quinolinecarboxylate (10a)
To a solution of 9 (1.00 g, 3.9 mmol) in dry acetone
(15 mL) was added anhydrous potassium carbonate
(2.72 g, 19.7 mmol) and the 1,3-dibromopropane
(2.40 g, 11.8 mmol). The reaction mixture was refluxed
in an oil bath for 48 h and the reaction was monitored
by TLC using ethyl acetate–hexane (8:2) as a solvent
system. The potassium carbonate was removed by suc-
tion filtration and the solvent removed under vacuum
to afford the crude product. This was further purified
by column chromatography using ethyl acetate–hexane
(6:4) as a solvent system to give pure product 10a
(1.17 g, 80%): IR (KBr) 1718 cmꢀ1 (ester), 1608 cmꢀ1
(keto); 1H NMR CDCl3: d (ppm) 1.42 (t, 3H,
J = 6.90 Hz), 1.85–2.06 (m, 2H), 3.40 (t, 2H, J =
6.13 Hz), 4.19 (t, 2H, J = 6.90 Hz), 4.37 (q, 2H,
J = 7.66 Hz), 7.44 (q, 1H, JH–F = 6.04 Hz), 8.15 (t, 1H,
JH–F = 9.06 Hz), 8.36 (s, 1H); MS (EI) m/z 374
(M+1)+. Anal. Calcd for C15H14BrF2NO3: C, 48.15;
H, 3.77; N, 3.74. Found: C, 48.02; H, 3.44; N, 3.64.
1H NMR spectra were recorded on Varian Gemini
200 MHz spectrometer using tetramethyl silane (TMS)
as an internal standard. Chemical shifts are reported
in parts per million (ppm) down field from tetramethyl
silane. Spin multiplicities are described as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet). Coupling
constants are reported in Hertz (Hz). Low-resolution
mass spectra were recorded on VG-7070H Micromass
mass spectrometer at 200 ꢁC, 70 eV with trap current
of 200 lA and 4 kV acceleration voltages. Optical rota-
tions are measured on Horiba, High Sensitive Polari-
meter, SEPA-300.
3.1. Synthesis of diethyl 2-(3,4-difluoroanilinomethyl-
ene)malonate (8)
3.4. Synthesis of ethyl 1-(4-bromobutyl)-6,7-difluoro-4-
oxo-1,4-dihydro-3-quinoline-carboxylate (10b)
The 3,4-difluoroaniline (2.54 g, 20 mmol) reacted with
diethyl ethoxymethylenemalonate (4.32 g, 21.6 mmol)
at 100 ꢁC for 80 min. During this time, a light nitrogen
Compound 10b was prepared according to the method
described for compound 10a employing compound 9

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A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
(1.00 g,
3.9 mmol)
1,4-dibromobutane
(2.57 g,
(0.69 g, 5.0 mmol) and compound 10a (0.39 g,
11.8 mmol) and K2CO3 (2.72 g, 19.7 mmol) to afford
the crude product, which was purified by column chro-
matography using ethyl acetate–hexane (8:2) to afford
10b (1.36 g, 90%): IR (KBr) 1718 cmꢀ1 (ester),
1.1 mmol). The reaction mixture was refluxed in an oil
bath for 48 h and 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 solvent was removed under vacuum to
afford the crude product. This was further purified by
column chromatography using ethyl acetate–hexane
(8:2) as a solvent system to give product 13a (0.61 g,
90%): 1H NMR CDCl3: d (ppm) 1.32–1.40 (m,
10H), 1.98–2.02 (m, 3H), 2.07–2.17 (m, 3H), 2.29–
2.31 (m, 1H), 2.65–2.87 (m, 4H), 3.20–3.27 (m, 2H),
3.94 (s, 3H), 4.17–4.23 (m, 2H), 4.29–4.40 (m, 4H),
4.62–4.68 (m, 1H), 4.80 (d, 1H, J = 3.77 Hz), 6.80 (s,
1H), 7.36 (q, 1H, JH–F = 6.79 Hz), 7.59 (s, 1H), 8.19 (t,
1H, JH–F = 9.06 Hz), 8.53 (s, 1H); MS (FAB) 693
(M+1)+. Anal. Calcd for C32H37F2N3O8S2: C, 55.40;
H, 5.38; N, 6.06. Found: C, 55.44; H, 5.43; F, 5.34; N,
5.98.
1
1608 cmꢀ1 (keto); H NMR CDCl3: d (ppm) 1.42 (t,
3H, J = 6.90 Hz), 1.85–2.06 (m, 2H), 3.40 (t, 2H,
J = 6.13 Hz), 4.19 (t, 2H, J = 6.90 Hz), 4.37 (q, 2H,
J = 7.66 Hz), 7.50 (q, 1H, JH–F = 6.04 Hz), 8.18, (t,
1H, JH–F = 9.06 Hz), 8.36 (s, 1H); MS (EI) m/z 389
(M+1)+. Anal. Calcd for C16H16BrF2NO3: C, 49.50;
H, 4.15; N, 3.61. Found: C, 49.53; H, 3.89; N, 3.45.
3.5. Synthesis of ethyl 1-(4-bromopentyl)-6,7-difluoro-4-
oxo-1,4-dihydro-3-quinoline-carboxylate (10c)
Compound 10c was prepared according to the method
described for compound 10a employing compound 9
(1.00 g,
3.9 mmol)
1,5-dibromopentane
(2.73 g,
11.8 mmol) and K2CO3 (2.72 g, 19.7 mmol) to afford
the crude product, which was purified by column chro-
matography using ethyl acetate–hexane (8:2) to afford
10c (1.41 g, 90%): IR (KBr) 1718 cmꢀ1 (ester),
3.8. Ethyl-1-(4-4-[2-di(ethylsulfanyl)methyl-(2S)-tetrahy-
dro-1H-1-pyrrolylcarbonyl]-2-methoxy-5-nitrophen-
oxybutyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-quinoline-
carboxylate (13b)
1
1608 cmꢀ1 (keto); H NMR CDCl3: d (ppm) 1.41 (t,
3H, J = 7.55 Hz), 1.55–1.65 (m, 2H), 1.87–1.98 (m,
4H), 3.39 (t, 2H, J = 6.04 Hz), 4.13 (t, 2H,
J = 7.55 Hz), 4.35 (q, 2H, J = 6.75 Hz), 7.17 (q, 1H,
JH–F = 6.04 Hz), 8.24 (t, 1H, JH–F = 9.06 Hz), 8.36 (s,
1H); MS (EI 70 eV) m/z 402 (M+1)+. Anal. Calcd for
C17H18BrF2NO3: C, 50.76; H, 4.51; N, 3.48. Found:
C, 50.82; H, 4.34; N, 3.23.
Compound 13b was prepared according to the method
described for compound 13a employing compound 12
(0.400 g, 1 mmol), 10b (0.41 g, 1.1 mmol) and K2CO3
(0.69 g, 5 mmol) to afford the crude product, which
was purified by column chromatography with ethyl
acetate–hexane (8:2) to afford compound 13b
(0.65 g, 95%): 1H NMR CDCl3: d (ppm) 1.32–1.38
(m, 10H), 1.92–2.00 (m, 3H), 2.09–2.19 (m, 3H), 2.32–
2.35 (m, 1H), 2.58–2.78 (m, 6H), 3.20–3.29 (m, 2H),
3.98 (s, 3H), 4.21–4.25 (m, 2H), 4.31–4.38 (m, 4H),
4.63–4.69 (m, 1H), 4.85 (d, 1H, J = 3.78 Hz), 6.82 (s,
1H), 7.38 (q, 1H, JH–F = 6.83 Hz), 7.61 (s, 1H), 8.19 (t,
1H, JH–F = 9.09 Hz), 8.55 (s, 1H); MS (FAB) 707
(M+1)+. Anal Calcd for: C33H39F2N3O8S2: C, 56.00;
H, 5.55; N, 5.94. Found: C, 55.86; H, 5.33; F, 5.39; N,
5.78.
3.6. 2-Di(ethylsulfanyl)methyl-(2S)-tetrahydro-1H-1-
pyrrolyl-4-hydroxy-5-methoxy-2-nitrophenylmethanone
(12)
To a stirred solution of EtSH (1.91 g, 19 mmol) and
BF3ÆOEt2 (1.41 g, 10 mmol) in DCM was added drop-
wise compound 11 (0.49 g, 1 mmol) in DCM (10 mL)
at rt. Stirring was continued until TLC indicated com-
pletion of the reaction. The solvent was evaporated in
vacuum. The residue was quenched with water
(25 mL) and then extracted with ethyl acetate
(3 · 25 mL). The combined organic phase washed with
brine (1 · 25 mL) dried over Na2SO4 and the solvent re-
moved in vacuum to afford the crude product. This was
further purified by column chromatography using ethyl
acetate–hexane (7:3) to afford the compound 12 (0.30 g,
75%); 1H NMR CDCl3: d (ppm) 1.20–1.40 (m, 6H),
1.75–2.35 (m, 4H), 2.70–2.88 (m, 4H), 3.20–3.32 (m,
2H), 3.95 (s, 1H), 4.60–4.70 (m, 1H), 4.85 (d, 1H,
J = 6.30 Hz), 6.20–6.32 (br s, 1H), 6.75 (s, 1H), 7.75 (s,
1H); MS (EI) m/z 400M+. Anal. Calcd for
C17H24N2O5S2: C, 50.98; H, 6.04; N, 6.99. Found: C,
50.63; H, 5.98; N, 6.58.
3.9. Ethyl-l-1-(5-4-[2-di(ethylsulfanyl)methyl-(2S)-tetra-
hydro-1H-1-pyrrolylcarbonyl]-2-methoxy-5-nitrophen-
oxypentyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-quinoline-
carboxylate (13c)
Compound 13c was prepared according to the method
described for compound 13a employing compound 12
(0.40 g, 1 mmol), 10c (0.42 g, 1.1 mmol) and K2CO3
(0.69 g, 5 mmol) to afford the crude product, which
was purified by column chromatography with ethyl
acetate–hexane (8:2) to afford compound 13c
1
(0.66 g, 94%); H NMR CDCl3: d (ppm) 1.32–1.36 (m,
10H), 1.93–2.02 (m, 3H), 2.10–2.19 (m, 3H), 2.31–
2.34 (m, 1H), 2.56–2.79 (m, 8H), 3.20–3.28 (m, 2H),
3.96 (s, 3H), 4.22–4.26 (m, 2H), 4.32–4.39 (m, 4H),
4.64–4.70 (m, 1H), 4.86 (d, 1H, J = 3.76 Hz), 6.85
(s, 1H), 7.40 (q, 1H, JH–F = 6.87 Hz), 7.63 (s, 1H), 8.21
(t, 1H, JH–F = 9.12 Hz), 8.58 (s, 1H), MS (FAB)
721 (M+1)+. Anal. Calcd for C34H41F2N3O8S2: C,
56.58; H, 5.72; N, 5.82; Found: C, 56.33; H, 5.43; N,
5.88.
3.7. Ethyl-1-(3-4-[2-di(ethylsulfanyl)methyl-(2S)-tetrahy-
dro-1H-1-pyrrolylcarbonyl]-2-methoxy-5-nitrophenoxy-
propyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarb-
oxylate (13a)
To a stirred solution of 12 (0.40 g, 1 mmol) in acetone
(15 mL) was added anhydrous potassium carbonate

A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
2027
3.10. Ethyl-1-(3-5-amino-4-[2-di(ethylsulfanyl)methyl-
(2S)-tetrahydro-1H-1-pyrrolylcarbonyl]-2-methoxyphen-
oxypropyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-quinoline-
carboxylate (14a)
3.14. Ethyl-6,7-difluoro-1-4-[7-methoxy-5-oxo-(11aS)-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaze-
pin-8-yloxy]butyl-4-oxo-1,4-dihydro-3-quinolinecarboxy-
late (6b)
Compound 13a (0.50 g, 0.74 mmol) dissolved in metha-
nol (10 mL) and added SnCl2Æ2H2O (0.83 g, 3.70 mmol)
was refluxed for 1.5 h. The progress of the reaction was
monitored by TLC. 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
(3 · 30 mL). The combined organic phase was dried
over Na2SO4 and evaporated under vacuum to afford
the crude product 14a.
Compound 6b was prepared according to the method
described for compound 6a employing 14b (0.20 g,
0.303 mmol), HgCl2 (0.19 g, 0.689 mmol) and CaCO3
(0.063 g, 0.637 mmol) to afford compound 6b (0.14 g,
1
68%): HNMR (CDCl3): d (ppm) 1.33–1.42 (m, 3H),
1.99–2.18 (m, 6H), 2.24–2.39 (m, 2H), 3.43–3.85 (m,
4H), 3.85–3.89 (m, 2H), 3.97 (s, 3H), 4.09–4.25 (m,
2H), 4.35–4.45 (m, 1H), 6.78 (s, 1H), 7.44 (t, 1H,
JH–F = 12.03 Hz), 7.54 (s, 1H), 7.63 (d, 1H, J =
4.4 Hz), 8.36 (q, 1H, JH–F = 11.2 Hz), 8.65 (s, 1H); MS
(FAB) 554 [M+1]+. Anal. Calcd for C29H29F2N3O6: C,
3.11. Ethyl-1-(4-5-amino-4-[2-di(ethylsulfanyl)methyl-
(2S)-tetrahydro-1H-1-pyrrolylcarbonyl]-2-methoxy-
phenoxybutyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-
quinolinecarboxylate (14b)
62.92; H, 5.28; N,7.59. Found: C, 62.71; H, 5.09; N,
7.44. ½aꢁ +368 (c 0.5, CHCl3).
26
D
3.15. Ethyl-6,7-difluoro-1-5-[7-methoxy-5-oxo-(11aS)-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaze-
pin-8-yloxy]pentyl-4-oxo-1,4-dihydro-3-quinolinecarb-
oxylate (6c)
Compound 14b was prepared according to the method
described for compound 14a employing compound 13b
(0.50 g, 0.72 mmol) and SnCl2Æ2H2O (0.81 g, 3.62 mmol)
to afford the amino diethyl thioacetal 14b.
Compound 6c was prepared according to the method
described for compound 6a employing 14c (0.20 g,
0.297 mmol), HgCl2 (0.19 g, 0.683 mmol) and CaCO3
(0.06 g, 0.65 mmol) to afford compound 6c (0.14 g,
70%): 1HNMR (CDCl3): d 1.35–1.45 (m, 3H), 1.96–
2.20 (m, 8H), 2.22–2.38 (m, 2H), 3.40–3.80 (m, 4H),
3.82–3.86 (m, 2H), 3.95 (s, 3H), 4.05–4.23 (m, 2H),
4.32–4.45 (m, 1H), 6.70 (s, 1H), 7.40 (t, 1H,
JH–F = 12.03 Hz), 7.50 (s, 1H), 7.60 (d, 1H, J =
4.4 Hz), 8.28 (q, 1H, JH–F = 11.2 Hz), 8.6 (s, 1H); MS
(FAB) 568 [M+1]+. Anal. Calcd for C30H31F2N3O6: C,
3.12. Ethyl-1-(5-5-amino-4-[2-di(ethylsulfanyl)methyl-
(2S)-tetrahydro-1H-1-pyrrolylcarbonyl]-2-methoxyphen-
oxypentyl)-6,7-difluoro-4-oxo-1,4-dihydro-3-quinoline-
carboxylate (14c)
Compound 14c was prepared according to the method
described for compound 14a employing compound 13c
(0.50 g, 0.71 mmol) and SnCl2Æ2H2O (0.80 g, 3.54 mmol)
to afford the amino diethyl thioacetal 14c.
63.48; H, 5.50; N, 7.40. Found: C, 63.23; H, 5.39; N,
26
D
3.13. Ethyl-6,7-difluoro-1-3-[7-methoxy-5-oxo-(11aS)-
2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaze-
pin-8-yloxy]propyl-4-oxo-1,4-dihydro-3-quinolinecarb-
oxylate (6a)
7.29. ½aꢁ +392 (c 0.5, CHCl3).
3.16. Thermal denaturation studies
Compounds were subjected to thermal denaturation
studies with duplex-form calf thymus DNA (CT-
DNA) using an adaptation of a reported procedure.24
Working solutions in aqueous buffer (10 mM NaH2-
PO4/Na2HPO4, 1 mM Na2EDTA, pH 7.00 + 0.01) con-
taining CT-DNA (100 lm in phosphate) and the PBD
(20 lm) were prepared by addition of concentrated
PBD solutions in MeOH to obtain a fixed [PBD]/
[DNA] molar ratio of 1:5. The DNA–PBD solutions
were incubated at 37 ꢁC for 0, and 18 h prior to analysis.
Samples were monitored at 260 nm using a Beckman
DU-7400 spectrophotometer fitted with high perfor-
mance temperature controller, and heating was applied
at 1 ꢁC minꢀ1 in the 40–90 ꢁC range. DNA helix coil
transition temperatures (Tm) were obtained from the
maxima in the (dA260)/dT derivative plots. Results are
given as the mean standard deviation from three
determinations and are corrected for the effects of
MeOH co-solvent using a linear correction term.25
Drug-induced alterations in DNA melting behaviour
are given by DTm = Tm(DNA + PBD) ꢀ Tm (DNA
alone), where the Tm value for the PBD-free CT-DNA
is 69.2 0.01. The fixed [PBD]/[DNA] ratio used did
A solution of 14a (0.15 g, 0.232 mmol), HgCl2 (0.15 g,
0.534 mmol) and CaCO3 (0.05 g, 0.488 mmol) in
acetonitrile–water (4:1, 12 mL) was stirred at room
temperature for 12 h until TLC (ethyl acetate), indi-
cates complete loss of starting material. The reaction
mixture was diluted with ethyl acetate (30 mL) and
filtered through
a Celite bed. The clear yellow
organic supernatant was washed with saturated
NaHCO3 (20 mL) and the combined organic phase is
dried over Na2SO4. The organic solvent was evaporated
in vacuum and purified by column chromatography
using ethyl acetate–methanol (9:1) to afford 6a
(0.09 g, 60%): 1H NMR (CDCl3): d (ppm) 1.35–
1.45 (m, 3H), 1.96–2.20 (m, 4H), 2.22–2.38 (m, 2H),
3.40–3.80 (m, 4H), 3.82–3.86 (m, 2H), 3.95 (s, 3H),
4.05–4.23 (m, 2H), 4.32–4.45 (m, 1H), 6.70 (s, 1H),
7.40 (t, 1H, JH–F = 12.03 Hz), 7.50 (s, 1H), 7.60
(d, 1H, J = 4.4 Hz), 8.30 (q, 1H, JH–F = 11.2 Hz), 8.6
(s, 1H); MS (FAB) 540 [M+1]+. Anal. Calcd
for C28H27F2N3O6: C, 62.33; H, 5.04; N, 7.79.
26
D
Found: C, 62.11; H, 4.99; N, 7.69. ½aꢁ +310 (c 0.5,
CHCl3).

2028
A. Kamal et al. / Bioorg. Med. Chem. 13 (2005) 2021–2029
Kamal, A.; Ramesh, G.; Srinivas, O.; Ramulu, P.;
Laxman, N.; Rehana, T.; Deepak, M.; Achary, M. S.;
Nagarajaram, H. A. Bioorg. Med. Chem. 2004, 12, 5427.
6. (a) Mascellino, M. T.; Farinelli, S.; Iegri, F.; Inoa, E.; De
Simone, C. Drug Exp. Clin. Res. 1998, 24, 139; (b) Petri,
W. A., Jr. In Goodman and Gilmanꢀs the Pharmacological
Basis of Therapeutics; 10th ed.; Hardman, J. G., Limbird,
L. E., Gilman, A. G., Eds.; McGraw-Hill: New York,
2001, pp 1179–1183; (c) Wang, J. C. Annu. Rev. Biochem.
1996, 6, 635; (d) Reece, R. J.; Maxwell, A. CRC Crit. Rev.
Biochem. Mol. Biol. 1991, 26, 335.
7. Hooper, D. C. Clin. Infect. Dis. 2001, 32(Suppl. 1), S9–
S15.
8. Wentland, M. P.; Lesher, G. Y.; Reuman, M.; Gruett, M.
D.; Singh, B.; Aldous, S. C.; Dorff, P. H.; Rake, J. B.;
Coughlin, S. A. J. Med. Chem. 1993, 36, 2801.
not result in binding saturation of the host DNA duplex
for any compound examined.
3.17. In vitro evaluation of cytotoxic activity
In routine compounds 6b–c have been evaluated for
their in vitro cytotoxicity in selected human cancer cell
lines of colon (HT-29, HCT-15), lung (A-549, HOP-
62) and cervix (SiHa) origin. A protocol of 48 h contin-
uous drug exposure has been used and a sulforhodamine
B (SRB) protein assay has been used to estimate cell via-
bility or growth.26 The results are expressed as percent
of cell growth inhibition determined relative to that of
untreated control cells.
9. Tomita, K.; Tsuzuki, Y.; Shibamori, K.-I.; Tashima, M.;
Kajikawa, F.; Sato, Y.; Kashimoto, S.; Chiba, K.; Hino,
K. J. Med. Chem. 2002, 45, 5564.
10. (a) Permana, P. A.; Snapka, R. M.; Shen, L. L.; Chu, D.
T. W.; Clement, J. J.; Plattner, J. J. Biochemistry 1994, 33,
11333; (b) Chu, D. T. W.; Hallas, R.; Alder, L.; Plattner, J.
J. Drug Exp. Clin. Res. 1994, 20, 177.
11. (a) Kamal, A.; Ramesh, G.; Ramulu, P.; Srinivas, O.;
Rehana, T.; Sheelu, G. Bioorg. Med. Chem. Lett. 2003, 13,
3451; (b) Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2003, 13, 3517; (c) Kamal, A.;
Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P.
Bioorg. Med. Chem. Lett. 2003, 13, 3577; (d) Kamal, A.;
Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg. Med. Chem.
Lett. 2004, 14, 471; (e) Kamal, A.; Reddy, P. S. M. M.;
Reddy, D. R. Bioorg. Med. Chem. Lett. 2004, 14, 2669; (f)
Kamal, A.; Reddy, K. L.; Reddy, G. S. K.; Reddy, B. S.
N. Tetrahedron Lett. 2004, 45, 3499.
3.18. Restriction endonuclease inhibition
Stock solutions of each PBD (100 lM) were prepared by
dissolving each compound in DMSO (Sigma). These
were stored at ꢀ20 ꢁC. Plasmid (pBR 322) containing
single BamH1 site was used in this assay. Restriction
endonuclease and the relevant buffer were obtained from
NEB. The DNA fragment (500 ng) was incubated with
each PBD (see Fig. 3 for PBD concentrations) in a final
volume of 16 lL for 16 h at 37 ꢁC. Next 10 · BamH1
buffer (2 lL) was added, and the reaction mixture was
made up to 20 lL with BamH1 (20 units) and then incu-
bated for 1 h at 37 ꢁC. Then loaded on to a 1% agarose
gel electrophoresis in Tris–acetate EDTA buffer at 80 V
for 2 h. The gels were stained with ethidium bromide
and photographed.
12. (a) Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.
Tetrahedron Lett. 2002, 43, 6629; (b) Kamal, A.; Laxman,
E.; Reddy, P. S. M. M. Tetrahedron Lett. 2000, 41, 8631;
(c) Kamal, A.; Laxman, E.; Arifuddin, M. Tetrahedron
Lett. 2000, 41, 7743; (d) Kamal, A.; Laxman, E.; Reddy,
P. S. M. M. Synlett 2000, 10, 1476.
13. (a) Kamal, A.; Reddy, G. S. K.; Raghavan, S. Bioorg.
Med. Chem. Lett. 2001, 11, 387; (b) Kamal, A.; Reddy, G.
S. K.; Reddy, K. L. Tetrahedron Lett. 2001, 42, 6969; (c)
Kamal, A.; Reddy, G. S. K.; Reddy, K. L.; Raghavan, S.
Tetrahedron Lett. 2002, 43, 2103; (d) Kamal, A.; Reddy,
K. L.; Devaiah, V.; Reddy, G. S. K. Tetrahedron Lett.
2003, 44, 4741; (e) Kamal, A.; Reddy, K. L.; Devaiah, V.;
Shankaraiah, N. Synlett 2004, 10, 1841; (f) Kamal, A.;
Reddy, K. L.; Devaiah, V.; Shankaraiah, N.; Reddy, Y.
N. Tetrahedron Lett. 2004, 45, 7667; (g) Kamal, A.;
Reddy, K. L.; Devaiah, V.; Shankaraiah, N. Synlett 2004,
14, 2533.
Acknowledgements
We thank Dr. A. K. Saxena, Division of Pharmacology,
Regional Research Laboratory, Jammu 180 001, India
for carrying out the in vitro anticancer assay in human
cancer cell lines. We are also grateful to CSIR, New
Delhi for the award of research fellowships to V.D.
and K.L.R.
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