Synthesis, DNA binding, and cytotoxicity studies of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates

Article abstract of DOI:10.1016/j.bmc.2007.08.026

A series of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates have been prepared and evaluated for their DNA binding ability as well as anticancer activity. Some of these molecules have shown significant anticancer activity in a number of cancer

Full text of DOI:10.1016/j.bmc.2007.08.026

Bioorganic & Medicinal Chemistry 15 (2007) 6868–6875
Synthesis, DNA binding, and cytotoxicity studies of
pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates
Ahmed Kamal,a,* R. Ramu,a Venkatesh Tekumalla,a G. B. Ramesh Khanna,a
Madan S. Barkume,b Aarti S. Juvekarb and Surekha M. Zingdeb
aDivision of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bAdvanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Khargar, Navi Mumbai 410 208, India
Received 16 July 2007; revised 16 August 2007; accepted 17 August 2007
Available online 23 August 2007
Abstract—A series of pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates have been prepared and evaluated for their DNA
binding ability as well as anticancer activity. Some of these molecules have shown significant anticancer activity in a number of can-
cer cell lines.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
H
N
OH
OH
O
O
HN
HN
OH
OH
11
DNA is one of the main molecular targets in the design
of anticancer compounds. Of late, the development of
hybrid ligands, combining two modes of binding to
DNA has received considerable attention. Such ligands
are capable of recognizing heterogeneous DNA
sequences. Anthraquinones represent an important class
of anticancer agents, which generally bind to DNA by
insertion and stacking between the base pairs of the
DNA double helix.1 DNA intercalation and sequential
inhibition of DNA topoisomerase by these compounds
is well known.2 Mitoxantrone (1), as shown in Figure 1,
is a lead compound in this series which is generally used
in the treatment of some hematological malignancies,
ovarian and breast cancers.3 Other anthraquinone-type
analogues such as bisantrene,4 chrysophanol, and emo-
dine5 have exhibited significant cytotoxic activity.
N
HO
H
8
N
H3CO
O
2
N
H
1
N
N
O
O
H
H
N
N
H3CO
OCH3
O
O
3
Figure 1. Chemical structures of mitoxantrone (1), DC-81 (2) and
DSB-120 (3).
and N2 of the guanine residue in the minor groove of
DNA.7 Studies like DNA footprinting and X-ray analy-
sis of these covalent adducts have demonstrated a high
sequence specificity for GC rich regions of DNA, in
particular for Pu-G-Pu triplets.8 Rational modifications
have been made to these natural products to improve
and extend the sequence selectivity. One approach is
the preparation of PBD dimers by coupling two PBD
units through different spacers at their A-C7/A0-C7,9
A-C8/A0-C8,10 C-C2/C0-C2,11 or A-C8/C-C2 posi-
tions.12 Among these, A-C8/A0-C8 linked PBD dimers
like DSB-120 (3) (Fig. 1) and SJG-136 have shown
promising cytotoxicity and efficient DNA cross-linking
properties. Alternative strategy involves the coupling
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
class of DNA binding anticancer compounds that
include the naturally occurring anthramycin and
DC-81 (2) (Fig. 1).6 The DNA-interactive ability and
consequential biological effects are a result of the cova-
lent bond formed between the N10/C11 carbinolamine/
imine moiety in the central B-ring of the PBD moiety
Keywords: Pyrrolobenzodiazepines; Anthraquinones; DNA binding;
Anticancer activity.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.026

A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
6869
of PBDs to other DNA-interactive moieties such as
cyclopropylbenzindole,13 distamycin, and netropsin.14
We have been interested in the development of new
PBD dimers and their hybrids.15 In this context, we have
recently synthesized and studied the biological proper-
ties of C8-linked PBD-anthraquinone hybrids.16 In con-
tinuation of these efforts, we report the full account of
the design, synthesis and biological evaluation of C8-
linked PBD-anthraquinone hybrids (11a–b) and a new
class of PBD dimers linked at their C8 positions through
an anthraquinone moiety (14a–c).
The deprotection of the thioacetal groups of 10a–b and
13a–c with HgCl2/CaCO3 resulted in the formation of
the target compounds 11a–b and 14a–c18 (Scheme 2).
3. DNA interactions: Thermal denaturation studies
The DNA binding affinity of the PBD-anthraquinone
hybrids (11a–b) and anthraquinone-PBD dimers
(14a–c) was investigated by thermal denaturation stud-
ies using calf thymus (CT) DNA. The studies have
been carried out at DNA/PBD molar ratios of 5:1.
The increase in melting temperature of DNA (DTm)
for each compound was examined at 0 h and 18 h of
incubation at 37 ꢁC (Table 1). The compounds 11a
and 11b elevated the helix melting temperature of
CT-DNA by 9.1 ꢁC and 5.2 ꢁC, respectively, after
incubation at 37 ꢁC for 18 h. In case of anthraqui-
none-PBD dimers, compound 14a has been found to
be an efficient stabilizing agent of double stranded
CT-DNA. This compound elevates the helix melting
temperature of CT-DNA by 17.9 ꢁC, whereas com-
pounds 14b and 14c have shown a DTm of 1.6 ꢁC
and 6.0 ꢁC, respectively, after incubation at 37 ꢁC for
18 h. It is interesting to observe that as the spacer
length increases from three to four carbons, there is
a decrease in the DNA binding ability, on further in-
crease from four to five carbons, the DNA binding
ability is enhanced. These results indicate the impor-
tance of spacer chain length in such PBD hybrids.
The naturally occurring DC-81 shows a DTm of
0.7 ꢁC, whereas DSB-120, the synthetic dimer of
DC-81, provides a D Tm of 15.1 ꢁC under similar con-
ditions. These results clearly demonstrate that com-
pound 14a has a significant DNA binding ability. It
is also interesting to note that PBD-anthraquinone hy-
brid 11a exhibited a higher DTm than the anthraqui-
none-PBD dimer 14b having same spacer length.
2. Chemistry
Initial attempts to couple 1-amino anthraquinone 4a
with (2S)-N-[4-[(n-carboxyalkyl)oxy]-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioac-
etal11 in the presence of EDCI and HOBt in different
solvents gave the intermediates 9a–b in poor yields, prob-
ably due to the low solubility of 1-amino anthraquinone
in the reaction solvent. Similarly, the coupling of 1,
4-dihydroxyanthraquinone 4b with (2S)-N-[4-(n-bro-
moalkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-car-
boxaldehyde diethyl thioacetal15a gave the desired
intermediates 12a–c in 15–20% yields. On the contrary,
the synthesis of the final molecules via 5a–b and 6a–c
intermediates made the coupling reactions more efficient
(Scheme 1). Reaction of 4a in the presence of pyridine in
toluene with bromo alkyl acid chloride gave compounds
5a–b in 80–82% yield. Reaction of 4b with dibromo
alkanes in the presence of K2CO3 in acetone gave the
intermediates 6a–c in high yields. The other precursor
(2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]pyrroli-
dine-2-carboxaldehyde diethyl thioacetal 8 was prepared
from compound 7, (2S)-N-[4-benzyloxy-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thi-
oacetal, which was synthesized starting from vanillin
according to the literature method.17 The coupling of
intermediates 5a–b and 6a–c with 8 provided the desired
intermediates 9a–b and 12a–c, respectively, in 70–92%
yield. Reductions of the nitro group using SnCl2Æ2H2O
in methanol gave amines 10a–b and 13a–c, respectively.
4. Cytotoxicity
Compounds 11a–b and 14a–c have been evaluated for
their in vitro cytotoxicity in selected human cancer cell
lines like Hop62 (lung), SiHa (cervix), MCF7 and ZR-
75-1 (breast), Colo205 (colon), PC3 (prostate), and
A2780 (ovarian), by employing the sulforhodamine B
(SRB) assay. The results described in Table 2. show
that all the new compounds are significantly cytotoxic,
with the concentration of the drug that produced 50%
inhibition of cell growth (IC50) ranging from 0.5 to
10 lM. Among all the compounds synthesized, com-
pound 14a displayed higher cytotoxicity with an IC50
value of <1 lM against all the cell lines examined.
Similarly, compound 14c has IC50 values of <1 lM
against SiHa, Colo205 and A2780 cell lines. Com-
pound 14b, comprising of two PBD units and four-
carbon spacer exhibited mild cytotoxicity against all
the cell lines tested. Compound 11a, which has one
PBD unit tethered to anthraquinone through a four-
carbon spacer has shown higher cytotoxicity against
ZR-75-1, A2780, HOP62, and PC3 cell lines with
IC50 values ranging from 0.5 to 0.8 lM. Compound
O
Br
( )n
O
NH
i
4a
4b
O
O
R1
O
5a-b
n = 3, 4
Br
Br
( )n
O
O
R2
4a: R1 = NH2, R2 = H
4b: R1 = R2 = OH
ii
O
O
( )n
6a-c n = 2-4
Scheme 1. Reagents and conditions: (i) bromo alkyl acid chloride,
pyridine, toluene, 60 ꢁC, 4 h; (ii) dibromoalkane, K2CO3, acetone,
reflux, 24 h.

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A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
NO2
BnO
MeO
CH(SEt)2
N
O
7
i
NO2
HO
CH(SEt)2
iii
N
MeO
ii
O
8
NO2
O
CH(SEt)2
CH(SEt)2
( )n
O
O
O
O
O
NO2
O
CH(SEt)2
N
( )n
MeO
9a-b
MeO
O
HN
O
N
O
( )n
NO2
O
O
O
N
MeO
iv
O
12a-c
O
NH2
O
CH(SEt)2
( )n
HN
iv
N
MeO
NH2
O
CH(SEt)2
CH(SEt)2
O
( )n
O
O
O
O
10a-b
O
O
N
MeO
v
O
O
( )n
NH2
N
O
O
( )n
H
HN
N
MeO
N
MeO
13a-c
O
O
v
11a: n = 3
11b: n = 4
O
N
O
( )n
O
O
O
O
H
H
N
N
MeO
O
( )n
N
O
14a: n = 2
14b: n = 3
14c: n = 4
MeO
O
Scheme 2. Reagents and conditions: (i) CH2Cl2, BF3-OEt/EtSH, 16 h; (ii) 5a–b, K2CO3, acetonitrile, reflux, 15 h; (iii) 6a–c, K2CO3, acetonitrile,
reflux, 24 h; (iv) SnCl2-2H2O, MeOH, reflux, 4 h; (v) HgCl2, CaCO3, CH3CN/H2O, rt, 12 h.
Table 1. Thermal denaturation data for PBD-anthraquinones (11a–b,
14a–c) with calf thymus (CT) DNA
11b has also shown similar cytotoxicity with IC50 val-
ues ranging from 0.5 to 0.8 lM against ZR-75-1,
A2780, MCF7, and PC3 cell lines.
a
Compound
[PBD]:[DNA]
molar ratiob
Induced DTm
after incubation
at 37 ꢁC (ꢁC)
0 h
18 h
5. Conclusion
11a
11b
14a
14b
14c
1:5
1:5
1:5
1:5
1:5
8.9
5.1
9.1
5.2
In conclusion, we have described the synthesis, DNA
binding ability, and anticancer activity of PBD-anthra-
quinone conjugates. Among all the compounds synthe-
sized, compound 14a, which has two PBD units
tethered to anthraquinone ring system through a three-
carbon spacer, exhibits higher DNA binding ability. In
addition, these compounds have also displayed promis-
ing anticancer activity in various cell lines and have pro-
vided an insight for future direction in the development
of such conjugates.
15.8
1.2
17.9
1.6
5.7
6.0
a For CT-DNA alone at pH 7.00 0.01, Tm = 69.4 ꢁ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 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].

A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
6871
Table 2. In vitro anticancer activity data for PBD-anthraquinone conjugates (11a–b, 14a–c)
Compound
IC50a (lM)
Hop62b
SiHac
MCF7d
ZR-75-1d
PC3e
Colo205f
A2780g
11a
11b
14a
14b
14c
0.8
1.0
1.8
1.2
0.8
9.0
0.8
1.5
1.4
0.6
0.6
9.0
1.2
0.5
0.5
0.5
0.5
0.8
0.8
0.6
7.0
1.0
0.5
1.8
<1.0
<1.0
10.0
<1.0
2.0
0.5
0.5
<1.0
h
—
0.6
h
—
10.0
0.5
1.4
1
1.5
0.6
ADR
0.5
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.
c Cervix cancer.
d Breast cancer
e Prostate cancer.
f Colon cancer.
g Ovarian cancer.
h IC50 value not attained at the concentrations used in the assay; ADR, adriamycin.
6. Experimental
2H), 3.48–3.60 (m, 2H), 7.65–7.80 (m, 3H), 7.96 (d,
1H, J = 6.70 Hz), 8.18–8.24 (m, 2H), 9.04 (d, 1H,
J = 8.90 Hz), 12.34 (s, 1H); MS (EI): m/z 371 [M]+.
6.1. Chemistry
Reaction progress was monitored by thin-layer chro-
matography (TLC) using GF254 silica gel with fluores-
cent indicator on glass plates. Visualization was
achieved with UV light and iodine vapour 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 (phospho-
rous pentoxide). 1H NMR spectra were recorded on
a Varian Gemini 200 MHz spectrometer using tetra-
methyl 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), br s (broad singlet), d (dou-
blet), t (triplet), q (quartet), and m (multiplet). Cou-
pling 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 accelera-
tion voltage. FABMS spectra were recorded on
LSIMS-VG-AUTOSPEC-Micromass. Melting points
were recorded on Electrothermal 9100 and are
uncorrected.
6.3. N-(9,10-Dihydro-9,10-dioxo-1-anthracenyl)-1-bromo-
pentanamide (5b)
The compound 5b was prepared following the method
described for the compound 5a employing 1-amin-
oanthraquinone (4a, 223 mg, 1 mmol) and 5-bromo pen-
tanoyl chloride (399 mg, 2 mmol) and the crude product
was purified by column chromatography to afford the
compound 5b as a yellow solid (313 mg, 81%). 1H
NMR (CDCl3): d 1.88–2.12 (m, 4H), 2.51–2.63 (m,
2H), 3.40–3.56 (m, 2H), 7.65–7.86 (m, 3H), 8.02 (d,
1H, J = 6.90 Hz), 8.20–8.32 (m, 2H), 9.12 (d, 1H,
J = 9.20 Hz), 12.36 (s, 1H); MS (EI): m/z 386 [M+1]+.
6.4. 1,4-Bis-(3-bromopropyloxy)anthracene-9,10-dione (6a)
To a solution of 1,4-dihydroxyanthraquinone (4b,
480 mg, 2 mmol) in acetone (30 mL) were added anhy-
drous potassium carbonate (1.1 g, 8 mmol) and 1,3-
dibromopropane (1.21 g, 6 mmol) and the mixture was
refluxed for 24 h. After completion of the reaction,
potassium carbonate was removed by filtration and the
solvent was evaporated under reduced pressure to get
the crude product. This was further purified by column
chromatography (30% EtOAc–hexane) to afford the
compound 6a as a yellow solid (791 mg, 82%); mp
1
6.2. N-(9,10-Dihydro-9,10-dioxo-1-anthracenyl)-1-bromo-
butanamide (5a)
117–119 ꢁC; H NMR (CDCl3): d 2.40–2.52 (m, 4H),
3.88 (t, 4H, J = 6.51 Hz), 4.26 (t, 4H, J = 5.53 Hz),
7.34 (s, 2H), 7.71–7.77 (m, 2H), 8.15–8.18 (m, 2H);
MS (FAB): 480 [M]+.
1-Aminoanthraquinone (4a, 223 mg, 1 mmol) was dis-
solved in 100 mL of toluene containing a catalytic
amount of pyridine. The mixture was warmed to 60 ꢁC
and 4-bromobutanoyl chloride (371 mg, 2 mmol) was
added dropwise. After the reaction mixture was stirred
for 4 h, the solvent was removed under reduced pressure
to get the crude product. The crude product was purified
by column chromatography (20% EtOAc–hexane) to af-
6.5. 1,4-Bis-(4-bromobutyloxy)anthracene-9,10-dione (6b)
The compound 6b was prepared following the method
described for the compound 6a, employing 1,4-
dihydroxyanthraquinone (4b, 480 mg, 2 mmol) and
1,4-dibromobutane (1.29 g, 6 mmol), and the crude
product was purified by column chromatography (30%
EtOAc–hexane) to afford the compound 6b as a yellow
1
ford compound 5a as a yellow solid (305 mg, 82%). H
NMR (CDCl3): d 2.22–2.36 (m, 2H), 2.62–2.78 (m,

6872
A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
solid (826 mg, 81%); mp 108–110 ꢁC; 1H NMR (CDCl3):
d 2.0–2.32 (m, 8H), 3.58 (t, 4H, J = 6.45 Hz), 4.10 (t, 4H,
J = 5.40 Hz), 7.25 (s, 2H), 7.62–7.77 (m, 2H), 8.12–8.18
(m, 2H); MS (FAB): 508 [M]+.
1 mmol) and 5b (425 mg, 1.1 mmol) and the crude prod-
uct was purified by column chromatography (50%
EtOAc–hexane) to afford the compound 9b (649 mg,
1
92%). H NMR (CDCl3): d 1.21–1.42 (m, 6H), 1.60–
2.40 (m, 8H), 2.62–2.85 (m, 6H), 3.15–3.30 (m, 2H),
3.95 (s, 3H), 4.10–4.25 (m, 2H), 4.65 (m, 1H), 4.84 (d,
1H, J = 3.64 Hz), 6.78 (s, 1H), 7.68 (s, 1H), 7.75–7.90
(m, 3H), 8.05 (d, 1H, J = 6.70 Hz), 8.20–8.35 (m, 2H),
9.15 (d, 1H, J = 8.90 Hz), 12.38 (br s, 1H); MS (FAB):
706 [M+1]+.
6.6. 1,4-Bis-(5-bromopentyloxy)anthracene-9,10-dione (6c)
The compound 6c was prepared following the method
described for the compound 6a, employing 1,4-
dihydroxyanthraquinone 4b (480 mg, 2 mmol) and 1,
5-dibromopentane (1.38 mg, 6 mmol) and the crude
product was purified by column chromatography (30%
EtOAc–hexane) to afford the compound 6c as a yellow
6.10. (2S)-N-{4-[N-(9,10-Dihydro-9,10-dioxo-1-anthrace-
nyl)-propane-3-carboxami-de]-oxy-5-methoxy-2-amino-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(10a)
1
solid (904 mg, 84%); mp 75–77 ꢁC; H NMR (CDCl3):
d 1.75–2.07 (m, 12H), 3.49 (t, 4H, J = 6.59 Hz), 4.08 (t,
4H, J = 6.13 Hz), 7.25 (s, 2H), 7.69–7.75 (m, 2H),
8.13–8.20 (m, 2H); MS (FAB): 536 [M]+.
The compound 9a (692 mg, 1 mmol) was dissolved in
methanol (20 mL) and added SnCl2Æ2H2O (1128 mg,
5 mmol) was refluxed for 4 h. The reaction mixture
was cooled and the methanol was evaporated under
reduced pressure. The residue was carefully adjusted
to pH 8 with saturated NaHCO3 solution and then
extracted with ethyl acetate (2· 30 mL). The combined
organic phase was washed with brine (15 mL), dried
over anhydrous Na2SO4, and evaporated under reduced
pressure to afford the amino diethyl thioacetal 10a as
yellow oil (503 mg, 76%). The compound was directly
used in the next step due to potential stability problem.
6.7. (2S)-N-[4-Hydroxy-5-methoxy-2-nitrobenzoyl]pyrroli-
dine-2-carboxaldehyde diethyl thioacetal (8)
To a stirred solution of EtSH (1.20 g, 19 mmol) and
BF3.OEt2 (1.419 g, 10 mmol) was added dropwise com-
pound 7 (491 mg, 1 mmol) in dichloromethane (20 mL)
at room temperature. Stirring was continued until the
reaction was completed and then the solvent was evapo-
rated under reduced pressure. The reaction mixture was
quenched with 5% NaHCO3 solution (20 mL), extracted
with dichloromethane (2· 20 mL) and dried over anhy-
drous Na2SO4. The solvent was removed under reduced
pressure and purified by column chromatography (50%
EtOAc–hexane) to afford compound 8 as pale yellow solid
(344 mg, 86%). 1H NMR (CDCl3): d 1.28–1.41 (m, 6H),
1.78–2.36 (m, 4H), 2.67–2.87 (m, 4H), 3.20–3.31 (m,
2H), 3.96 (s, 3H), 4.62–4.70 (m, 1H), 4.82 (d, 1H,
J = 3.64 Hz), 6.77(s, 1H), 7.67(s, 1H);MS(FAB):400 [M]+.
6.11. (2S)-N-{4-[N-(9,10-Dihydro-9,10-dioxo-1-anthrace-
nyl)-butane-4-carboxamide]-oxy-5-methoxy-2-amino-
benzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(10b)
The compound 10b was prepared following the method
described for the compound 10a, employing the com-
pound 9b (706 mg, 1 mmol) to afford the amino diethyl
thioacetal 10b (500 mg, 74%).
6.8. (2S)-N-{4-[N-(9,10-Dihydro-9,10-dioxo-1-anthracenyl)-
propane-3-carboxami-de]-oxy-5-methoxy-2-nitrobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (9a)
6.12. 7-Methoxy-8-[N-(9,10-dihydro-9,10-dioxo-1-anth-
racenyl)-propane-3-carboxamide]-oxy-(11aS)-1,2,3,11a
tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one (11a)
To a solution of 8 (401 mg, 1 mmol) in dry acetonitrile
(30 mL) were added anhydrous K2CO3 (553 mg,
4 mmol) and 5a (409 mg, 1.1 mmol). The reaction mix-
ture was refluxed for 15 h. After completion of reaction,
K2CO3 was removed by filtration and the solvent was
evaporated under reduced pressure, the crude product
was purified by column chromatography (50% EtOAc–
hexane) to afford compound 9a (623 mg, 90%). 1H
NMR (CDCl3): d 1.21–1.38 (m, 6H), 1.53–2.42 (m,
6H), 2.62–2.81 (m, 6H), 3.10–3.28 (m, 2H), 3.91 (s,
3H), 4.20–4.30 (m, 2H), 4.60–4.70 (m, 1H), 4.80 (d,
1H, J = 3.62 Hz), 6.74 (s, 1H), 7.68 (s, 1H), 7.71–7.85
(m, 3H), 8.02 (d, 1H, J = 6.80 Hz), 8.20–8.30 (m, 2H),
9.15 (d, 1H, J = 8.70 Hz), 12.38 (br s, 1H); MS (FAB):
692 [M+1]+.
A solution of amino thioacetal 10a (662 mg, 1 mmol),
HgCl2 (597 mg, 2.2 mmol) and CaCO3 (240 mg,
2.4 mmol) in 15 mL of acetonitrile/water (4:1) was stir-
red slowly at room temperature overnight. The reaction
mixture was diluted with ethyl acetate (30 mL) and fil-
tered through celite. The clear yellow organic superna-
tant was extracted with ethyl acetate (2· 20 mL). The
organic layer was washed with saturated NaHCO3
(20 mL), brine (20 mL), and the combined organic phase
was dried over anhydrous Na2SO4. The organic layer
was evaporated under reduced pressure and the crude
product was purified by column chromatography (90%
EtOAc–hexane) to afford the compound 11a as a pale
26
yellow solid (301 mg, 56%). mp 75–76 ꢁC; ½aꢀ þ 349:5
D
6.9. (2S)-N{4-[N-(9,10-Dihydro-9,10-dioxo-1-anthracenyl)-
butane-4-carboxamide]-oxy-5-methoxy-2-nitrobenzoyl}pyr-
rolidine-2-carboxaldehyde diethyl thioacetal (9b)
(c 0.5, CHCl3); 1H NMR (CDCl3): d 1.95–2.10 (m,
2H), 2.20-2.40 (m, 4H), 2.78–2.86 (m, 2H), 3.50–3.81
(m, 3H), 3.91 (s, 3H), 4.15–4.26 (m, 2H), 6.76 (s, 1H),
7.42 (s, 1H), 7.55 (d, 1H, J = 4.25 Hz), 7.70–7.82 (m,
3H), 8.0 (d, 1H, J = 6.90 Hz), 8.20–8.30 (m, 2H), 9.15
(d, 1H, J = 8.70 Hz), 12.38 (br s, 1H); MS (FAB): 538
The compound 9b was prepared following the method
described for the compound 9a, employing 8 (401 mg,

A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
6873
[M+1]+. Anal. Calcd for C31H27N3O6: C, 69.26; H, 5.06;
N, 7.82. Found: C, 69.29; H, 5.02; N, 17.90.
(m, 20H), 2.65–2.85 (m, 8H), 3.12–3.28 (m, 4H), 3.93
(s, 6H), 4.05–4.20 (m, 8H), 4.60–4.71 (m, 2H), 4.83 (d,
2H, J = 3.66 Hz), 6.77 (s, 2H), 7.24 (s, 2H), 7.60–7.73
(m, 4H), 8.02–8.18 (m, 2H); MS (FAB): 1177 [M+1]+.
6.13. 7-Methoxy-8-[N-(9,10-Dihydro-9,10-dioxo-1-anth-
racenyl)-butane-4-carboxamide]-oxy-(11aS)-1,2,3,11a tet-
rahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one (11b)
6.17. 1,4-Bis-{3-[(2S)-N-(4-oxy-5-methoxy-2-amino-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]propyloxy}anthracene-9,10-dione (13a)
The compound 11b was prepared following the method
described for the compound 11a employing 10b (676 mg,
1 mmol) to afford the compound 11b as a pale yellow so-
The compound 12a (1.12 g, 1 mmol) was dissolved in
methanol (40 mL) and added SnCl2Æ2H2O (2.256 g,
10 mmol) was refluxed for 4 h. The reaction mixture
was cooled and the methanol was evaporated under vac-
uum. The residue was carefully adjusted to pH 8 with
saturated NaHCO3 solution and then extracted with
ethyl acetate (2· 30 mL). The combined organic phase
was washed with brine (15 mL), dried over anhydrous
Na2SO4, and evaporated under vacuum to afford the
amino diethyl thioacetal 13a as yellow oil (895 mg,
82%) and due to potential stability problems directly
used in the next step.
26
lid (287 mg, 52%). mp 79–81 ꢁC; ½aꢀ þ 277 (c 0.5,
D
CHCl3); 1H NMR (CDCl3): d 1.85–2.40 (m, 8H),
2.60–2.78 (m, 2H), 3.51–3.80 (m, 3H), 3.93 (s, 3H),
4.15–4.20 (m, 2H), 6.78 (s, 1H), 7.42 (s, 1H), 7.60 (d,
1H, J = 4.34 Hz), 7.65–7.83 (m, 3H), 8.0 (d, 1H,
J = 6.90 Hz), 8.20–8.25 (m, 2H), 9.15 (d, 1H,
J = 8.80 Hz), 12.38 (br s, 1H); MS (FAB): 552 [M+1]+.
Anal. Calcd for C32H29N3O6: C, 69.68; H, 5.30; N,
7.62. Found: C, 69.64; H, 5.33; N, 7.59.
6.14. 1,4-Bis-{3-[(2S)-N-(4-oxy-5-methoxy-2-nitro-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]propyloxy}anthracene-9,10-dione (12a)
6.18. 1,4-Bis-{4-[(2S)-N-(4-oxy-5-methoxy-2-amino-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]butyloxy}anthracene-9,10-dione (13b)
To a solution of 6a (482 mg, 1 mmol) in dry acetonitrile
(30 mL) were added anhydrous K2CO3 (829 mg,
6 mmol) and 8 (801 mg, 2 mmol). The reaction mixture
was refluxed for 24 h. After completion of reaction,
K2CO3 was removed by filtration and the solvent was
evaporated under reduced pressure, the crude product
was purified by column chromatography (80% EtOAc–
hexane) to afford compound 12a (907 mg, 81%). 1H
NMR (CDCl3): d 1.24–1.40 (m, 12 H), 1.92–2.15 (m,
12H), 2.62–2.82 (m, 8H), 3.12–3.25 (m, 4H), 3.92 (s,
6H), 4.05–4.32 (m, 8H), 4.60–4.72 (m, 2H), 4.82 (d,
2H, J = 3.66 Hz), 6.75 (s, 2H), 7.24 (s, 2H), 7.61–7.74
(m, 4H), 8.02–8.18 (m, 2H); MS (FAB): 1121 [M+1]+.
The compound 13b was prepared following the method
described for the compound 13a, employing the com-
pound 12b (1.15 g, 1 mmol) to afford the amino diethyl
thioacetal 13b (895 mg, 80%).
6.19. 1,4-Bis-{5-[(2S)-N-(4-oxy-5-methoxy-2-amino-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]pentyloxy}anthracene-9,10-dione (13c)
The compound 13c was prepared following the method
described for the compound 13a, employing the com-
pound 12c (1.18 g, 1 mmol) to afford the amino diethyl
thioacetal 13c (963 mg, 84%).
6.15. 1,4-Bis-{4-[(2S)-N-(4-oxy-5-methoxy-2-nitro-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]butyloxy}anthracene-9,10-dione (12b)
6.20. 1,4-Bis-{3-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tet-
rahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]pro-
pyloxy}anthracene-9,10-dione (14a)
The compound 12b was prepared following the method
described for the compound 12a, employing 6b (510 mg,
1 mmol) and 7 (810 mg, 2 mmol) and the crude product
was purified by column chromatography (80% EtOAc–
hexane) to afford the compound 12b (872 mg, 76%); H
NMR (CDCl3): d 1.25–1.40 (m, 12H), 2.0–2.32 (m,
16H), 2.62–2.80 (m, 8H), 3.12–3.25 (m, 4H), 3.92 (s,
6H), 4.08–4.40 (m, 8H), 4.60–4.71 (m, 2H), 4.82 (d,
2H, J = 3.62 Hz), 6.77 (s, 2H), 7.23 (s, 2H), 7.62–7.78
(m, 4H), 8.02–8.18 (m, 2H); MS (FAB): 1149 [M+1]+.
A solution of amino thioacetal 13a (1.19 g, 1 mmol),
HgCl2 (1.19 mg, 4.4 mmol), and CaCO3 (480 mg,
4.8 mmol) in acetonitrile–water (4:1) was stirred slowly
at room temperature overnight. The reaction mixture
was diluted with ethyl acetate (30 mL) and filtered
through celite. The clear yellow organic supernatant
was extracted with ethyl acetate (2· 20 mL). The organic
layer was washed with saturated NaHCO3 (20 mL),
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 (15% MeOH–
6.16. 1,4-Bis-{5-[(2S)-N-(4-oxy-5-methoxy-2-nitro-
benzoyl)pyrrolidine-2-carboxaldehyde diethyl thioac-
etal]pentyloxy}anthracene-9,10-dione (12c)
EtOAc) to afford the compound 14a as a yellow solid
26
D
The compound 12c was prepared following the method
described for the compound 12a, employing 6c (538 mg,
1 mmol) and 8 (801 mg, 2 mmol) and the crude product
was purified by column chromatography (80% EtOAc–
hexane) to afford the compound 12c (824 mg, 70%);
1H NMR (CDCl3): d 1.23–1.42 (m, 12H), 1.83–2.18
(414 mg, 51%); mp 125–126 ꢁC; ½aꢀ þ 304:5 (c 1,
CHCl3); 1H NMR (CDCl3): d 1.96–2.05 (m, 4H),
2.20–2.50 (m, 8H), 3.55–3.85 (m, 6H), 3.92 (s, 6H),
4.20–4.32 (m, 4H), 4.42–4.52 (m, 4H), 6.85 (s, 2H),
7.22 (s, 2H), 7.40 (s, 2H), 7.52 (d, 2H, J = 4.39 Hz),
7.65–7.71 (m, 2H), 8.02–8.15 (m, 2H); MS (FAB): 813

6874
A. Kamal et al. / Bioorg. Med. Chem. 15 (2007) 6868–6875
[M+1]+. Anal. Calcd for C46H44N4O10: C, 67.97; H,
5.46; N, 6.89. Found: C, 68.01; H, 5.35; N, 6.86.
cer, breast cancer, prostate cancer, colon cancer, ovarian
cancer). For each compound, dose–response curves
against each cell line were measured. Sulforhodamine
B (SRB) protein assay21,22 has been used to estimate cell
viability or growth.
6.21. 1,4-Bis-{4-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tet-
rahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]butyl-
oxy}anthracene-9,10-dione (14b)
The cell lines were grown in RPMI 1640 medium con-
taining 10% fetal bovine serum and 2 mM L-glutamine
and were inoculated into 96-well microtiter plates in
90 lL at plating densities depending on the doubling
time of individual cell lines. The microtiter plates were
incubated at 37 ꢁC, 5% CO2, 95% air, and 100% relative
humidity for 24 h prior to addition of experimental
drugs. Aliquots of 10 lL of the drug dilutions were
added to the appropriate microtiter wells already con-
taining 90 lL of cells, resulting in the required final drug
concentrations.
The compound 14b was prepared following the method
described for the compound 14a employing 13b
(1.12 mg, 1 mmol) to afford the compound 14b as a yel-
26
low solid (386 mg, 46%); mp 120–122 ꢁC; ½aꢀ þ 143:5
D
(c 0.75, CHCl3); 1H NMR (CDCl3): d 1.96–2.35 (m,
16H), 3.50–3.84 (m, 6H), 3.92 (s, 6H), 4.12–4.38 (m,
8H), 6.82 (s, 2H), 7.23 (s, 2H), 7.42 (s, 2H), 7.56 (d,
2H, J = 4.39 Hz), 7.62–7.72 (m, 2H), 8.02–8.18 (m,
2H); MS (FAB): 841 [M+1]+. Anal. Calcd for
C48H48N4O10: C, 68.56; H, 5.75; N, 6.66. Found: C,
68.59; H, 5.77; N, 6.58.
Plates were incubated further for 48 h and assay was ter-
minated by the addition of 50 lL of cold trichloro acetic
acid (TCA) (final concentration, 10% TCA) and incu-
bated for 60 min at 4 ꢁC. The plates were washed five
times with tap water and air-dried. Sulforhodamine B
(SRB) solution (50 lL) at 0.4% (w/v) in 1% acetic acid
was added to each of the wells, and plates were incu-
bated for 20 min at room temperature. The residual
dye was removed by washing five times with 1% acetic
acid. The plates were air-dried. Bound stain was subse-
quently eluted with 10 mM trizma base, and the absor-
bance was read on an ELISA plate reader at a
wavelength of 540 nm with 690 nm reference wave-
length. Percent growth was calculated on a plate-by-
plate basis for test wells relative to control wells.
Percentage growth was expressed as the (ratio of average
absorbance of the test well to the average absorbance of
the control wells) · 100.
6.22. 1,4-Bis-{5-[7-Methoxy-8-oxy-(11aS)-1,2,3,11a tet-
rahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepin-5-one]pen-
tyloxy}anthracene-9,10-dione (14c)
The compound 14c was prepared following the method
described for the compound 14a employing 13c
(1.15 mg, 1 mmol), to afford the compound 14c as a yel-
26
low solid (417 mg, 48%); mp 92–94 ꢁC; ½aꢀ þ 424 (c 0.5,
D
CHCl3); 1H NMR (CDCl3): d 1.95–2.32 (m, 20H), 3.53–
3.82 (m, 6H), 3.92 (s, 6H), 4.07–4.13 (m, 8H), 6.76 (s,
2H), 7.23 (s, 2H), 7.44 (s, 2H), 7.58 (d, 2H,
J = 4.40 Hz), 7.65–7.69 (m, 2H), 8.02–8.16 (m, 2H);
MS (FAB): 869 [M+1]+. Anal. Calcd for C50H52N4O10:
C, 69.11; H, 6.03; N, 6.45. Found: C, 69.07; H, 5.59;
N, 6.51.
6.23. Thermal denaturation studies
The DNA binding affinity of the novel anthraquinone-
PBD conjugates (11a–b and 14a–c) has been evaluated
through thermal denaturation studies with duplex-form
calf thymus DNA (CT-DNA) using modified reported
procedure.19,20 Working solutions in aqueous buffer
(10 mM NaH2PO4/Na2HPO4, 1 mM Na2EDTA, pH
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 max-
ima 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 69.4 ꢁC 0.01.
Acknowledgments
The authors are grateful to the Department of Biotech-
nology (BT/PR/7037/Med/14/933/2006), New Delhi for
financial assistance. 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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