Synthesis of C8-linked pyrrolo[2,1-c][1,4]benzodiazepine-benzimidazole conjugates with remarkable DNA-binding affinity

Article abstract of DOI:10.1016/j.bmcl.2004.06.069

Two types of benzimidazoles have been synthesized and linked to DC-81 at C8-position through different alkyl chain spacers. These PBD conjugates have exhibited remarkable DNA-binding affinity, and a representative compound shows promising in vitro antican

Full text of DOI:10.1016/j.bmcl.2004.06.069

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4791–4794
Synthesis of C8-linked pyrrolo[2,1-c][1,4]benzodiazepine–
benzimidazole conjugates with remarkable DNA-binding affinity
Ahmed Kamal,* P. Ramulu, O. Srinivas, G. Ramesh and P. Praveen Kumar
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received 8 April 2004; accepted 14 June 2004
Available online 22 July 2004
Abstract—Two types of benzimidazoles have been synthesized and linked to DC-81 at C8-position through different alkyl chain
spacers. These PBD conjugates have exhibited remarkable DNA-binding affinity, and a representative compound shows promising
in vitro anticancer activity.
ꢀ 2004 Published by Elsevier Ltd.
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a
family of low molecular weight DNA-interactive antitu-
mour antibiotics originally isolated from Streptomyces
species,1 well known examples include anthramycin, sib-
iromycin and DC-81 (1). These compounds are known
to exert their cytotoxic potency effect by covalently
binding to the exocyclic C2–NH2 of guanine residues
within the minor groove of DNA. Naturally occurring
and synthetic derivatives possessing PBD ring system
are generally of interest as potential anticancer and gene
targeting agents due to their sequence specificity while
binding to duplex DNA with a preference for 50-Pu–
G–Pu motifs.2 In the literature, large number of PBD
conjugates have been synthesized and evaluated for their
biological activity, particularly for their antitumour po-
tential.3 Recently, Thurston and co-workers have syn-
thesized C8-cyclic amine PBD conjugates and
evaluated for their antitumour activity and also demon-
strated for their DNA-binding affinity.4 We have de-
signed and synthesized noncross-linking mixed imine–
amine PBD dimers that have significant DNA-binding
ability and potent antitumour activity,5 and also synthe-
sized a number of PBD conjugates that have shown po-
tent anticancer activity and significant DNA-binding
ability.6
thought to result from their inhibition of microtubule
formations.7 Substituted benzimidazoles have proven
as drug leads, which have exhibited pharmacological
interest.8 In addition, 2-substituted benzimidazoles
cover a broad range of biological activities, including
antitumour (2). A series of 2-phenylbenzimidazole-4-
carboxamides have shown in vitro and in vivo antitu-
mour activity and DNA-binding affinity.9
10
N
11a
N
9
6
11
HO
R
H
8
N
H
1
2
CH3
CH3
H
N
N
7
H3CO
5
C
O
(CH2)2 N
4
3
O
2
1 (DC-81)
R = Ph, 2-Furyl, 2, 3-thienyl, 2-Pyrrolyl
H
N
NH
N
N
N
N
H3C
OH
3 (Hoechst-33258)
H
N
(CH2)n
O
N
O
H
N
R
N
H3CO
The benzimidazoles are potent antitumour, antifungal
and antiparasitic agents, whose mode of action is
4a-c; R = H,
5a-c; R = N-Methylpiperazine
O
n = 3-5
Keywords: Pyrrolobenzodiazepine; Benzimidazoles; Hoechst-33258;
DNA-binding ability; Cytotoxicity.
Further, some 2,5-disubstituted benzimidazoles are
known to act as topoisomerase I poisons and also shown
cytotoxic activity against human lymphoblastoma, and
*
Corresponding author. Tel.: +91-40-27193157; fax: +91-40-
27193189; e-mail: ahmedkamal@iict.res.in
0960-894X/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.06.069

4792
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4791–4794
RPMI 8402 cells.10 Several groups have explored the
possibility of utilizing the pharmacophore like benzimi-
dazoles motif derived from the well established bis-benz-
imidazole dye Hoechst-33258.11,12 This compound not
only shows in vitro antitumour activity but also act as
an inhibitor of DNA topoisomerase I. It has been re-
vealed from the foot printing and related structural
methods that Hoechst-33258 recognizes 3–4 consecutive
A/T base pairs by hydrogen bonding to base edges in 1:1
complexes.13
H
N
NO2
Br(CH2)n
O
CH(SEt)2
+
OH
N
R
N
H3CO
O
8a-c
6 R = H
7 R = N-Methylpiperazine
i
H
N
(CH2)n
O
NO2
N
O
CH(SEt)2
N
R
This is in continuation to our efforts towards the struc-
tural modifications of PBD ring system and also the
development of new synthetic strategies14 for this ring
system. We herein report the synthesis of substituted
benzimidazole analogues that have been linked through
different alkyl chain spacers at C8-position of DC-81 (1).
These PBD conjugates have shown remarkable DNA-
binding affinity and promising in vitro anticancer activ-
ity for a typical example.15
H3CO
O
9 a-c
10 a-c
ii
H
N
(CH2)n
O
NH2
O
CH(SEt)2
N
R
N
H3CO
R = H, N-Methylpiperazine
Synthesis of benzimidazole–PBD conjugates has been
carried out by employing the PBD precursors (8a–c)
and 4-(benzimidazol-2-yl)phenol (6) or 4-[6-(4-methyl-
1-piperazinyl)-benzimidazol-2-yl]phenol (7). The precur-
sor 6 has been prepared by the condensation of o-phenyl-
enediamine and 4-hydroxybenzaldehyde. While the
precursor 7 has been prepared by replacing the chlorine
of 5-chloro-2-nitroaniline with N-methylpiperazine fol-
lowed by reduction of nitro group and is further con-
densed with 4-hydroxybenzaldehyde as shown in
Scheme 1.
O
n = 3-5
11 a-c
12 a-c
iii
4 a-c; R = H
5 a-c; R = N-Methylpiperazine
Scheme 2. Reagents and conditions: (i) K2CO3, DMF, rt, 48h, 72–
76%; (ii) SnCl2Æ2H2O, MeOH, reflux, 6–8h, 80–85%; (iii) HgCl2,
CaCO3, CH2CN–H2O (4:1), rt, 12h, 55–61%.
Whereas, the preparation of other PBD precursors 8a–c
has been carried out by employing the commercially
available vanillin by earlier reported methods16 that in-
volves oxidation of vanillin followed by esterification,
monoalkylation with dibromoalkanes, nitration fol-
lowed by ester hydrolysis and coupling of 2(S)-pyrroli-
dine carboxaldehyde diethylthioacetal afforded 8a–c.
The key intermediates 9a–c and 10a–c have been pre-
pared by linking compounds of 6 and 7 to compounds
8a–c, and these nitro compounds upon reduction by
SnCl2Æ2H2O in methanol gives the aminothioacetal pre-
cursors 11a–c and 12a–c which upon deprotection of thi-
oacetal by using HgCl2/CaCO3 affords the desired PBD-
conjugates 4a–c and 5a–c (Scheme 2).17
The DNA-binding activity for these substituted benzim-
idazoles and C8-linked PBD conjugates has been exam-
ined by thermal denaturation studies using calf thymus
H
N
NH2
NH2
i
OH
N
6
H
NO2
NH2
N
+
NO2
NH2
ii
Cl
N
N
N
CH3
N
N
H3C
iii
H
N
NH2
NH2
i
OH
N
N
N
H3C
H3C
7
Scheme 1. Reagents and conditions: (i) 4-hydroxy benzaldehyde, Na2S2O5, EtOH/H2O, reflux, 80ꢁC; (ii) K2CO3, DMF, 90ꢁC; (iii) H2, 10% Pd/C,
EtOH, rt.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4791–4794
4793
Table 1. Thermal denaturation data for PBD-benzimidazole conju-
gates with calf thymus (CT) DNA
Table 2. In vitro anticancer activity of 5a in selected human cancer cell
lines
PBD
conjugates
[PBD]/[DNA]
molar ratiob
DTm (ꢁC)a after
incubation at 37ꢁC
Cancer panel/cell line
LC50 (lM)
Nonsmall cell lung
NCI-H522
EKVX
0h
18h
0.01
25.2
17.4
23.5
7
4a
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
2.9
2.9
11.4
4.0
HOP-62
NCI-H226
11.0
3.8
4b
4c
10.3
16.6
15.5
22.4
0.3
11.0
22.5
15.5
22.6
0.7
Colon
KM12
CNS
5a
5b
45.2
5c
DC-81
SF-539
SF-68
SNB-19
0.01
27.8
47.5
a For CT-DNA alone at pH7.00 0.01, Tm =69.2 ꢁC 0.01 (mean
value from 20 separate determinations), all DTm values are 0.05–
0.1ꢁC.
Melanoma
SK-MEL-2
SK-MEL-5
UACC-62
MALME-3M
M14
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion=100lM and ligand concentration=20lM in aqueous sodium
phosphate buffer (10mM sodium phosphate+1mM EDTA,
pH7.00 0.01).
0.01
0.01
0.01
34.4
0.01
(CT) DNA. Melting studies show that these compounds
stabilize the thermal helixficoil or melting stabilization
(DTm) for the CT-DNA duplex at pH7.0, incubated at
37ꢁC, where PBD/DNA molar ratio is 1:5. In this assay,
compound 6 has not shown any significant DTm while
DTm of compound 7 is 2.9ꢁC. Interestingly, in this study
PBD conjugates have shown significantly high melting
temperature values (4.0–22.6ꢁC). The DTm of com-
pound 5a is 16.6ꢁC at 0h while the melting temperature
increases to 22.5ꢁC upon incubation for 18h at 37ꢁC.
However, compound 5c elevates the helix melting tem-
perature of CT-DNA by 22.4ꢁC at 0h and there is not
much difference in the melting temperatures even after
incubation for 18h (22.6ꢁC). Data for compound 7,
4a–c, 5a–c and DC-81 are included in Table 1. It is inter-
estingly to observe that both the PBD-conjugates 4 and
5 the DTm values are higher when the length of alkyl
chain spacers is 3 or 5. Further, it is to be noted that
in case of 5c the DTm value is remarkably significant
even at 0h of incubation at 37ꢁC. The typical DNA
thermal denaturation profiles for compounds 5a–c after
18h of incubation period have been shown in Figure 1.
Another aspect which has been seen from the DNA
melting data is the difference in the DTm values after
incubation for 18h is not noticeable except in case of 5a.
Ovarian
IGROVI
OVCAR-3
OVCAR-5
OVCAR-8
45.3
34.6
33.9
21.0
Renal
A 498
RXF 393
786-0
0.01
0.01
31.2
18.7
39.7
SN12-C
TK-10
Prostate
PC-3
DV-145
16.4
33.7
Breast
MDA-MB-435
MDA-MB-231/ATCC
BT-549
0.01
13.5
12.6
42.1
T47D
tency in a wide spectrum of cell lines with LC50 values
ranging from 0.01 to 50lM (Table 2), especially this
compound is more potent in case of melanoma. More-
over, this compound 5a exhibits less than 10nM potency
as seen from the GI50 values in almost all the 60 cancer
cell lines. The in vitro cytotoxicity (IC50) for the
As a representative member compound 5a has been
evaluated in the standard 60 cell line cancer screen of
NCI. This compound shows significant cytotoxic po-
Figure 1. Thermal melting curves for CT-DNA and its ligand complexes: (a) CT-DNA with no ligand, (b) CT-DNA+5a, (c) CT-DNA+5b and (d)
CT-DNA+5c.

4794
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4791–4794
naturally occurring DC-8118 is 0.38 and 0.33lM in
L1210 and PC6 cell lines, respectively.
6. (a) Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.;
Ramulu, P. Bioorg. Med. Chem. Lett. 2002, 12, 1917; (b)
Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G.
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In conclusion, C8-linked PBD–benzimidazole conju-
gates have been synthesized that exhibit remarkable
DNA-binding ability. Moreover, 5a exhibits potential
anticancer activity in a number of cancer cell lines. This
investigation further reveals the significance of combin-
ing a noncovalent DNA-binding component (substi-
tuted benzimidazoles) to the covalent binding PBD
moiety. The detailed mechanistic and molecular model-
ing studies for these PBD conjugates are in progress.
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We thank the National Cancer Institute (NCI), Mary-
land for the primary anticancer assay in human cancer
cell lines. We are also grateful to CSIR, New Delhi for
the award of research fellowships to P.R., O.S., G.R.
and P.P.K.
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17. Selected data for compound 4c: 1H NMR (CDCl3) d 1.60–
2.19 (m, 8H), 2.15–2.20 (m, 2H), 3.55–3.90 (m, 6H), 3.99–
4.20 (m, 4H), 6.74–6.90 (m, 3H), 7.19–7.30 (m, 3H), 7.59–
7.70 (mÅ, 3H), 7.90–8.0 (d, 2H, J=8.6Hz); MS (FAB) 525
[M+1]+ . Compound 5c: 1H NMR (CDCl3) d 1.60–2.10
(m, 10H), 2.30 (s, 3H), 2.55–2.62 (m, 4H), 3.08–3.19 (m,
4H), 3.60–3.79 (m, 3H), 3.82 (s, 3H), 3.92–4.05 (m, 4H),
6.64 (s, 1H), 6.79–6.90 (m, 3H), 6.96 (s, 1H), 7.35–7.88 (d,
1H, J=4.4Hz), 7.92–7.96 (d, 2H, J=9.0Hz); MS (FAB)
623 [M+1]+Å.
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