Synthesis of C8-linked pyrrolo[2,1-c][1,4]benzodiazepine-acridone/acridine hybrids as potential DNA-binding agents

Article abstract of DOI:10.1016/j.bmcl.2004.04.087

Pyrrolobenzodiazepine hybrids linked to acridone/acridine ring systems at C8-position have been designed and prepared that exhibit significant DNA-binding affinity, and a representative compound shows promising in vitro anticancer activity.

Full text of DOI:10.1016/j.bmcl.2004.04.087

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4107–4111
Synthesis of C8-linked pyrrolo[2,1-c][1,4]benzodiazepine-acridone/
acridine hybrids as potential DNA-binding agents
Ahmed Kamal,* O. Srinivas, P. Ramulu, G. Ramesh and P. Praveen Kumar
Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received 1 March 2004; accepted 13April 2004
Available online
Abstract—Pyrrolobenzodiazepine hybrids linked to acridone/acridine ring systems at C8-position have been designed and prepared
that exhibit significant DNA-binding affinity, and a representative compound shows promising in vitro anticancer activity.
Ó 2004 Elsevier Ltd. All rights reserved.
There is a considerable interest in the development of
low molecular weight DNA-binding agents towards
their application on various biological responses par-
ticularly anticancer activity. Pyrrolo[2,1-c][1,4]benzo-
diazepines (PBDs) are a group of potent DNA-
interactive antitumour antibiotics derived from Strep-
tomyces species,1 well known members include DC-81
(1), anthramycin, chicamycin and tomaymycin. Their
interaction with DNA has been extensively investigated
and it is considered unique since they bind within the
minor groove of duplex DNA forming a covalent ami-
nal bond with N2-amino group of guanine base,2 giving
rise to preference for Pu–G–Pu sequences.3 The PBDs
have also been used as a scaffold to attach ethylene-
diaminetetraacetic acid (EDTA),4 epoxide,5 polyamide,6
oligopyrrol7 and cyclic amine8 moieties leading to novel
hybrids of PBD. Recently, we have also designed and
synthesized a number of PBD hybrids that have signif-
icant DNA-binding ability and potent anticancer activ-
ity.9
10
9
6
11
N
HO
H
8
11a
1
2
N
N
7
H3CO
5
4
N
3
O
O
N
H
1 (DC-81)
(DACA)
2
O
N
N
H
O
N
O
N
( )n
H
( )n
H
O
N
H
O
N
H
N
N
H3CO
H3CO
O
O
3 a-b n = 1, 2
4 a-b
n = 1, 2
Keywords: Pyrrolobenzodiazepine; Acridone; Acridine; DNA-binding affinity; In vitro anticancer activity.
* Corresponding author. Tel.: +91-40-27193157; fax: +91-40-27193189; e-mail addresses: ahmedkamal@iict.res.in; ahmedkamal@
ins.iictnet.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.087

4108
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4107–4111
O
CO2H
i
OH
+
Cl
H2N
N
H
OH
O
OH
O
ii
7
5
6
O
iii
N
N
H
OH
O
OH
O
9
8
Scheme 1. Reagents and conditions: (i) K2CO3, Cu, pentylalcohol, reflux, 36 h, 85%; (ii) H2SO4, 100 °C, 4 h, 80%; (iii) Al–Hg, KOH, aqueous
ethanol, reflux, then FeCl3, 60%.
CH(SEt)2
CH(SEt)2
BnO
NO2
N
HO
NO2
N
i
H3CO
H3CO
O
O
11
10
ii
H
N
CH(SEt)2
CH(SEt)2
H2N
O
NO2
O
NO2
N
( )n
( )
n
iii
BOC
N
H3CO
H3CO
O
O
12 a, b
n = 2, 3
13 a, b
iv
O
N
H
CH(SEt)2
O
NO2
N
( )n
N
H
O
H3CO
O
14 a, b
v
O
N
H
CH(SEt)2
O
NH2
N
( )n
N
H
O
H3CO
vi
O
15 a, b
3 a, b n = 1, 2
Scheme 2. Reagents and conditions: (i) EtSH–BF3OEt2, CH2Cl2, 12 h, rt, 70%; (ii) Boc-protected bromo alkylamine, K2CO3, DMF, rt, 24 h, 85–
88%; (iii) TFA, CH2Cl2, 8 h, rt; (iv) EDCl–HOBt, compound 8, DMF, 24 h, rt, 50–54%; (v) SnCl2–2H2O, MeOH, reflux, 2 h, 70–73%; (vi) HgCl2–
CaCO3, CH3CN–H2O, 12 h, rt, 48–52%.
Acridine and acridone based compounds comprise of an
important class of DNA-intercalating anticancer drugs,
and are structurally characterized by the presence of a
planar and semiplanar chromophore portion possibly

A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4107–4111
4109
capable of intercalation into DNA. These compounds
are known to possess a broad range of biological
activities with different modes of action, but all have in
common the ability to bind tightly but reversibly to
DNA by intercalation between the base pairs of the
double helix. In fact, the concept of intercalation has
been first introduced to explain the reversible and non-
covalent binding interactions between acridines and
DNA.10 Some noticeable examples that are clinically in
use and in clinical trials are constituted by amsacrine,11
its derivative CI-92112 and N-[(2-dimethyl amino)-
ethyl]acridine-4-carboxamide (DACA).13 DACA (2) is a
lipophilic mono-intercalator, which entered phase I
clinical trial on the basis of its mixed inhibition of
both DNA topoisomerase-I and topoisomerse-II, and
excellent activity in experimental solid tumour models.14
Further, DACA has shown to retain activity in cell lines
resistant due to alterations in topo-I, topo-II and
p-glycoprotein expressions.15 A number of acridine
derivatives have exhibited interesting antitumour
properties, including the acridone-4-carboxamide,16 and
synthetic strategies19 for this ring system. We herein
report the synthesis and DNA-binding ability of C8-
linked acridone/acridine PBD hybrids with different
alkoxyamide spacers.
Synthesis of these acridone/acridine linked PBD
hybrids has been carried out by employing the corre-
sponding acridone/acridine 4-carboxylic acids as start-
ing materials. Acridine-4-carboxylic acid 9 has been
prepared by reduction of the corresponding acridone-4-
carboxylic acid 8 with aluminium mercury amalgam,
followed by FeCl3 reoxidation of the resulting acri-
dane.13 Jourdan–Ullmann copper-catalyzed condensa-
tion of 2-chloro benzoic acid 5 with anthranilic acid12
6
followed by cyclization of the resulting diphenyl amine-
2,20-dicarboxylic acid20 7 in H2SO4 gives the required
acridone 4-carboxylic acid 8 (Scheme 1). Whereas,
(2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)pyrroli-
dine-2-carboxaldehyde diethyl thioacetal 10 has been
prepared by literature method,21 which upon debenzy-
lation gives 11. Linker components N-(tert-butoxycar-
bonyl)bromoalkyl amines have been prepared by
treatment of bromoalkyl amines with Boc-anhydride.
These have been linked to 11 through an ether linkage
using K2CO3, in DMF at room temperature to give
12a–b. Their deprotection with TFA affords the re-
quired precursor amines (13a–b), followed by amida-
tion with acridone or acridine-4-carboxylic acids using
EDCI/HOBT affords the corresponding nitrothioac-
etal intermediates 14a–b/16a–b. Further, these upon
the
bis-functionalized
acridone/acridine-4-carbox-
amides.17
Therefore, it has been considered of interest to design
and synthesize C8-linked PBD-acridone/acridine
hybrids that could not only improve the DNA-binding
ability but also the biological activity.18 This is in con-
tinuation of our efforts in structural modifications of
PBD ring system and also the development of new
CH(SEt)2
H2N
O
NO2
N
( )n
H3CO
+
N
O
OH
9
O
13 a, b n = 2,3
i
N
CH(SEt)2
O
NO2
( )n
N
H
O
N
H3CO
ii
O
16 a, b
N
CH(SEt)2
O
NH2
N
( )n
N
H
O
H3CO
O
17 a, b
iii
4 a, b n = 1,2
Scheme 3. Reagents and conditions: (i) EDCl–HOBt, DMF, 24 h, rt, 56–58%; (ii) SnCl2–2H2O, MeOH, reflux, 2 h, 68–72%; (iii) HgCl2–CaCO3,
CH3CN–H2O, 12 h, rt, 49–53%.

4110
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4107–4111
reduction by SnCl2Æ2H2O in methanol and followed by
deprotection of aminothioacetal precursors 15a–b/17a–
b using HgCl2/CaCO3 affords the acridone/acridine
linked PBD hybrids 3a–b/4a–b (Schemes 2 and 3).22
exhibits less than 10 nM potency at the GI50 level against
most of the cancer cell lines. The in vitro cytotoxicity
(IC50) for the naturally occurring DC-8124 is 0.38 and
0.33 lM in L1210 and PC6 cell lines, respectively.
Interestingly this compound has promising in vitro
anticancer activity in a number of human cancer cell
lines.
The DNA-binding ability of these C8-linked PBD-acri-
done/acridine hybrids has been investigated by thermal
denaturation studies using calf thymus (CT) DNA23 at
pH 7.0, incubated at 37 °C. It is interesting to observe
that these hybrid molecules elevate the helix melting
temperature of the CT-DNA significantly (Table 1).
One of these hybrids (3b) elevates the helix melting
temperature of CT-DNA by 12.5 °C after incubation for
18 h. In the same experiment the naturally occurring
DC-81 having one imine group exhibits DTm of 0.7 °C.
The enhancement of DNA-binding ability of these
hybrids can be correlated to other interactions produced
by the acridone/acridine component in addition to
covalent linkage of the imine component. Moreover, as
the carbon chain increases from two to three in case of
3b there is a substantial increase in the DNA-binding
affinity.
In conclusion, C8-linked PBD-acridone/acridine hybrids
have been synthesized that exhibit enhanced
DNA-binding ability particularly for 3b and promising
anticancer activity for this representative example in
certain cancer cell lines. This investigation further
reveals the significance of combining a noncovalent
DNA-binding component (acridone) to the covalent
binding PBD moiety. The detailed mechanistic and
molecular modelling studies for these PBD hybrids are
in progress.
Acknowledgements
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 O.S., P.R., G.R.
and P.P.K.
One of the representative compound 3b has been eval-
uated for in vitro anticancer activity in the standard 60-
cancer cell line screen of NCI (Table 2). The LC50 value
of compound 3b against nonsmall cell lung cancer NCI-
H23cell line is 0.07 lM. It exhibits activity in melanoma
cancer panel, in which M14 and UACC-62 cell lines are
affected, with LC50 values of <0.01 lM. It also exhibits
cytotoxic potency against renal cancer cell line A498
with LC50 value of 0.05 lM. Further, this analogue
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1:5
1:5
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1:5
4.7
5.7
12.5
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11.0
3.1
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22. Selected data for compound 3a. 1H NMR (CDCl3) d 1.90–
2.35 (m, 4H), 3.35–3.70 (m, 4H), 3.80 (s, 3H), 4.10–4.30
(m, 3H), 6.80 (s, 1H), 7.20–7.35 (m, 3H), 7.50–7.70 (m,
3H), 8.20 (d, 2H, J ¼ 8:18 Hz), 8.40 (d, 1H, J ¼ 8:2 Hz),
9.05–9.10 (m, 1H), 12.60 (s, 1H); MS (FAB) 511 [M+H]þꢀ.
Compound 3b. 1H NMR (CDCl3) d 2.0–2.40 (m, 6H),
3.50–3.75 (m, 4H), 3.80 (s, 3H), 4.20–4.40 (m, 3H), 6.80 (s,
1H), 7.20–7.30 (m, 2H), 7.40–7.45 (m, 1H), 7.50 (s, 1H),
7.60–7.80 (m, 3H), 8.10 (d, 1H, J ¼ 8:18 Hz), 8.40 (d, 1H,
J ¼ 8:2 Hz), 8.70 (d, 1H, J ¼ 7:6 Hz), 12.30 (s, 1H); MS
(FAB) 525 [M+H]þꢀ.
Compound 4a. 1H NMR (CDCl3) d 1.90–2.35 (m, 4H),
3.40–3.80 (m, 4H), 3.90 (s, 3H), 4.10–4.40 (m, 3H), 6.80 (s,
1H), 7.45–7.80 (m, 5H), 7.95 (d, 1H, J ¼ 8:18 Hz), 8.10 (d,
1H, J ¼ 8:18 Hz), 8.20 (d, 1H, J ¼ 8:92 Hz), 8.80 (s, 1H),
8.90 (d, 1H, J ¼ 6:69 Hz), 12.30 (s, 1H); MS (FAB) 495
[M+H]þꢀ. Compound 4b. 1H NMR (CDCl3) d 2.0–2.45 (m,
6H), 3.50–3.70 (m, 2H), 3.80–4.0 (m, 5H), 4.25–4.45 (m,
3H), 6.80 (s, 1H), 7.50–7.80 (m, 5H), 8.0–8.10 (m, 2H),
8.20 (d, 1H, J ¼ 8:18 Hz), 8.9 (s, 1H), 9.0 (d, 1H,
J ¼ 7:43Hz), 12.0 (s, 1H); MS (FAB) 509 [M+H] þꢀ
.
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