Pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates. Synthesis, DNA binding and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2004.07.036

New pyrrolobenzodiazepine-anthraquinone hybrids have been designed and synthesized, found to effectively bind to DNA and also exhibit cytotoxicity against many cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2004.07.036

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4907–4909
Pyrrolo[2,1-c][1,4]benzodiazepine–anthraquinone
conjugates. Synthesis, DNA binding and cytotoxicity
a
a
b
Ahmed Kamal,a, R. Ramu, G. B. Ramesh Khanna, Ajit Kumar Saxena,
*
M. Shanmugavelb and Renu Moti Panditab
aDivision of Organic Chemistry-I, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bDivision of Pharmacology, Regional Research Laboratory, Jammu 180 001, India
Received 25 May 2004; accepted 15 July 2004
Available online 7 August 2004
Abstract—New pyrrolobenzodiazepine–anthraquinone hybrids have been designed and synthesized, found to effectively bind to
DNA and also exhibit cytotoxicity against many cancer cell lines.
Ó 2004 Published by Elsevier Ltd.
In recent years, combination chemotherapy employing
antineoplastic agents with different mechanisms of
action is one of the methods that is being adopted to
combat cancer. Therefore a single molecule containing
two functional groups, each with different type of action
could also be beneficial in the treatment of cancer. Inter-
calating agents such as anthracenediones represent an
important class of antineoplastic agents.1,2 These com-
pounds contain a planar chromophore that inserts
between two base pairs in the DNA helix, resulting in
miscoding and possible cell death. Mitoxantrone, which
is a lead compound in this series is generally used in
clinic for treatment of some haematological malignan-
cies, ovarian and breast cancers.3 Other anthraqui-
none-type analogues, such as bisantrene,4 chryso-
phanol and emodine5 have exhibited significant in vivo
cytotoxic activities.
C-11 is responsible for the biological activities of PBDs.6
X-ray and foot-printing studies on covalent DNA–PBD
adducts have demonstrated a high sequence specificity
for GC-rich DNA regions in particular, for Pu–G–Pu
motifs.7 In the past few years, several hybrid com-
pounds, in which known antitumour compounds or sim-
ple active moieties of known antitumour agents tethered
to PBD, have been designed, synthesized and evaluated
for their biological activity.8–11 Recently, we have been
involved in the development on new synthetic strate-
gies12 for the preparation of PBD ring systems and also
H
N
OH
OH
O
O
HN
HN
OH
OH
N
H
O
H
11
N
8
H3CO
The pyrrolo[2,1-c][1,4]benzodazepines (PBDs) are a
group of naturally occurring antitumour antibiotics pro-
duced by various Streptomyces species and examples of
which include anthramycin, tomaymycin, sibiromycin
and DC-81. The formation of a covalent bond in the
minor groove of DNA by nucleophilic attack of 2-amino
group of a guanine base to form an aminal linkage to
O
N
H
DC-81
mitoxantrone
O
N
O
( )n
O
O
O
NH
H
N
H3CO
Keywords: Anthraquinones; Pyrrolobenzodiazepines; Cytotoxicity;
DNA binding.
n=3-4
*
Corresponding author. Tel.: +91-40-27193157; fax: +91-40-
27193189; e-mail: ahmedkamal@iict.res.in
1a-b
0960-894X/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.07.036

4908
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4907–4909
in the design of structurally modified PBDs and their hy-
brids.13 In this pursuit some novel PBD hybrids and
conjugates have been prepared by linking the PBD moi-
ety to the DNA interactive compounds through suitable
alkane spacers. In continuation of these efforts, it has
been considered of interest to combine features of inter-
calating agent like that of anthraquionone family with
the DNA binding agent that is, PBD moiety, which
led to the synthesis of the compounds 1a and 1b. The
anthraquinone has been linked through its amino func-
tionality to the C8 position of the A-ring of PBD. It has
been envisaged that the combination of DNA intercalat-
ing and binding properties in such conjugates could be
attractive targets for their DNA binding potential and
antitumour activity.
CH(SEt)2
CH(SEt)2
NO2
NO2
BnO
HO
i
N
N
H3CO
H3CO
O
O
4
3
ii
O
CH(SEt)2
NO2
( )n
O
O
O
NH
N
H3CO
O
5
iii
Synthesis of these novel anthraquinone–PBD conjugates
has been carried out by employing (9,10-dihydro-9,10-
dioxo-1-anthracenyl)-1-bromo-alkanamide 2 as one of
the starting materials, which has been obtained by
the reaction of an appropriate acid chloride with
amino anthraquinone (Scheme 1). The other precursor
(2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)pyrrol-
idine-2-carboxaldehyde diethyl thioacetal 3 has been
prepared by literature method,14 which on debenzyl-
ation gives (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzo-
yl)pyrrolidine-2-carboxaldehyde diethyl thioacetal 4.
Etherification of 4 by (9,10-dihydro-9,10-dioxo-1-anthra-
cenyl)-1-bromo-alkanamide gives the desired intermedi-
ate 5. This nitrothioacetal has been reduced with
SnCl2Æ2H2O to give 6. The deprotection of the thioacetal
group afforded the desired compounds 1a–b (Scheme
2).15
O
CH(SEt)2
NH2
( )n
O
O
O
NH
N
H3CO
O
6
iv
1a-b
n = 2, 3
Scheme 2. Reagents and conditions: (i) EtSH–BF3OEt2, CH2Cl2, 12h,
rt, 75%; (ii) compound 2, K2CO3, acetone, 15h, reflux, 90–92%; (iii)
SnCl2Æ2H2O, MeOH, 4h, reflux, 76%; (iv) HgCl2, CaCO3, CH3CN/
H2O, 12h, rt, 52–56%.
Compounds 1a–b 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), cervix (SiHa)
origin by using SRB method. Usually, when the concen-
tration of the compound solution is 10À6 mol/L, the
inhibition of the solution is more than 50%, then
compound is considered as an effective agent. According
to this standard, it has been observed from Table 1 that
both 1a and 1b exhibit a strong effect to HT-29,
HCT-15, HOP-62 cell lines. However, the in vitro
cytotoxicity (IC50) for naturally occurring DC-8116
is 0.38 and 0.33lM in L1210 and PC6 cell lines,
respectively, and in case of 1,4-disubstituted anthra-
quinones, for example, mitoxantrone17 the activity is
0.1lM in L1210 cells. Therefore, the in vitro cytotoxic-
ity exhibited by these new PBD–anthraquinones is
highly significant.
The DNA binding ability for these hybrids has been
examined by using thermal denaturation assay with calf
thymus (CT) DNA. Interestingly, the compounds 1a
and 1b elevates the helix melting temperature of CT-
DNA by 9.1 and 5.2°C after incubation for 18h at
37°C. On the other hand the naturally occurring DC-
81 exhibits a DTm of 0.7°C. This demonstrates that
these PBD–anthraquinone conjugates have significant
DNA binding ability as illustrated in Table 2. In the lit-
erature,17 some substituted amido anthraquinones have
shown DTm values in the range of 8.5–10.0°C. However,
mitoxantrone gave a DTm of 15.9°C with calf thymus
DNA at phosphate-drug ratio 10:1. It is observed that
the other hybrids such as chrysene13c linked PBD
have exhibited DTm values in the range of 1.8–
2.8°C. Whereas, the higher Tm values in the case
of anthraquinone–PBD hybrids are probably because
of the presence of additional carbonyl groups, which
could enable to produce additional noncovalent interac-
tions.
O
( )n
O
O
NH2
O
O
NH
Br
In conclusion, these new conjugates synthesized by
the combination of both DNA binding pyrrolo-
benzodiazepines and DNA intercalating anthraquinones
have exhibited significant in vitro antitumour
activity and DNA binding affinity. The detailed
biological and molecular modelling studies are in pro-
gress.
i
2a-b n=3-4
Scheme 1. Reagents and conditions: (i) bromo alkionyl chloride,
pyridine, toluene, 60°C, 82%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4907–4909
Table 1. The percentage growth inhibition data for anthraquinone–PBD hybrids
4909
Compd (mol/L)
Cell lines
A-549
10À5
HT-29
10À5
HCT-15
10À5
HOP-62
10À5
SiHa
10À5
10À4
10À6
10À4
10À6
10À4
10À6
10À4
10À6
10À4
10À6
1a
1b
93
93
86
87
68
73
NT
NT
66
82
59
61
93
93
27
10
47
56
NT
NT
90
97
74
73
57
73
52
49
41
40
NT: Not tested.
Table 2. Thermal denaturation data for anthraquinone–PBD hybrids
with CT-DNA
8. (a) Thurston, D. E.; Morris, S. J.; Hartley, J. A. Chem.
Commun. 1996, 563; (b) Wilson, S. C.; Howard, P. W.;
Forrow, S. M.; Hartley, J. A.; Adams, L. J.; Jenkins, T.
C.; Kelland, L. R.; Thurston, D. E. J. Med. Chem. 1999,
42, 4028.
Compound
Induced DTm °C after incubation at 37°C
0h
18h
9. Tercel, M.; Stribbling, S. M.; Sheppard, H.; Siim, B. G.;
Wu, K.; Pullen, S. M.; Botting, K. J.; Wilson, W. R.;
Denny, W. A. J. Med. Chem. 2003, 46, 2132.
1a
1b
8.9
5.1
0.3
9.1
5.2
0.7
DC-81
10. Baraldi, P. G.; Balboni, G.; Cacciari, B.; Guiotto, A.;
Manfredini, S.; Romagnoli, R.; Spalluto, G.; Thurston, D.
E.; Howard, P. W.; Bianchi, N.; Rutigliano, C.; Mischiati,
C.; Gambari, R. J. Med. Chem. 1999, 42, 5131.
11. Damayanthi, Y.; Reddy, B. S. P.; Lown, J. W. J. Org.
Chem. 1999, 64, 290.
12. (a) Kamal, A.; Laxman, E.; Reddy, P. S. M. M.
Tetrahedron Lett. 2000, 41, 7743; (b) Kamal, A.; Reddy,
G. S. K.; Raghavan, S. Bioorg. Med. Chem. Lett. 2001, 13,
387; (c) Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.
Tetrahedron Lett. 2003, 44, 2557.
For CT-DNA alone at pH7.00 0.01, Tm = 69.8°C 0.01 (mean
value from 10 separate determinations), all DTm values are 0.1–
0.2°C. For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA con-
centration = 100lM and ligand concentration = 20lM in aqueous
sodium phosphate buffer [10mM sodium phosphate + 1mM EDTA,
pH7.00 0.01].
Acknowledgements
13. (a) Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.;
Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.;
Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679; (b)
Kamal, A.; Reddy, B. S. N.; Reddy, G. S. K.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2002, 12, 1933; (c) Kamal, A.;
Ramesh, G.; Ramulu, P.; Srinivas, O.; Rehana, T.; Sheelu,
G. Bioorg. Med. Chem. Lett. 2003, 13, 3451; (d) Kamal,
A.; Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg. Med.
Chem. Lett. 2003, 13, 3517; (e) Kamal, A.; Srinivas, O.;
Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med.
Chem. Lett. 2003, 13, 3577; (f) Kamal, A.; Reddy, P. S. M.
M.; Reddy, D. R. Bioorg. Med. Chem. Lett. 2004, 14,
2669.
Two of the authors R.R., G.B.R.K. are grateful to
CSIR, New Delhi for the award of research fellowship.
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