Synthesis of novel non-cross-linking pyrrolobenzodiazepines with remarkable DNA binding affinity and potent antitumour activity

Article abstract of DOI:10.1039/b009195m

Mixed imine-amide pyrrolobenzodiazepine dimers have been prepared which exhibit potent antitumour activity and have significant DNA binding affinity; one of them, 1c, has been shown to cause a remarkable rise in the melting temperature of calf thymus DNA.

Full text of DOI:10.1039/b009195m

Synthesis of novel non-cross-linking pyrrolobenzodiazepines with remarkable
DNA binding affinity and potent antitumour activity
Ahmed Kamal,*a N. Laxman,a G. Ramesh,a K. Neelimab and Anand K. Kondapib
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India.
E-mail: ahmedkamal@iict.ap.nic.in
b Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
Received (in Cambridge, UK) 16th November 2000, Accepted 23rd January 2001
First published as an Advance Article on the web 13th February 2001
Mixed imine–amide pyrrolobenzodiazepine dimers have
been prepared which exhibit potent antitumour activity and
have significant DNA binding affinity; one of them, 1c, has
been shown to cause a remarkable rise in the melting
temperature of calf thymus DNA.
position by a suitable alkane spacer. Interestingly, the larger
sized spacer (n = 5) increases the melting temperature of CT
DNA by a significant 17 °C after 18 h incubation at a
(PBD)+(DNA) ratio of 1+5 for 1c.
There has been increasing interest in discovering and develop-
ing small molecules that are capable of interacting with nucleic
acids in a sequence-selective manner.1–3 Such compounds have
potential in the therapy of genetic-based diseases (some
cancers),4 diagnostics, and validation of DNA sequences. The
pyrrolo[2,1-c][1,4]benzodiazepines (PBD’s) are a well known
class of antitumour antibiotics with sequence-selective DNA
binding ability that are derived from various Streptomyces
species. Their interaction with DNA has been extensively
studied and is considered unique since they bind within the
minor groove of DNA, forming a covalent aminal bond between
the C11 position of PBD B-ring and the N2-amino group of a
guanine base.5 Further, the requirement of (S)-stereochemistry
at C11a for these compounds enables a snug fit in the minor
groove of DNA.6 It has also been shown that PBD’s inhibit both
endonuclease activity and in vitro transcription in a sequence-
selective manner.7
Synthesis of the imine–amide mixed dimers of PBD has been
carried out employing the commercially available vanillin.
Oxidation of vanillin followed by benzylation and nitration by
literature methods17 provides the starting material 4.
-Proline
L
The naturally occurring PBD’s (e.g. anthramycin and
tomaymycin) span approximately 3 base pairs with a preference
for purine–guanine–purine sequences. Thurston and co-work-
ers8 have synthesized C8 diyldioxy ether-linked PBD dimers
(DSB-120), that span approximately six base pairs of DNA and
in which the sequence selectivity is also increased (e.g. purine–
GATC–pyrimidine for DSB-120). Moreover, the cytotoxic
potency and large change in calf thymus (CT) DNA melting
temperature has been attributed to its ability to irreversibly
cross-link DNA via guanine residues on opposite strands
because of the presence of two active sites (i.e. two imine
functionalities). Recently, another new cross-linking PBD
dimer (SJG-136) having C2/C2A-exo unsaturation has been
prepared by the same group and exhibits extraordinary DNA
binding affinity.9 It has been established that the imine
functionality or its equivalent methanolamine form is a primary
requirement for the covalent binding, whereas the non-covalent
interactions of PBD’s with DNA bases help in rationalising the
sequence selectivity and drug orientations.10 Therefore, it has
been considered of interest to design and synthesize C8 linked
PBD-dimers, wherein one ring of PBD has the imino function
while the other has an amide group. It has been envisaged that
such a mixed dimer could offer more insight into not only the
covalent binding but also the role played by non-covalent
interactions with DNA bases.
methyl ester has been coupled to 4 followed by debenzylation
with BF3·OEt2–EtSH to give the nitro ester 6 (Scheme 1).
Another precursor has been prepared from vanillic acid methyl
ester 7 by its etherification with dibromoalkanes to afford 8. The
mono alkylation of 7 has been achieved by using 3 molar
equivalents of the dibromo alkanes. Nitration of 8 followed by
ester hydrolysis and coupling (2S)-pyrrolidinecarbaldehyde
diethyl thioacetal gives 11. The key intermediate has been
prepared by linking 6 and 11. Reduction of both nitro groups
provides 13 which has a dilactam moiety at one end and the
amino functionality on the other. Deprotection of the thioacetal
group by using the method of Thurston and coworkers8 affords
the target molecules 1a–c (Scheme 2).
Interestingly, the data presented in Table 1 show that as the
size of the linker spacer increases from n = 3 to 5 the DNA
stabilization is also enhanced. In this assay for a 1+5 molar ratio
of (PBD)+(DNA), one of these mixed imine–amide PBD dimers
(1c) elevates the helix melting temperature of CT DNA by a
remarkable to 17.0 °C after incubation for 18 h at 37 °C. Under
similar conditions, the dimer having two imino functionalities
i.e. DSB-120 provides a DTm of 15.4 °C. On the other hand, the
naturally occurring DC-81 having only one imino group
In recent years a number of hybrid molecules containing the
PBD ring system have been synthesized to improve upon the
DNA binding ability and sequence selectivity.11 We have been
interested in the structural modifications of the PBD ring system
and the development of new synthetic strategies.12–16 In
continuation of these efforts, we report a new synthesis of novel
mixed dimers of PBD containing the imino function in one of
the PBD rings and an amido group in the other, linked at the C8
Scheme 1 Reagents and conditions: i, SOCl2
hydrochloride, Et3N, H2O, 0 °C, 30 min, 85%; ii, BF3·OEt2–EtSH, CH2Cl2,
rt, 8 h, 88%.
L-proline methyl ester
DOI: 10.1039/b009195m
Chem. Commun., 2001, 437–438
This journal is © The Royal Society of Chemistry 2001
437

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cancer assays carried out on three human cell lines; lung (NCI-
H460), breast (MCF7) and CNS (SF-268), exhibit significant
anticancer activity for these compounds as illustrated in Table
1.
In summary, the synthesis of 1a–c† reported here describes
the importance of non-covalent interactions for increasing the
DNA binding affinity and potent antitumour activity of the non-
cross-linking mixed imine–amide PBD dimers. These findings
may allow researchers to design newer analogues with
improved therapeutic potential, particularly for antitumour
activity. The sequence selectivity, endonuclease activity and
detailed anticancer activity of these non-cross-linking PBD
compounds will be reported elsewhere.
We thank the National Cancer Institute, Maryland for the
primary anticancer assay in human cell lines. We are also
thankful to CSIR, New Delhi for the award of research
fellowship to two of us (N. L. and G. R.).
Notes and references
† Selected data for compound 1a: dH(200 MHz, DMSO-d6 + CDCl3)
1.89–2.5 (m, 10H), 3.4–4.05 (m, 13H), 4.1–4.4 (m, 3H), 6.6 (s, 1H), 6.82 (s,
1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable);
nmax (KBr)/cm21 3450–3460 (br), 2993, 2345, 1686, 1654, 1606, 1518,
1491, 1437, 1384, 1265, 1228, 1119, 1021, 839, 784; [a]D30 +202.6 (c 0.5,
CHCl3); m/z (FAB): 549 (M + H)+ (calc. for C29H31N4O7).
Scheme 2 Reagents and conditions: i, Br(CH2)nBr, K2CO3, CH3COCH3,
reflux, 48 h, 82–86%; ii, SnCl4–HNO3, CH2Cl2, 225 °C, 5 min, 88–91%;
iii, 1 M LiOH, THF, MeOH, H2O (3+1+1), rt, 12 h, 89–93%; iv, SOCl2 then
DMF, THF, H2O, 2(S)-pyrrolidinecarbaldehyde diethyl thioacetal, Et3N,
3 h, 89–92%; v, 6, K2CO3, CH3COCH3, reflux, 48 h, 85–90%; vi,
SnCl2·2H2O, MeOH, reflux, 40 min, 80–85%; vii, HgCl2, CaCO3, CH3CN–
H2O (4+1), 3–8 h, 55–61%.
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Table 1 Thermal denaturation with calf thymus DNA,a at a [PBD]+[DNA]
molar ratio of 1+5b and in vitro one dose primary anticancer assayc in the
NCI-H460, MCF 7 and SF-268 for 1a–c
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Induced DTm/°Cab after incubation
at 37 °C for
Growth percentages
(Lung)
(Breast) (CNS)
Compound
0 h
18 h NCI-H460
MCF7
SF-268
1a
1b
1c
DC-81(3)
DSB-120
6.5
5.0
7.0
8.5 216
4
10
241
7
11
281
221
—
14.0 17.0 239
0.3
10.2 15.4
0.7
—
—
—
—
—
a For CT-DNA at pH 7.00 ± 0.01, DTm = 66.5 °C ± 0.01 (mean value from
60 separate determinations), all DTm values ± 0.1–0.2 °C. b For a 1+5 molar
ratio of [ligand]+[DNA], where CT-DNA concentration = 100 mM in
aqueous buffer [10 mM sodium phosphate + 1 M EDTA, pH 7.00 ± 0.01].
c One dose of 1a–c at 1024 molar concentration.
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exhibits a DTm of 0.7 °C. This demonstrates that compound 1c
containing a single imino functionality has a very significant
DNA binding affinity. To the best of our knowledge, this is the
first synthetic non-cross-linking molecule to exhibit a remark-
able DNA binding effect similar to the naturally occurring
sibiromycin (DTm = 16.3 °C at 18 h).11 These data indicates
that non-covalent interactions play an important role for the
enhancement of DNA binding affinity. The preliminary anti-
438
Chem. Commun., 2001, 437–438