Synthesis and DNA-binding ability of pyrrolo[2,1-c][1,4]benzodiazepine- azepane conjugates

Article abstract of DOI:10.1016/j.bmcl.2005.11.090

A series of pyrrolobenzodiazepine-azepane conjugates linked through different alkane spacers have been prepared and their DNA thermal denaturation studies have been carried out. One of the compound (4b), elevates the DNA helix melting temperature of the CT-DNA by 2.0°C after incubation for 36 h at 37°C.

Full text of DOI:10.1016/j.bmcl.2005.11.090

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1160–1163
Synthesis and DNA-binding ability
of pyrrolo[2,1-c][1,4]benzodiazepine-azepane conjugates
q
*
Ahmed Kamal, D. Rajasekhar Reddy, P. S. Murali Mohan Reddy and Rajendar
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 14 October 2005; revised 17 November 2005; accepted 25 November 2005
Available online 19 December 2005
Abstract—A series of pyrrolobenzodiazepine–azepane conjugates linked through different alkane spacers have been prepared and
their DNA thermal denaturation studies have been carried out. One of the compound (4b), elevates the DNA helix melting temper-
ature of the CT-DNA by 2.0 °C after incubation for 36 h at 37 °C.
Ó 2005 Elsevier Ltd. All rights reserved.
In recent years, there has been considerable interest
in DNA-binding molecules, particularly due to their
involvement in carcinogenesis and their use as
antitumour agents and probes of DNA structure.
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), a group of
potent naturally occurring antibiotics isolated from
Streptomyces species, are of considerable interest due
to their potential as antitumour agents, gene regulators
of guanine (H-bond donor) which point into the minor
groove (Fig. 1).
The primary advantage of the level of hydroxylation in
hydroxyazepanes is their high water solubility, allowing
them to circumvent the problem of poor bioavailability
seen with many other MGBLs and the chirality allows
improved sequence selectivity. Recently, polyhydroxy-
lated bis-azepanes have been reported as new motifs
2
and DNA probes. The PBDs interact within the minor
groove of DNA by forming a covalent bond between
6
for DNA-minor groove-binding agents. Further, there
are many instances where the PBD-based molecules
their electrophilic C11-position and the exocyclic
3
7
C2-NH moiety of a guanine residue. Bonding occurs
2
have problems of solubility and bioavailability. There-
in a sequence-specific fashion with a preference for
PuGPu motifs. Typical examples of PBD natural prod-
fore, design of conjugates like PBD-azepanes could not
only address this issue but moreover these compounds
will have both the covalent and non-covalent moieties.
Inspired by these observations and on the basis of these
presumptions and our earlier efforts on the design and
4
ucts are DC-81 (1) and sibiromycin (2). A survey of
established DNA-binding antibiotics reveals that a
significant number of them have minor groove-binding
5
8
elements composed of oligosaccharides. Hydroxyaze-
panes have several properties that make them potential-
development of structurally modified PBDs, we herein
report the synthesis of PBD-azepanes linked through
different alkane spacers to evaluate their DNA-binding
potential.
6
ly useful as minor groove-binding ligands (MGBLs).
The flexibility of the seven-membered ring (compared
with that of a five- or six-membered ring) would allow
the hydroxyl groups to adopt a variety of positions
increasing the probability of their forming hydrogen
bonds with the N-3 of the purine, the urea carbonyl of
the pyrimidine bases (H-bond acceptors), or the 2-amino
The desired final target molecules 4a–d have been
obtained from the key intermediates 13a–d and 14.
The intermediates 13a–d have been obtained from the
compounds 7 and 10. The bis-epoxide (7) has been
obtained from D-mannitol (5) as the starting material
and the other compound 4-acetoxyphenylamine (10)
has been obtained from 4-nitrophenol (8) as the starting
material. The precursor 14 has been obtained by litera-
Keywords: Pyrrolobenzodiazepine; Hydroxyazepane; DNA-binding
ability.
9
q
ture methods. One of the key intermediates bis-epoxide
(7) has been obtained by converting D-mannitol (5) to its
triacetonide using acetone and catalytic amount of
See Ref. 1.
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189;
e-mail addresses: ahmedkamal@iict.res.in; ahmedkamal@iictnet.org
*
0
960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.090

A. Kamal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1160–1163
1161
OH
HO
N
H
OH
H
H
H C
3
N
H CO
3
N
O
N
O
CH
3
CH
3
O
H C
H CHN
3
O
1
2
3
OH
N
OH
HO
HO
OH
HO
HO
OH
OH
N
.2HCl
O
O
3
OH
H
HO
HO
O
N
n
N
O
O
O
O
H CO
3
n = 3-5, 8
4
a-d
Figure 1. DC-81 (1), sibiromycin (2), bis-azepane (3), azepane-PBDs (4a–d).
H SO , followed by removal of terminal acetonides
2
the intermediate compounds 7 and 10 in water at
1
4
1
using aq acetic acid, providing the monoacetonide (6).
The monoacetonide (6) on tosylation using pyridine
and tosyl chloride in dichloromethane at 0 °C for 4 h
provides the 1,6-di-O-tosyl-3,4-O-isopropylidene-D-
mannitol which on treatment with anhydrous K CO
95 °C (Scheme 3).
The compounds 15a–d have been obtained by coupling
of compounds 13a–d and 14. These upon reduction of
the nitro group employing H /Pd-C in ethanol followed
2
3
2
in methanol for 2.5 h at 25 °C affords the bis-epoxide
by deprotection of thioacetal group afford the target
12
compounds 4a–d (Scheme 4).
1
0
(
7) (Scheme 1).
Another intermediate, 4-acetoxyphenylamine (10), has
been obtained from 4-nitrophenol (8) by simultaneous
acetylation (9) employing acetyl chloride, triethylamine
and DMAP in dichloromethane and nitro group reduc-
The DNA-binding affinity of the novel azepane-linked
PBDs (4a–d) was investigated by thermal denaturation
studies using calf thymus (CT) DNA. The studies
for these compounds (4a–d) were carried out by DNA/li-
gand molar ratios of 5:1. The increase in the helix melt-
1
3,14
tion employing H /Pd-C in methanol (Scheme 2).
2
ing temperature (DT ) for each compound was
m
Refluxing the compound 12 and dibromoalkanes in
acetone employing K CO as the base gave the
compounds 13a–d. The compound 12 has been obtained
by deacetylation of the compound 11 using 2M NaOH
and methanol, which inturn have been obtained from
examined after 0, 18 and 36 h incubation at 37 °C. Data
for compound 1 are included in Table 1 for comparison.
The compound 4b elevates the helix melting temperature
of the CT-DNA by 2.0 °C after incubation at 37 °C for
2
3
36 h. The naturally occurring DC-81 (1) gives a DT of
m
0
.7 °C under identical experimental conditions. This re-
sult illustrates the effect of introducing the azepane moi-
ety through different alkane spacers at C8-position of
the DC-81 (Table 1).
O
O
O
O
i
ii
HO
HO
OH iii
D-mannitol
O
iv
O
5
OH
It is observed from the thermal denaturation study data
that when azepane is linked to PBD through a three-car-
bon spacer (4a) there is no DNA-binding. However, in
case of compound 4b when the azepane sub-unit is
linked to the PBD through a four-carbon alkane spacer
it exhibits maximum DNA-binding which eventually
increases to 2.0 °C upon incubation for 36 h at 37 °C.
Probably the azepane sub-unit attains a proper align-
ment for the non-covalent interactions that could take
place when the alkane spacer comprises of four carbon
units. In case of compounds with five and eight alkane
spacers, the DTm value is marginally enhanced to
6
7
Scheme 1. Reagents and conditions: (i) acetone, H
2
4
SO ; (ii) 70%
AcOH; (iii) TsCl, pyridine, CH
2
Cl
2
; (iv) K
2
3
CO , MeOH.
NO2
NH2
NO2
i
ii
O
O
OH
O
O
1.1 °C after incubation for 36 h at 37 °C compared to
8
9
10
DC-81 (1), which exhibits 0.7 °C under identical experi-
mental conditions (Table 1). Therefore, similar to other
hybrids of PBD the linker length plays an important role
Scheme 2. Reagents and conditions: (i) acetyl chloride, NEt
CH Cl ; (ii) H /Pd-C, MeOH.
3
, DMAP,
2
2
2

1162
A. Kamal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1160–1163
O
O
O
O
O
O
HO
OH
HO
OH
HO
OH
N
O
N
N
O
i
ii
iii
7
+10
OH
Br
(
)n
O
1
1
12
13a-d
n = 3-5, 8
Scheme 3. Reagents and conditions: (i) H
2
2 3
O, 95 °C; (ii) 2M NaOH, MeOH (iii) dibromoalkanes, K CO
, reflux.
in these compounds as well. However, the bis-azepane
(
pH 5.0 after incubation for 12 h.
3) exhibits the helix melting temperature of 0.7 °C at
6
HO
NO2 CH(SEt)2
N
1
3a-d
+
H CO
3
In conclusion, synthesis and DNA thermal denatur-
ation studies of pyrrolobenzodiazepine–azepane conju-
gates linked through different alkane spacers are
described. The biological screening of these com-
pounds is under investigation. Further research will
involve the preparation of azepanes with more number
of hydroxyl groups, or with the hydroxyls modified as
their methyl ethers, and are linked to the PBD moiety
which may exhibit better minor groove-binding abili-
ties. These compounds may form stable complexes
with the double-stranded DNA and are expected to
possess improved bioavailability.
O
1
4
i
HO
O
O
N
O
nO
NO2 CH(SEt)2
N
HO
HO
H CO
3
O
ii
15a-d
O
N
O
nO
NH2 CH(SEt)2
N
O
HO
Acknowledgments
H CO
3
O
The authors D.R.S.R., P.S.M.M.R. and Rajendar are
grateful to CSIR, New Delhi, for the award of Research
Fellowships.
iii
16a-d
4
a-d
n = 3-5, 8
Scheme 4. Reagents and conditions: (i) K
(ii) H /Pd-C, EtOH, 24 h; (iii) HgCl , CaCO
2
CO
3
, acetone, reflux, 48 h;
CN-H O (4:1), 8 h.
References and notes
2
2
3
, CH
3
2
1
2
. Part of this work was presented in an International
Conference: Organic Synthesis & Process Chemistry
(
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calf thymus (CT) DNA
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b
molar ratio
DT
m
(°C) after incubation
at 37 °C for
1
4
0
h
18 h
36 h
4
4
4
4
a
b
c
1:5
1:5
1:5
1:5
1:5
0.0
1.1
0.0
1.0
1.0
1.1
0.7
0.0
2.0
1.1
1.1
0.7
2
01.
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À0.8
0.2
d
DC-81 (1)
0.3
a
For CT-DNA alone at pH 7.00 ± 0.01, T
value from 10 separate determinations), all DT
.2 °C.
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phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH
m
= 69.6 °C ± 0.01 (mean
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1
H
1.40 (s, 6H), 1.90–2.20 (m,
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3
,
d
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1H), 7.40 (d, 1H, J = 4.3), 7.60–7.70 (m, 1H); MS
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