Synthesis of novel C2 and C2-C8 linked pyrrolo[2,1-c][1,4]benzodiazepine-naphthalimide hybrids as DNA-binding agents

Article abstract of DOI:10.1016/S0960-894X(03)00718-2

Synthesis of C2 and C2-C8 linked pyrrolobenzodiazepine-naphthalimide hybrids have been prepared that exhibit significant DNA-binding affinity and cytotoxicity.

Full text of DOI:10.1016/S0960-894X(03)00718-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3577–3581
Synthesis of Novel C2 and C2–C8 Linked
Pyrrolo[2,1-c][1,4]benzodiazepine-naphthalimide Hybrids
as 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 2 May 2003; accepted 24June 2003
Abstract—Synthesis of C2 and C2-C8 linked pyrrolobenzodiazepine-naphthalimide hybrids have been prepared that exhibit
significant DNA-binding affinity and cytotoxicity.
# 2003 Elsevier Ltd. All rights reserved.
The pyrrolo[2,1-c][1,4]benzodiazepine (PBD) anti-
tumour antibiotics have generated interest as potential
anticancer and gene-targeting agents.1 These com-
pounds show their biological activity by forming a
covalent bond between the C11 position of the PBD and
the exocyclic N2 group of guanine, giving rise to pref-
erence for Pu-G-Pu sequences.2 Typical examples of
PBD natural products such as tomaymycin and DC-81,
possess the same aromatic substitution pattern. More-
over, tomaymycin possesses C2-exo unsaturation and its
cytotoxic activity more than DC-81.3,4 Thurston and co-
workers have synthesized a series of novel C ring modified
C2-exo and C2-C3-endo unsaturated pyrrolo[2,1-c][1,4]-
benzodiazepines, that have shown interesting DNA
binding ability and in vitro cytotoxicity.5 Recently, C2-
aryl substituted pyrrolobenzodiazepines have also been
prepared with potent antitumour activity.6 We have
also designed and synthesized non-cross-linking mixed
imine–amide PBD dimers that have significant DNA-
binding ability and promising anticancer activity.7
reported the design, synthesis and biological evaluation
of C8 linked PBD-naphthalimide hybrids as new anti-
cancer agents.11 Therefore, it has been considered of
interest to design and synthesize C2 linked PBD-naph-
thalimide hybrids that could improve the DNA binding
ability and biological activity. In this endeavor we have
also synthesized novel C2–C8 linked PBD-naphthali-
mide hybrid to explore its DNA binding potential. This
is in continuation to our efforts in the structural modi-
fication of PBD ring system12 and also the development
of new synthetic strategies13 for this ring system. We
herein report the synthesis, DNA-binding ability and
antitumour activity of novel C2 and C2–C8 linked
PBD–naphthalimide hybrids with different alkylamide
spacers.
Synthesis of C2 linked PBD-naphthalimide hybrids has
been carried out by employing 4-benzyloxy-5-methoxy-
2-nitrobenzoic acid 7 as the starting material, which has
been obtained by the procedure described in the litera-
ture.14 trans-4-Hydroxy l-proline methylester hydro-
chloride has been coupled to compound 7 to give the
nitro ester 8. The hydroxy group is protected with
TBDMS-Cl followed by reduction with DIBAL-H to
produce the corresponding aldehyde, which is protected
with EtSH/TMS-Cl. Surprisngly, in this reaction pro-
tection of aldehyde to diethyl thioacetal and deprotec-
tion of TBDMS takes place in the same step to afford
the compound 11. Then C2 hydroxy group is oxidized
with TPAP/NMO to give compound 12, which upon
Horner-Emmons olefination with methyl (diethyl phos-
phono) acetate affords compound 13. In this reaction,
Naphthalimides are DNA interclating agents with
potential anti-cancer activity and two members of this
class amonafide and mitonafide are in clinical trails.8
These mono interclating chromophores have been
linked by a flexible alkylamine chain to bis-naphthali-
mides (DMP-840,9 LU 7955310), which are more potent
than the corresponding monomers. Recently, we have
*Corresponding author. Tel.: +91-40-2719-3159; fax: +91-40-2719-
3189; e-mail: ahmedkamal@iict.ap.nic.in
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00718-2

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A.Kamal et al./ Bioorg.Med.Chem.Lett.13 (2003) 3577–3581
(E) ester15 (13) has been obtained exclusively which
upon hydrolysis affords the corresponding acid 14.
Further amidation of the compound 24 with 6b pro-
vides compound 25. Then subsequent reduction fol-
lowed by deprotection of diethyl thioacetal group
affords the desired compound 317 (Scheme 3).
The key intermediates 15a–b have been prepared by
amidation of compound 14 with 6a–b, that have been
obtained from commercially available 1,8-naphthali-
mide as shown in Scheme 1. The compounds 15a–b are
Compounds 2a–b and 3 (Table 1) have been evaluated
for the primary anticancer activity in the standard three-
cell line panel consisting of the MCF7 (breast), NCI-
H460 (lung) and SF-268 (CNS). Amongst these hybrids
2b has promising anticancer activity in a 60-cell line
panel. The GI50 value of compound 2b (Table 2) against
leukemia cancer CCRF-CEM, HL-60 (TB), MOLT-4
and SR cell lines are 0.062, 0.091, 0.055 and <0.01 mM
respectively. Compound 2b exhibits a cytotoxic potency
in CNS cancer panel, in which SNB-19 cell line affected,
with GI50 value of 0.080 mM. Compound 2b exhibited
cytotoxic potency against breast cancer cell line T-47D
with GI50 value of 0.034 mM.
.
reduced with SnCl2 2H2O to afford the corresponding
amino diethyl thioacetal (16a–b). Deprotection of
amino diethyl thioacetal with HgCl2/CaCO3 provides
the target molecules 2a–b16 (Scheme 2).
Compound 17 has been prepared by the methodology
described in our previous report.7 This upon protection
with TBDMS-Cl, reduction followed by ethanethiol
protection affords compound 20. The coupling of this
compound with 1,8-naphthalimide affords the inter-
mediate 21. This upon oxidation, Horner–Emmons ole-
fination and ester hydrolysis gives compound 24.
The DNA binding ability of these novel C2 and C2–C8
linked PBD-naphthalide hybrids has been investigated
by thermal denaturation studies using calf thymus (CT)
DNA 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 3). One of these hybrids (2b) elevates the helix
melting temperature of CT-DNA by 13.5 ꢀC after incu-
bation for 18 h. In the same experiment the naturally
occurring DC-81 having one imine group exhibits a
ÁTm of 0.7 ꢀC. Further, synthetically designed C2-exo
unsaturated PBDs have been reported5 to exhibit lower
Table 1. In vitro one-dose primary anticancer assaya of C2 and C2–
C8 linked PBD-naphthalimide hybrids
PBD hybrids
Growth percentages
(Lung) NCI-H460 (Breast) MCF7 (CNS) SF-268
2a
2b
3
101
0
102
89
0
86
95
1
97
aOne dose of 2a–b and 3 at 10À4 molar concentration.
Scheme 1. (i) Br–(CH2)n–NHBOC, K2CO3, DMF, 12 h, rt, 94%; (ii) TFA, DCM, 0 ꢀC, 8 h, 70%.

A.Kamal et al./ Bioorg.Med.Chem.Lett.13 (2003) 3577–3581
3579
ÁTm values (1.33–2.38 ꢀC) and, for example, ÁTm of
compound 1 is 2.38 ꢀC. Therefore, the enhancement of
DNA binding ability of these hybrids can be correlated
to other interactions produced by the naphthalimide
component in addition to covalent linkage of the imine.
Moreover, as the carbon chain increases from two to
three as in the case of 2b, there is a substantial increase
in the DNA binding affinity, whereas, 3, that is C2–C8
linked PBD to two naphthalimide moieties, also shows
substantial DNA binding ability.
Scheme 2. (i) SOCl2, C6H6, trans-4-hydroxy l-proline methylester hydrochloride, Et3N, H2O, THF, 0 ꢀC, 1 h, 75%; (ii) TBDMS-Cl, imidazole,
DCM, 12 h, rt, 92%; (iii) DIBAL-H, DCM, À78 ꢀC, 45 min, 64%; (iv) EtSH-TMS-Cl, CHCl3, 18 h, rt, 72%; (v) TPAP–NMO, DCM–MeCN, 2.5 h,
rt, 78%; (vi) methyl (diethyl phosphono) acetate, NaH, THF, 0 ꢀC, 2 h, 85%; (vii) 1 N LiOH, THF–H2O–MeOH, 2 h, rt, 83%; (viii) EDCI-HOBt,
.
compound 6a–b, DCM–H2O, 24h, rt, 60–62%; (ix) SnCl 2 2H2O, MeOH, reflux, 2 h, 75–78%; (x) HgCl2–CaCO3, CH3CN–H2O, 12 h, rt, 56–58%.
Table 3. Thermal denaturation data for C2 and C2–C8 linked PBD–
nathphalimide hybrids with CT-DNA
Table 2. In vitro cytotoxicity of compound 2b in selected cancer cell
lines
PBD hybrids
[PBD]/[DNA]
molar ratiob
ÁTm (ꢀC)a after 18 h
incubation at 37 ꢀC for
Cancer panel/cell line
GI50 (mM)
2a
2b
3
DC-81
1:5
1:5
1.5
1:5
8.7
13.5
10.5
0.7
Leukemia
CCRF-CEM
HL60 (TB)
MOLT-40.055
SR
0.062
0.091
<0.010
0.08
aFor CT-DNA alone at pH 7.00Æ0.01, Tm=69.2 ꢀCÆ0.01 (mean value
from 10 separate determinations), all ÁTm values areÆ0.1–0.2 ꢀC.
bFor a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concen-
tration=100 mM and ligand concentration=20 mM in aqueous
sodium phosphate buffer [10 mM sodium phosphate+1 mM EDTA,
pH 7.00Æ0.01].
CNS
SNB-19
Breast
T-47D
0.034

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A.Kamal et al./ Bioorg.Med.Chem.Lett.13 (2003) 3577–3581
Scheme 3. (i) TBDMS-Cl, imidazole, DCM, 12 h, rt, 88%; (ii) DIBAL-H, DCM, À78 ꢀC, 60 min, 68%; (iii) EtSH–TMS–Cl, CHCl3, 18 h, rt, 85%;
(iv) compound 4, K2CO3, DMF, 12 h, rt, 90%; (v) TPAP–NMO, DCM–MeCN, 2.5 h, rt, 72%; (vi) methyl (diethyl phosphono) acetate, NaH, THF,
.
0
ꢀC, 2 h, 71%; (vii) 1 N LiOH, THF–H2O–MeOH, 2 h, rt, 78%; (viii) EDCI–HOBt, compound 6b, DCM–H2O, 24h, rt, 62%; (ix) SnCl 2 2H2O,
MeOH, reflux, 2 h, 72%; (x) HgCl2–CaCO3, CH3CN–H2O, 12 h, rt, 52%.
In conclusion, novel C2 and C2–C8 linked PBD-naph-
thalimide hybrids have been synthesized that exhibit
significant DNA-binding ability. Further, these com-
pounds exhibit promising anticancer activity in certain
cancer cell lines. The detailed mechanistic and molecular
modeling studies for these PBD hybrids are in progress.
References and Notes
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UK, 1993; Vol. 1, p 54.
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Acknowledgements
We thank the National Cancer Institute, Maryland for
the primary anticancer assay in human 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.
L. R.; Thurston, D. E. Bioorg.Med.Chem.Lett.
2000, 10,

A.Kamal et al./ Bioorg.Med.Chem.Lett.13 (2003) 3577–3581
3581
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16. Selected data for compound 2b. 1H NMR (CDCl3) d 1.80–
2.2 (m, 2H), 3.2–3.35 (m, 4H), 3.98 (s, 3H), 4.20–4.30 (m, 2H),
4.45–4.65 (m, 3H), 5.20 (s, 2H), 6.0 (s, 1H), 6.85 (s, 1H), 6.92
(m, 1H), 7.25–7.45 (m, 5H), 7.55 (s, 1H), 7.75 (m, 2H), 7.84 (d,
J=3.8 Hz, 1H), 8.23 (d, J=7.8 Hz, 2H), 8.56 (d, J=7.0 Hz,
2H); MS (FAB) 629 [M+H]+Á
1
17. Selected data for compound 3. H NMR (CDCl3) d 1.94–
2.40 (m, 4H), 3.20–3.40 (m, 4H), 3.45 (s, 3H), 4.20–4.60 (m,
9H), 6.05 (s, 1H), 6.80 (s, 1H), 6.93 (m, 1H), 7.32 (s, 1H), 7.70–
7.85 (m, 4H), 7.88 (d, J=4.08 Hz, 1H), 8.18–8.30 (m, 4H),
12. Kamal, A.; Laxman, N.; Ramesh, G.; Srinivas, O.;
Ramulu, P. Bioorg.Med.Chem.Lett. 2002, 12, 1917.
8.56 (d, J=7.2 Hz, 4H); MS (FAB) 776 [M+H]+Á
.