Five-member thio-heterocyclic fused naphthalimides with aminoalkyl side chains: Intercalation and photocleavage to DNA

Article abstract of DOI:10.1016/j.bmcl.2004.12.011

Novel five-member thio-heterocyclic fused naphthalimides with aminoalkyl side chains were designed, synthesized and evaluated. These compounds have high Scatchard binding constants. They could damage DNA (supercoiled pBR322) from form I (closed) to II (ni

Full text of DOI:10.1016/j.bmcl.2004.12.011

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1139–1142
Five-member thio-heterocyclic fused naphthalimides with
aminoalkyl side chains: intercalation and photocleavage to DNA
a
Yufang Xu,a Baoyuan Qu,a Xuhong Qiana,b, and Yonggang Li
*
aShanghai Key Lab. Chem. Biology, East China University of Science and Technology, Shanghai 200237, China
bState Key Lab. Fine Chemicals, Dalian University of Technology, Dalian 116012, China
Received 20 September 2004; accepted 6December 2004
Available online 7 January 2005
Abstract—Novel five-member thio-heterocyclic fused naphthalimides with aminoalkyl side chains were designed, synthesized and
evaluated. These compounds have high Scatchard binding constants. They could damage DNA (supercoiled pBR322) from form
I (closed) to II (nicked) at a concentration as low as 10 lM and from form I to form III at a concentration of 50 lM. The results
implied that the influence of intercalating ability of chromophores on photocleaving ability of photocleavers depended on the mech-
anism of photocleavage.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Synthetic photochemical DNA cleaving reagents are of
R
R
N
great interest in chemistry, biology and medicine. These
reagents can site-selectively or nonselectively cleave
DNA triggered by near-UV light.1 Although many
photocleavers can damage DNA (supercoiled pBR322)
from form I (closed) to II (nicked), only a few of them
at a lower concentration, are able to photocleave
DNA from form I to III (linear). It was known that
many naphthalimide derivatives are famous anti-cancer
agents or DNA photocleavers, but there are few hetero-
cyclic fused naphthalimide as intercalative photocle-
avers for DNA.1g,2 Large planar chromophore might
show good DNA intercalative ability, some believe that
the high intercalation would increase the photocleaverÕs
affinity to DNA and promote photocleavage. We
reported two isomers, six- and five-member heterocyclic
fused naphthlimides with hydroperoxyl group (C and D,
Fig. 1).1m,n D with high intercalating ability (binding
constant 8.72 · 105 MÀ1) could damage DNA from
form I to form II at a concentration as low as 1 lM
and C with lower intercalating ability (binding constant
4.26 · 103 MÀ1) damaged DNA from form I to form II
at a concentration of 5 lM. For these DNA photocleav-
ing agents of radicals, it seemed that the intercalation
was the main factor to affect photocleaving abilities.
O
O
1 R=CH2CH2N(CH3)2
O
O
2 R=CH2CH2CH2N(CH3)2
3 R=
CH2CH2N
NH
S
S
S
4 R=CH2CH2CH2CH3
(A)
(B)
O
O
O
OOH
OOH
N
S
O
(D)
(C)
Figure 1. Structures of reported (A), (C), (D) and novel (B)
photocleavers.
However, we wondered if the case is the same for those
of nonradicals, which damaged DNA mainly through
electron transfer mechanism. Therefore, we hope to
investigate photocleavers of five- and six-member het-
erocyclic fused naphthlimides with aminoalkyl side
chains.
Keywords: Five-member; Thio-heterocyclic fused naphthalimide; Inter-
calation; Photocleavage.
Previously, we already reported that six-member A1–A4
could photodamage the circular supercoiled pBR322
DNA from form I to II at a concentration as low as
*
Corresponding author. Tel.: +8621 46253589; fax: +8621
64252603; e-mail: xhqian@ecust.edu.cn
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.011

1140
Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1139–1142
R
O
O
N
O
O
O
a
b
c
d
e
S
Br
Scheme 1. Synthesis of novel naphthalimide-derived photocleavers.
Reagents and conditions: (a) HNO3/H2SO4; (b) benzenethiol, Pyridine,
ethanol, reflux, 5 h, 87% yield; (c) SnCl2/HCl, 85 ꢁC, 1 h, 96% yield; (d)
NaNO2, HOAc, H2SO4; CuSO4, HOAc, H2O, 86% yield; (e) RNH2,
ethanol, reflux, 2–3 h, 85% yield.
a,b
Table 1. Spectra data of compounds B1–B4
Compd
UV kmax/nm (lge)
FL kmax/nm (/)
B1
B2
B3
B4
384 (4.24)
383 (4.27)
382 (4.15)
380 (4.08)
436(0.0215)
433 (0.0213)
429 (0.0151)
426(0.0333)
a In absolute ethanol.
b With fluorescein as standard (/ = 0.90).
0.5 lM1l,o and to form III at a concentration of 50 lM.
For comparison, we would present here the isomers of
A, novel photocleavers of five-member thio-heterocyclic
fused naphthalimides with aminoalkyl side chains at the
imide (B), and study the influence of the intercalation on
the photocleavage.
Figure 2. Photocleavage of the supercoiled pBR322 DNA. The
cleavage activities were evaluated using the supercoiled circular
pBR322 DNA (form I) (30 ng/lL) with a compound in the buffer
Tris–HCl (pH 7.5) under photoirradiation (2300 W/cm2) through a
transluminator (366 nm) in the distance of 20 cm at 0 ꢁC and then
analyzed on a 1% agarose gel. (a) Photocleavage of compounds B1–B4.
Photoirradiation: 2 h; lane 1–4: DNA and compounds B3, B1, B2, B4 at
the concentration of 50 lM, respectively; lane 5: DNA alone; lane 6:
DNA alone (no hm). (b) Concentration dependent of B3Õs photoclea-
vage. Lane 1–7: DNA and B3 at the concentration of 100, 50, 20, 10, 5,
2, 0.5 lM, respectively; lane 8: DNA alone (no hm). (c) pH dependence
of photocleavage lane 2, 4, 6, 8: DNA alone (hm, 60 min), pH = 8.5,
8.0, 7.5, 7.0; lane 1, 3, 5, 7: DNA and B3 (50 lM), pH = 8.5, 8.0, 7.5,
7.0, respectively. Lane 9: DNA alone (no hm). (d) Time dependence of
photocleavage for B3 lane 1–4: DNA and B3 (50 lM) (hm, 120, 90, 60,
30 min), respectively; lane 5: DNA alone (hm, 75 min); lane 6: DNA
alone (no hm).
Therefore, several novel photocleavers B1–4 were
designed (Fig. 1). These compounds were synthesized
from 4-bromonaphthalic anhydride shown in Scheme
1.3 After the separation through silica gel column
chromatography with the eluent of chloroform–acetone
(1:1, v/v), their structures were confirmed by IR,
1H NMR, MS and element analysis.4 It was obvious
from Table 1 that their absorptions were around
382 nm with the similar intensities. They also had the
emission at 430 nm with weak fluorescent intensities.
All of these compounds were shown to possess high
intercalating abilities rather than their isomers,
compound A (Fig. 1).
The affinities to DNA of these compounds were very
strong, binding constants were about ꢀ105 MÀ1. With
B1 as an example, the Scatchard binding constant5
between B1 and CT-DNA was determined to be
2.8 · 105 MÀ1, which is very similar to that of D. It indi-
cated that B1–4 bind DNA mainly via intercalation
exerted by the chromophore of thio-heterocyclic fused
naphthalimides.
Further experiment indicated that B3 could cleave the
closed supercoiled DNA to the form II at a concentra-
tion as low as 10 lM and to the form III at a concentra-
tion of 50 lM. However, no cleavage was observed in
the control reactions run in the dark or without com-
pounds (Fig. 2b). In addition, the bufferÕs pH value
did not obviously affected its DNA cleaving actions
(Fig. 2c), and it exhibited better DNA damage abilities
under prolonged photoirradiation (Fig. 2d). It was ob-
served previously that A3, the isomer of B3, could cleave
the closed supercoiled DNA to the nicked form at a con-
centration as low as 0.5 lM and to the linear form at a
concentration of 50 lM. It means that A (six-member
heterocyclic isomer) have a slight higher DNA photoc-
leavage than B (five-member heterocyclic isomer), which
is much different from the case for C and D.
Photocleaving abilities of compounds B1–B4 were then
examined with the closed supercoiled pBR322 DNA un-
der the photoirradiation at 366 nm as shown in Figure
2a. The cleaving efficiency was defined by the degree
of the relaxation of supercoiled DNA. It was apparent
that B3 exhibited the greatest DNA cleaving ability over
B1 and B4, while B4 could hardly damage DNA under
the same condition. The order of their photocleaving
abilities was as follows: B3 > B2 > B1 > B4.

Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1139–1142
1141
the approximation of the photocleaver to DNA and
facilitated the radicals to damage DNA. However, in
the case of A and B, which photodamaged DNA in elec-
tron transfer mechanism, the high intercalating ability
resulted in the isolation of the photocleaver within the
hydrophobic inner of DNA from oxygen in surrounding
solution might prevent them from undergoing electron
transfer7 to some extent between the amino groups
and oxygen. These two cases implied that the intercala-
tion did not always promote photocleavage and the
influence of intercalating ability of chromophores on
the cleaving ability of photocleavers depended on their
damage mechanisms.
Figure 3. The effect of different additives on the photocleavage of
supercoiled pBR322 DNA (30 ng/lL) in the buffer Tris–HCl (pH 7.5)
under photoirradiation (2300 W/cm2) with a transluminator (366 nm)
for 2 h in the distance of 20 cm at 0 ꢁC; lane 1: DNA and B3 in the
presence of ethanol (1.7 M); lane 2: DNA and B3 in the presence of
superoxide dismutase (SOD, 100 lg/mL); lane 3: DNA and B3 in the
presence of dithiothreitol (DTT, 30 mM); lane 4: DNA and B3 in the
presence of histidine (6mM); lane 5: DNA and B3 at the concentration
of 50 lM; lane 6: DNA alone; lane 7: DNA alone (no hm).
In summary, the present work demonstrated the design
and evaluation of novel and high intercalating photo-
cleavers with five-member thio-heterocyclic fused naph-
thalimides containing aminoalkyl side chains at the
imide. They could damage DNA (supercoiled pBR322)
from form I (closed) to II (nicked) at a concentration
as low as 5 lM and from form I to form III at a concen-
tration of 50 lM. The comparison of these two types of
isomers implied that the influence of intercalating abili-
ties of chromophores on the cleaving abilities of photo-
cleavers depended on their damage mechanisms. The
experimental results suggested that the aminoalkyl side
chain connected with chromophore is also very impor-
tant for photodamage in electron transfer mechanism.
The anti-cancer studies on these photocleavers are also
in progress.
Mechanism experiment was also performed with the
addition of histidine, dithiothreitol, superoxide dismu-
tase and ethanol (Fig. 3). It was found that histidine
(singlet oxygen quencher) ethanol (radical quencher)
had no effect on the cleavage reaction, However, dithio-
threitol (DTT, superoxide anion radical scavenger) re-
tarded the reaction efficiently. It should be pointed out
that superoxide dismutase (SOD, superoxide radical
killer) accelerated the rate of DNA-cleaving reaction,
because the hydrogen peroxide produced by SOD from
superoxide anion radical, could lead to DNA damage in
the presence of UV light or after reduction by the trace
of metal ions. Obviously, superoxide anion radical was
involved in the DNA cleavage. It was supposed by Eri-
ksson and co-workers6 that the reactive superoxide an-
ion radicals formed in the photoirradation had two
pathways. The most common way was reduction of
the excited triplet by an electron donor (e.g., one of
the DNA bases), followed by electron transfer from
the reduced photosensitizer to molecular oxygen. SaitoÕs
research work2d has proved this way. The other way was
a direct ionization of the photosensitizer by radiation,
and electron uptake by molecular oxygen (Ôdirect elec-
tron transferÕ). In our experiment, without using piper-
idine treatment, we could exclude DNA damage by G
oxidation and we found that compounds A4, B4 had
no any cleaving ability in the absence of aminoalkyl side
chain. It implied that the superoxide anion radical was
possibly formed through the electron transfer from
nitrogen in aminoalkyl side chain to oxygen.
Acknowledgements
The National Key Project for Basic Research
(2003CB114400), National Natural Science Foundation
of China, The Science and Technology Foundation of
Shanghai and The Education Commission of Shanghai
partially supported this study.
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