N-Aroyloxylthioxo-naphthalimides as DNA photocleavers of aroyloxyl oxygen radicals: Synthesis, evaluation, and substituents' effect

Article abstract of DOI:10.1016/j.bmc.2004.02.013

Novel N-Aroyloxylthioxo-naphthalimides as highly efficient 'time-resolved' DNA photocleavers of aroyloxyl radicals type were designed and synthesized. The substituents at the aroyloxyl moiety have an important and unusual influence on the DNA photocleavage, and DNA photodamages of the compounds were unusually not depended on the electronic effects of substituents on the corresponding oxygen-centered radicals. With AM1 semi-empirical quantum calculation, it was found that their photocleaving activities were correlated with the densities of electron clouds on the N-O bonds in the triplet state. N-(m-Dichloro-benzoyloxy) -thioxo-naphthalimide could photodamage DNA effectively at less than the concentration of 2μM.

Full text of DOI:10.1016/j.bmc.2004.02.013

Bioorganic & Medicinal Chemistry 12 (2004) 2335–2341
N-Aroyloxylthioxo-naphthalimides as DNA photocleavers of
aroyloxyl oxygen radicals: synthesis, evaluation, and substituentsꢀ
effect
Yufang Xu,a Xiayu Huang,a Xuhong Qianb,* and Wei Yaoa
aShanghai Key Laboratory of Chemical Biology, Institute of Pesticides & Pharmaceuticals, East China University of Science and
Technology, Shanghai 200237, China
bState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
Received 27 October 2003; accepted 5 February 2004
Abstract—Novel N-Aroyloxylthioxo-naphthalimides as highly efficient ꢁtime-resolvedꢀ DNA photocleavers of aroyloxyl radicals
type were designed and synthesized. The substituents at the aroyloxyl moiety have an important and unusual influence on the DNA
photocleavage, and DNA photodamages of the compounds were unusually not depended on the electronic effects of substituents on
the corresponding oxygen-centered radicals. With AM1 semi-empirical quantum calculation, it was found that their photocleaving
activities were correlated with the densities of electron clouds on the N–O bonds in the triplet state. N-(m-Dichloro-benzoyloxy)-
thioxo-naphthalimide could photodamage DNA effectively at less than the concentration of 2 lM.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
centered radicals derived from novel photonucleases of
N-aroyloxylthioxo-naphthalimides.
Oxygen-centered radicals are very useful in chemistry,
biology, and medicine.1–3 In recent years, the design and
study of DNA ꢁartificial photonucleasesꢀ of the oxygen-
centered radical type with no need of a metal or external
oxidant, have attracted much attention and have led to
the development of both specific gene-targeted drugs and
artificial restriction enzymes.4;5 Recently, highly efficient
ꢁtime-resolvedꢀ photonuclease with releasing hydroxyl
radical and aroyloxyl radicals were developed.6 Previ-
ously we ever reported the remarkable promoting action
to photocleaving activity of thio-moiety on heterocycles,
which was attributed to the enhancement of the elec-
tronic transition from n to pꢀ for thio-counterparts by
compared with oxo-counterparts.6 There as yet have
been few reports about substituent effects of oxygen-
centered radicals on their DNA photocleaving activity,
although ones knew that the electron-donating group
was favorable to the formation and stability of oxygen-
centered radicals in a chemical system. Here we report
important and unusual substituent effects of oxygen-
It was known that N-hydroxypyridinethiones (A), N-
aroyloxypyridine-2-thiones (B) were DNA photoclea-
vers through release of hydroxyl or aroyloxyl radicals
(Scheme 1).7–9 The alkylacyl oxygen radicals derived
from N-alkylacyloxypyridine-2-thiones almost had not
photocleaving activity by comparison with highly effi-
cient aroyl counterparts,10–12 it was probably caused by
that the oxygen-centered radical with aroyl moiety has
better stability and was easy to form. In this paper, in
order to study the role of substituents on oxygen-cen-
tered radicals in photonucleases, we synthesized novel
N-aroyloxylthioxo-naphthalimides (C) through the
introduction of monosubstituted, disubstituted benzoyl
or heterocyclic aroyl groups to that oxygen atom of N–
O bond for obtaining the corresponding oxygen-cen-
tered radicals and evaluated their DNA photocleaving
activities (Scheme 1).
2. Results and discussion
2.1. Synthesis and spectra
Keywords: Aroyloxylthioxonaphthalimides; Substituent effect; DNA
photocleaver; Oxygen centered radical.
We have tried three synthetic routes to the target N-
aroyloxythionaphthalimides C. It was found that the
* Corresponding author. Tel.: +86-21-64253589; fax: +86-21-642526-
03; e-mail addresses: xhqian@dlut.edu.cn, xhqian@ecust.edu.cn
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.013

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Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
S
R2
R1= Ph, C6H11, Ph(CH2)2, naphthyl
R2=H, NHCO(CH2)4NH-acridine
N
S
N
O
S
N
OH
R1
OH
B
A2
A1
O
X
N
O
O
O
C0 X=O,Y=H
C1 X=S, Y=H
C2 X=S, Y=F
C3 X=S, Y=Cl
C4 X=S, Y=CH3
C5 X=S, Y=OCH3
C6 X=S, Y=m-Cl2
C7 X=S, Y=m-Me2
C8 X=S, Y=o-F, p-Cl
C9 X=S, Y=m, p-OMe2
C10 X=S, Y=o-Cl, m-Pyridine
C11 X=S, Y=o-furanyl
Y
C
Scheme 1.
route i was unsuccessful, in which all of the three car-
bonyl groups in the intermediate have the possibility to
react with the Lawessonꢀs reagent (LR, 2,4-bis(p-meth-
oxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide),
the reaction mixture always gave a tar-like oil from
which no component could be isolated by silica gel or
basic alumina preparative TLC. The route ii was also
unattractive, as the hydroxyl group in the intermediate
could react with LR reagent and resulted in the
decomposition of LR, the total yield was very low. Only
the route iii with one-pot procedure gave high yield, in
which the target compounds were synthesized with 1,8-
naphthalic anhydride as the starting material by the
dithionation with LR reagent, the imidation with
hydroxylamine hydrochloride at refluxing in acetonitrile
and the condensation with substituted benzoyl chlor-
ides. The structures of the target compounds were
Meanwhile, the intercalation experiments of these
compounds to the Calf thymus DNA with C1 and C6 as
examples, were carried out by using an electronic
absorption spectra technique instead of the fluorescence
quenching method,14 as C1–C11 only have very weak
fluorescence. During the addition of Calf thymus DNA
the absorption intensities of C1, C6 became weaker with
the increase of the concentration of DNA (Fig. 1).
It was widely accepted that the DNA photocleaving
activities of N-hydroxy-(A) and N-aroyloxypyridine-2-
thione (B) are attributable to the formation of persistent
hydroxyl or aroyloxyl radicals during the photolysis,
and that they are able to give ESR signals through the
spin trapping method, whilst the concomitant part, a
thiyl radical signal was not detected under similar con-
ditions.7;13;15 In addition, the similar sulfur-centerd
thiopyridyl radical is known not to contribute to the
DNA damage.10;12;13 In our case, PBN (N-tert-butyl-a-
phenylnitrone) trapping ESR spectroscopy identified
that C1–C11 in benzene could generate a free aroyloxyl
radicals signal under irradiation (Fig. 2), which should
be responsible for the DNA damage.
1
identified by using H NMR, EI-MS, IR, and elemental
analysis (Scheme 2).
All thioxo-compounds (C1–C11) had long-wavelength
absorption and very weak fluorescence (Table 1). This
implies that for the latter their excitation energy might
be more easily transferred from the singlet excited state
to cleave the N–O bond,13 besides being dissipated as
heat in the system by internal conversion processes.
The photocleaving ability for DNA double strand-scis-
sion was evaluated for all the novel N-aroyloxylthioxo-
OH
i
O
N
O
O
O
O
N
O
R
a
c
.
b
S
O
O
c
O
O
O
R
ii
N
O
OH
N
c
a
O
S
O
S
S
iii
b
Scheme 2. Reagents and conditions: (a) RCOCl, NEt3, rt, 45 min; (b) NH2OHÆHCl, NEt3, CH3CH, reflux, 2 h; (c) Lawessonꢀs reagent, C6H5–Cl-p,
reflux, 1 h.

Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
Table 1. Photocleaving activities, spectra data,a;b and calculated parametersc;d
2337
Compound
UV kmax/nm (log ꢀ)
FL kmax/nm (/)
Photocleaving activity (100 lM, hm1 h)
Electronic density
of oxygen radicalc
Polar surface area
2
d
ꢀ
(A )
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
392 (4.29)
400 (4.37)
390 (4.34)
390 (4.23)
390 (4.21)
397.5 (4.19)
397.0 (4.17)
398.5 (4.21)
398.5 (4.18)
398.0 (4.15)
397.5 (4.16)
491 (<0.001)
454 (<0.001)
466 (<0.001)
466 (<0.001)
454 (<0.001)
435.6 (0.0014)
435.6 (0.0016)
435.4 (0.0014)
436.0 (0.0016)
466.0 (<0.001)
468.3 (<0.001)
50.4
100
49.6
0
)0.069
)0.067
)0.066
)0.071
)0.072
)0.060
)0.070
)0.066
)0.068
)0.068
)0.058
66.958
66.930
67.005
65.515
76.744
65.628
65.211
66.526
84.946
79.875
78.771
86.2
63.4
49.5
29.7
39.1
39.5
60.2
41.6
42.2
13.8
36.7
51.5
70.3
60.9
60.5
39.8
58.4
57.8
a In absolute ethanol.
b With quinine sulfate in sulfuric acid as quantum yield standard.
c By semi-empirical SCF AM1 method (hyperchem 5.5).
d The polar surface area of the aroyl acid corresponding to a radical calculated by molecular modeling MMX-E method (Pcmodel 6.0).
40
0.35
0um
50um
100um
200um
0.30
0.25
0.20
0.15
0.10
0.05
30
20
10
0
DNA
A
350 360 370 380 390 400 410 420 430 440 450
360
380
400
420
440
460
Wavelength (nm)
¦Ë (nm)
Figure 1. Interaction of C1, C6, and DNA. Absorption changes from 300 to 500 nm of C1, C6 (20 lM) during the addition of Calf thymus DNA
(0, 50, 100, 200 lM) in THF (10 mM), Tris–HCl (pH 7.4) solution (3:10, v/v).
naphthalimides (Fig. 3). Obviously, the DNA damages
were caused by the aroyloxyl radicals with different
substituents,16 which were formed from the corre-
sponding N-aroyloxylthioxo-naphthalimides under
photo-irradiation. It was found that the DNA photoc-
leaving abilities of these compounds were in the order:
C6 > C7 ꢁ C8 > C10 ꢁ C11 > C5 > C1 > C9 > C4 > C3 >
C2. The photodamage abilities of these compounds
should be correlated with the formation, stabilities, and
reactivities of the corresponding oxygen-centered
Figure 2. ESR spectroscopy of the radical released by C1 and C6. ESR
with PBN (N-tert-butyl-a-phenylnitrone) trapping in benzene under
light. Microwave frequency 9.782 GHz, mid range 3430 G, scan range.
Figure 3. Photocleavage on pBR 322 DNA. The cleavage activities were evaluated using supercoiled circular pBR322 (form I) DNA (50 lM/bp) with
the compound in the buffer Tris–HCl (pH 7.6) under photoirradiation (2300 lW/cm2) with a transluminator (366 nm) at a distance of 20 cm at 0 °C
and then analyzed on a 1% agarose gel. DNA photocleavage efficiency was defined as the degree of relaxation of the supercoiled DNA, relaxed
circular DNA (single-stranded cleavage) as form II. (a) Effects of different substituents on photocleavage (100 lM; hm, 45 min). Lane 1: DNA alone (no
hm); Lane 2: DNA and C1; Lane 3: DNA and C2; Lane 4: DNA and C3; Lane 5: DNA and C4; Lane 6: DNA and C5; Lane 7: DNA alone (hm). (b)
Effects of different substituents on photocleavage (100 lM; hm, 60 min). Lane 1: DNA alone (hm); Lane 2–8: DNA and C6–C11, C5, respectively; Lane
7: DNA alone (no hm).

2338
Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
radicals. It was known that in radical chemistry, elec-
tron-donating substituents could improve the stabilities
of oxygen-, and heteroatom-centered radicals, while
electron-withdrawing substituents do not.17 However, in
our case the electron-donating effect was not so impor-
tant here, for instance, the compound with the m-di-
chlorobenzoyl group (C6) showed the highest activity,
while that with strong electron-donating m, p-di-
methoxybenzoyl group (C9) was the worst, this observa-
tion implied that these radicalsꢀ stabilities were not only
or important factor to influence on their photobio-
activities.
photocleaving activity was very low (Form II 3.5%,
100 lM).6 While for C1, with the same benzoyloxyl
radical part, its density on the N–O bond at excited
triplet state is low, so itꢀs easy to break and its photoc-
leaving activity is high (Form II 54.8%, 100 lM). A very
similar conclusion could be obtained with other com-
pounds, for example C6, its very low density is corre-
sponding to very high photocleaving activity (Form II
70.3%, 100 lM). These suggested that the formation of
these radicals was important factor to influence on their
photobioactivities, as the radicals usually were very
reactive.
From the Table 1 it could be found that the order of
photocleaving activities of these radicals derived from C,
is also not consistent with that of the electronic densities
of aroyloxyl radicals at excited singlet state, which
were calculated by semi-empirical SCF AM1 method
(Hyperchem 5.5). In addition, no relationships were
obtained between the photocleaving activities and the
calculated steric parameters or others (Table 1). But, it
seems that there is some correlation between the electron
densities on the N–O bonds of the compounds at excited
triplet state (not excited singlet state) and their photoc-
leaving abilities (Fig. 4). For example, the density on the
N–O bond of C0 (oxo-compound) at excited triplet state
is very high, it implies that the breakage of the N–O
bond at excited triplet state is very difficult, so its
Figure 5a indicates that the aroyloxyl radical was gen-
erated from C6 at a relatively linear rate, this continuous
generation of reactive oxygen-centered radicals provides
an alternative to Fenton-based chemistry, where oxygen
radicals are formed in a rapid burst. This kind of reagent
is attractive for ꢁtime-resolvedꢀ DNA cleavage studies.18
Figure 5b also demonstrate that C6 could damage DNA
effectively at a concentration of 2 lM. It probably was
the most potent compound in N-aroyloxypyridine-2-
thione derivatives.9 Figure 5c shows that pH had little
influence on the photocleaving activity of C6, which
means that these aroyloxyl radicals are quite stable in a
wide range of pH values. In addition, the experiments
showed that the solvents did not have influence on its
photocleaving activity.
Figure 4. The distribution of electron clouds for the compounds.

Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
2339
Figure 5. (a) Time-dependant photocleavage of DNA using C6 (100 lM). Lane 1: DNA alone (no hm); Lane 2: C6 and DNA (hm, 90 min); Lane 3: C6
and DNA (hm, 75 min); Lane 4: C6 and DNA (hm, 60 min); Lane 5:C6 and DNA (hm, 30 min); Lane 6: DNA alone (hm, 1.5 h). (b) Concentration-
dependant photocleavage of DNA using C6 (hm, 1 h). Lane 1: DNA alone (no hm); Lane 2–8: DNA and C6 at concentrations of 2, 5, 10, 20, 50, 100, and
200 lM, respectively. Lane 9: DNA alone (hm). (c) pH effect on DNA photocleavage of C6 (100 lM; hm, 1 h). Lane 1, 3, 5, 7: DNA alone, pH ¼ 8.5,
8.0, 7.5, 7.0, respectively; Lane 2, 4, 6, 8: DNA and C6, pH ¼ 8.5, 8.0, 7.5, 7.0, respectively.
3. Conclusion
Reagent under Ar protection, refluxed 24 h, then cooled,
filtered, and dried to afford the crude product 4 g
(17 mmol, 69%). The recrystallization in ethanol and
chlorobenzene to give brown solid, mp: 211–212 °C (lit.
211–212 °C.). EI-MS: m=z 230 (Mþ, 100).
Novel N-aroyloxylthioxo-naphthalimides as highly effi-
cient ꢁtime-resolvedꢀ DNA photocleavers of aroyloxyl
radicals type were designed and synthesized. It was
found that substituents at the oxygen-centered radicals
had important and unusual influence on the DNA
photocleaving activity, the photocleaving activity of
radical is correlated with the density of electron clouds
on the N–O bond in the triplet state. It suggested that
the formation of these radicals was important factor to
influence on their photobioactivities. N-(m-Dichloro-
benzoyloxy)-thioxo-naphthalimide could photodamage
DNA effectively at less than the concentration of 2 lM,
our study probably provides a novel ꢁtime-resolvedꢀ and
the most efficient DNA photocleaver in N-aroyloxy-
pyridine-2-thione derivatives.
5.3. 2-Hydroxy-benzo[de]isoquinoline-1,3-dithione
To a solution of hydroxylamine hydrochloride (0.24 g)
in acetonitrile (25 mL), was added 1,8-thionaphthlimide
(0.8 g), refluxed 2 h, cooled, filtered, the orange-red
crude product was recrystallized in ethanol. EI-MS
(m=z,%): 229 (Mþ, 7.3), 213 ([M+Hþ)OHꢂ], 56.5), 197
([M)S]þ, 100).
5.4. Benzoic acid 1-oxo-3-thioxo-1H,3H-benzo[de]iso-
quinolin-2-yl ester (C1)
4. Experimental
To the solution of 2-hydroxy-benzo[de] isoquinoline-1,3-
dithione, ice/salt bath cooled, was added drops of tri-
ethylamine and benzoyl chloride (0.49 g), the mixture
reacted 1 h, filtered, concentrated, and silca gel purifi-
cation (Rf ¼ 0:54, petroleum ether: dichlorometh-
ene ¼ 1:4, v/v) to give orange-red product C1. Mp: 189–
190 °C EI-MS (m=e, %): 333 (Mþ, 1.9), 213
([M+Hþ)PhCOOÆ], 2.8), 105 (PhCOþ, 100); IR (KBr):
4.1. Materials and methods
Melting points were taken on a digital melting point
apparatus WRS-1 made in Shanghai and it was uncor-
rected. Infrared spectra were recorded on a Nicolet FT
1
IR-20SX, mass spectra on a Hitachi M80, H NMR on
1
a Bruker AM-300 or AM-500 using TMS as an internal
standard. Combustion analysis for elemental composi-
tion was done on Italy MOD.1106 analyzer. Absorption
spectra were measured on Shimadzu UV-265, fluores-
cence spectra on a Hitachi 850.
3030, 1780, 1715, 1610, 1580, 1270, 780, 740 cmꢃ1; H
NMR (CDCl3): d 9.01 (dd, J1 ¼ 7:6 Hz, J2 ¼ 0:9 Hz, 1H,
7-H), 8.67 (dd, J1 ¼ 7:2 Hz, J2 ¼ 1:1 Hz, 1H, 2-H), 8.27
(m, 4H, 3–6H), 7.72 (m, 3H, 20-, 40-, 60-H), 7.57 (m, 2H,
30-, 50-H); C19H11NO3S required: C, 68.46; H 3.32; N,
4.20. Found: C, 68.51, H, 3.49, N, 4.22.
5. Synthesis
5.5. 4-Fluoro-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C2)
5.1. Preparation of substituted benzoyl chloride
Preparation and purification of this compound (and the
other compounds as follows) was accomplished follow-
ing the procedure described for C1,4-fluorobenzoyl
chloride (0.55 g) was used here. Orange product
(Rf ¼ 0:56). Mp: 186–187 °C, EI-MS (m=z, %): 351 (Mþ,
3.6), 213 (4.6), 123 (p-FPhCOþ, 100); IR (KBr): 3040,
To a solution of substituted-benzoic acid was added
thionyl chloride. The mixture was refluxed for 3 h until
no hydrogen chloride gas produced and concentrated
under reduced pressure to obtain the substituted benzoyl
chloride.
1
1780, 1710, 1600, 1580, 1500, 1270, 840 cmꢃ1; H NMR
(DMSO-d6): d 8.92 (d, J ¼ 7:5 Hz, 1H, 2-H), 8.64 (m,
3H, 7-, 4-, 5-H), 8.32 (m, 2H, 3-, 6-H), 7.97 (m, 2H, -H,
30-, 50-H), 7.56 (m, 2H, 20,60-H); C19H10FNO3S required:
C, 64.95; H, 2.87; N, 3.99. Found: C, 64.59; H, 2.87; N,
4.02.
5.2. Benzo[de]isochromene-1,3-dithione19
To a solution of 1,8-naphthlimide (5 g, 25 mmol) in
chlorobenzene (90 mL), was added the Lawessonꢀs

2340
Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
5.6. 4-Chloro-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C3)
(DMSO-d6): 8.92 (d, J32 ¼ 7:2 Hz, 1H, 2-H), 8.64 (m,
0 0
3H, 4-, 5-, 7-H), 8.24 (m, JF–HðmÞ ¼ 8:0 Hz, J5 6 ¼ 8:3 Hz,
1H, 30-H), 7.96 (m, 2H, 3-, 6-H), 7.88 (dd,
JF–HðoÞ ¼ 10:8 Hz, J5 3 ¼ 1:8 Hz, 1H, 30-H), 7.65 (dd,
0
0
The similar preparation and purification procedure as
the above, 4-chlorobenzoyl chloride (0.61 g) was used
here. Orange-red product (Rf ¼ 0:62). Mp: 182–183 °C.
EI-MS (m=e, %): 367 (Mþ, 1.7), 213 (2.5), 139 (p-
ClPhCOþ, 100); IR (KBr): 3035, 1780, 1710, 1650, 1580,
JF–HðpÞ ¼ 1:8 Hz, J6 5 ¼ 8:3 Hz, 1H, 50-H); EI-MS (m=z,
%): 385 (Mþ, 16.93), 213 ([M+Hþ)2-F-4-ClC6H3COOꢂ],
80.43), 157 (2-F-4-ClC6H3COþ, 100); IR (KBr): 3040,
0
0
1780, 1710, 1610, 1580, 1500, 1280, 840 cmꢃ1
;
1270, 840 cmꢃ1
.
1H NMR (DMSO-d6): d 8.91 (d,
C19H9ClFNO3S required: C, 59.15; H, 2.35; N, 3.63.
Found: C, 59.34; H, 2.13; N, 3.52.
J ¼ 7:5 Hz, 1H, 2-H), 8.64 (m, 3H, 7-, 4-, 5-H), 8.23 (m,
2H, 3-, 6-H), 7.96 (m, 2H, 30-, 50-H), 7.79 (m, 2H, 20,60-
H); C19H10ClNO3S required: C, 62.04; H, 2.74; N, 3.81.
Found: C, 62.38; H, 2.90; N, 3.55.
5.11. 3,5-Dimethyl-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C8)
5.7. 4-Methyl-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C4)
3,5-Dimethylbenzoyl chloride (0.59 g) was used here.
1
Brown product. Mp: 216–218 °C; H NMR d (DMSO-
d6): 8.92 (d, J ¼ 7:7 Hz, 1H, 2-H), 8.63 (m, 3H, 4-, 5-, 7-
H), 7.95 (m, 2H, 3-, 6-H), 7.81 (s, 2H, 20-, 60-H), 7.50 (s,
1H, 40-H), 2.42 (s, 6H, 2CH3); ESI-MS (m=z, %): 384
(Mþ+Na), 745 (2Mþ+Na), 1106 (3Mþ+Na); IR (KBr):
4-Methylbenzoyl chloride (0.54 g) was used here. Orange
product (Rf ¼ 0:55). Mp: 204–205 °C. EI-MS (m=z, %):
347 (Mþ, 2.3), 213 (2.8), 119 (p-MePhCOþ, 100); IR
(KBr): 3040, 1770, 1715, 1610, 1585, 1510, 1270,
3040, 1780, 1710, 1610, 1580, 1510, 1280, 840 cmꢃ1
;
1
840 cmꢃ1; H NMR (CDCl3): d 9.01 (dd, J1 ¼ 7:6 Hz,
C21H15NO3S required: C, 69.79; H, 4.18; N, 3.88.
Found: C, 69.83; H, 4.30; N, 3.70.
J2 ¼ 0:9 Hz, 1H, 2-H), 8.70 (dd, J1 ¼ 7:3 Hz,
J2 ¼ 1:1 Hz, 1H, 7-H), 8.22 (m, 2H, 4-, 5-H), 8.18 (m,
2H, 3-, 6-H), 7.80 (m, 2H, 20-, 60-H), 7.40 (m, 2H, 30-5-
H), 2.50 (s, 3H, –CH3); C20H13NO3S required: C, 69.15;
H, 3.77; N, 4.03. Found: C, 68.95; H, 3.86; N, 3.99.
5.12. 3,4-Dimethoxy-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C9)
5.8. 4-Methoxy-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C5)
3,5-Dimethoxylbenzoyl chloride (0.70 g) was used here.
1
Brown product. Mp: 221–223 °C, H NMR d (DMSO-
d6): 8.91(d, J32 ¼ 7:5 Hz, 1H, 2-H), 8.61 (m, 3H, 4-, 5-, 7-
H), 7.91 (m, 3H, 3-, 6-, 60-H), 7.59 (d, J6 2 ¼ 1:7 Hz, 1H,
0
0
4-Methoxylbenzoyl chloride (0.60 g) was used here. Or-
ange product (Rf ¼ 0:36). Mp: 200–201 °C. EI-MS (m=z,
%): 363 (Mþ, 1.6), 213 (1.5), 135 (p-MeOPhCOþ, 100);
IR (KBr): 3040, 1780. 1710, 1600, 1580, 1510, 1270,
20-H), 7.25 (dd, J6 5 ¼ 8:6 Hz, 1H, 50-H), 3.93 (s, 3H, 30-
OCH3), 3.87 (s, 3H, 40-OCH3); EI-MS (m=z, %): 393
(Mþ, 79.55), 213 ([M+Hþ)3,4-2OCH3C6H3COOÆ],
94.21), 165 (3,4-2OCH3C6H3COþ, 100); IR (KBr): 3040,
0
0
1
845 cmꢃ1; H NMR (DMSO-d6): 8.92 (dd, J1 ¼ 7:6 Hz,
1770, 1710, 1600, 1580, 1515, 1280, 840 cmꢃ1
;
J2 ¼ 1:0 Hz, 1H, 2-H), 8.63 (m, 3H, 7-, 4-, 5-H), 8.16 (m,
2H, 3-, 6-H), 7.96 (m, 2H, 20-, 60-H), 7.22 (m, 2H, 30-, 50)-
H), 3.92 (s, 3H, –OCH3); C20H13NO4S required: C, 66.10;
H, 3.61; N, 3.85. Found: C, 66.57; H, 3.78; N, 3.65.
C21H15NO5S required: C, 64.12; H, 3.84; N, 3.56.
Found: C, 64.87; H, 4.03; N, 3.48.
5.13. 2-Chloro-nicotinic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C10)
5.9. 3,5-Dichloro-benzoic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C6)
2-Chloronicotinic chloride (0.61 g) was used here.
1
3,5-Dichlorobenzoyl chloride (0.73 g) was used here.
Orange-red product (Rf ¼ 0:54). Mp: 212–214 °C; 1H
NMR d (DMSO-d6): 8.93 (dd, J32 ¼ 7:7 Hz, 1H, 2-H),
Brown product. Mp: 210–212 °C; H NMR d (DMSO-
d6): 8.94 (d, J32 ¼ 7:5 Hz, 1H, 2-H), 8.68 (m, 3H, 4-, 5-,
0
0
7-H), 7.97 (m, 2H, 3-, 6-H), 8.83 (dd, J3 4 ¼ 4:8 Hz,
8.62 (m, 3H, 7-, 4-, 5-H), 8.22 (m, J2 4 ¼ J6 4 ¼ 1:9 Hz, 40-
J2 4 ¼ 1:3 Hz, 1H, 40-H), 8.72 (dd, J3 2 ¼ 7:7 Hz,
0
0
0 0
0
0
0 0
H), 8.19 (d, J4 2 ¼ J4 6 ¼ 1:9 Hz, 2H, 20-, 60-H), 7.97 (m,
2H, 3-, 6-H); EI-MS (m=e, %): 401 (Mþ, 16.78), 213
([M+Hþ)3,5-2ClC6H3COOÆ], 56.69), 173 (3,5-2ClC6H3-
COþ, 100); IR (KBr): 3040, 1780, 1715, 1610, 1580, 1270,
770, 740 cmꢃ1; C19H9Cl2NO3S required: C, 56.73; H,
2.26; N, 3.48. Found: C, 56.53; H, 2.30; N, 3.67.
J4 2 ¼ 1:3 Hz, 1H, 20-H), 7.80 (q, J2 3 ¼ 7:7 Hz,
0
0
0 0
0
0
0 0
J4 3 ¼ 4:8 Hz, 1H, 30-H); EI-MS (m=z, %): 368 (Mþ,
11.52), 213 ([M+Hþ)2-ClC5H3COOÆ], 28.33), 140 (2-
ClC5H3COþ, 100); IR (KBr): 3040, 1780, 1710, 1600,
0
0
1580, 1500, 1270, 840 cmꢃ1
;
C18H9ClN2O3S: 368.0012. Found: 368.0022.
HRMS: calcd for
5.10. 2-Fluoro-4-chloro-benzoic acid 1-oxo-3-thioxo-
1H,3H-benzo[de]isoquinolin-2-yl ester (C7)
5.14. Furan-2-carboxylic acid 1-oxo-3-thioxo-1H,3H-
benzo[de]isoquinolin-2-yl ester (C11)
2-Fluoro-4-chlorobenzoyl chloride (0.67 g) was used
1
Furan-2-carboxylic chloride (0.46 g) was used here. Red
product. Mp: 209–211 °C, 1H NMR d (DMSO-d6): 8.90
here. Orange product. Mp: 208–210 °C; H HNMR d

Y. Xu et al. / Bioorg. Med. Chem. 12 (2004) 2335–2341
2341
(d, J32 ¼ 7:7 Hz, 1H, 2-H), 8.62 (m, 3H, 4-, 5-, 7-H), 7.94
Acknowledgements
(m, 2H, 3-, 6-H), 8.29 (d, J4 2 ¼ 0:9 Hz, 1H, 40-H), 7.85
0
0
(d, J3 2 ¼ 3:6 Hz, 1H, 20-H), 6.94 (q, J2 3 ¼ 3:6 Hz,
This was partially supported by the National Natural
Science Foundation of China, the National Key Project
for Basic Research (2003CB114400), Shanghai Com-
mittee of Science and Technology, and Shanghai Com-
mission of Education.
0
0
0 0
J4 3 ¼ 1:7 Hz, 1H, 30-H); EI-MS (m=z, %): 323 (Mþ,
14.53), 213 ([M+Hþ)C4H3OCOOÆ], 8.32), 95
(C4H3OCOþ, 100); IR (KBr): 3040, 1780, 1710, 1600,
1580, 1500, 1270, 840; HRMS: calcd for C17H9NO4S:
323.0252. Found: 323.0260.
0
0
5.15. Spectroscopic measurements
References and notes
The compounds were dissolved in absolute ethanol to
give 10ꢃ5 M solutions, which were read with Shimadzu
UV-265 for absorption spectra and with Perkin Elmer
LS 50 using quinine sulfate in sulfuric acid as quantum
yield for fluorescence spectra.
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5.16. Intercalation studies of compounds C to DNA
0.1 mL of solution of a compound in DMSO (10ꢃ3–10ꢃ4
M) mixed with 0.1 M Tris–HCl buffer (pH 7.4) to 10 mL.
Then, two groups of samples were prepared in the con-
centration of chemical at 10ꢃ5–10ꢃ6 M, one contained
Calf thymus DNA 50 lM, the other contained no DNA
but had the same concentration of chemical as control.
All the above solution was shaken for 3 days at 25 °C in
the dark. Fluorescence wavelength and intensity area of
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12. Epe, B.; Adam, W.; Ballmaier, D.; Grimm, G. N.; Saha-
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5.17. Photocleavage of supercoiled DNA using
compounds C
14. Gupta, M.; Ali, R. J. Biochem. 1984, 95, 1253.
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300 ng pBR322 DNA (form I), 1 lL of solution of
chemical in DMSO and 10 mM Tris–HCl buffer (pH 7.6)
were mixed to 10 lL and stood for 10 min at 0 °C, then
irradiated for 30 min or more with light (2.3 mW/cm2,
365 nm) using lamp placed at 20 cm from sample.
Supercoiled DNA runs at position I, nicked DNA at
position II, and linear DNA at position III. The samples
were analyzed by gel electrophoresis in 1% Agarose and
gel was stained with ethidium bromide.