Synthesis and properties of benzothioxanthene dicarboximide hydroperoxide: An efficient 'time-resolved' DNA photocleaver with long-wavelength

Article abstract of DOI:10.1016/S0040-4039(02)00442-2

A novel hydroperoxide of benzothioxanthene dicarboximide B4 was synthesized through naphthyl radical-induced aromatic 1,5-hydrogen transfer in an unusual Pschorr cyclization and photooxygenation. It was evaluated as an excellent 'time-resolved' DNA photocleaver with long-wavelength absorption, which can photonick the duplex DNA at a micromolar concentration of 5 μM upon irradiation at 450 nm.

Full text of DOI:10.1016/S0040-4039(02)00442-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2995–2998
Synthesis and properties of benzothioxanthene dicarboximide
hydroperoxide: an efficient ‘time-resolved’ DNA photocleaver
with long-wavelength
Xuhong Qian,a,* Ping Mao,b,c Wei Yaob,c and Xiangfeng Guoa
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, PO Box 40, 158 Zhongshan Road, Dalian 116012,
China
bInstitute of Pesticides and Pharmaceuticals, East China University of Science and Technology, Shanghai 200237, China
cShanghai Key Laboratory of Chemical Biology, PO Box 544, Shanghai 200237, China
Received 5 December 2001; revised 20 February 2002; accepted 28 February 2002
Abstract—A novel hydroperoxide of benzothioxanthene dicarboximide B4 was synthesized through naphthyl radical-induced
aromatic 1,5-hydrogen transfer in an unusual Pschorr cyclization and photooxygenation. It was evaluated as an excellent
‘time-resolved’ DNA photocleaver with long-wavelength absorption, which can photonick the duplex DNA at a micromolar
concentration of 5 mM upon irradiation at 450 nm. © 2002 Published by Elsevier Science Ltd.
In recent years, the development of artificial photonu-
cleases has received increasing interest due to their
significant importance in molecular biology and human
medicine.1–4 Many kinds of DNA cleavage agents have
been developed. Organic hydroperoxides that can
efficiently generate hydroxyl radicals5 by photoirradia-
tion with long-wavelength light (>300 nm) without
using metal ions is an extremely useful hydroxyl radical
source, particularly for the design of DNA-cleaving
molecules.5 Long-wavelength UV-light (>350 nm) or
more preferably visible light irradiation is an attractive
cofactor, since it is safer and easy to manipulate. While
up until now, most DNA photocleavers releasing oxy-
gen-centered radicals showed photo-bioactivity around
350 nm,6–9 few of them showed photocleavage under
irradiation by visible light, and then only in the pres-
ence of metal cations.10–12 In addition, those photo-
cleavers with ‘time-resolved’ properties were very
rare.10–13 In fact, no organic hydroperoxide, with ‘time-
resolved’ photocleaving properties or with photocleav-
ing ability under irradiation with long-wavelength
visible light, has been reported.
compounds. Encouraged by these results and special
promotion action of sulfur to DNA intercalation or
photocleavage,9e,13c benzothioxanthene hydroperoxide
B4 was synthesized, and its DNA photocleaving prop-
erties were evaluated.
OH
OH
O O
O O
S
N
N
O
2
1
O
Compound B4, as an isomer of 2, was obtained via
three reaction steps: (1) 3-diazonium-4-phenylthio-
naphthalic anhydride A afforded isomers A2 and B215,16
through an unusual Pschorr cyclization; (2) the imida-
tion of B2 to give B3; (3) the photooxygenation of B3
to produce B4. The structures of all compounds were
established via 1H NMR, IR, EI MS and elemental
analyses.20 FT-IR spectra of hydroperoxide B4 dis-
played the characteristic absorption peaks of the OꢀO
band vibration around 800 cm−1.
The hydroperoxides 1 and 2 were reported as novel and
efficient intercalating DNA cleavers.9,14 The high DNA
cleavage efficiencies of these reagents might be
attributed to the large conjugated planarity of these
Usually, in Pschorr cyclization reactions a new bond is
formed between two aromatic rings next to a het-
eroatom, at the diazonium substituted positions to give
multinuclear aromatic hydrocarbons, especially for the
derivatives of phenanthrene. Only one example of the
* Corresponding author.
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)00442-2

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X. Qian et al. / Tetrahedron Letters 43 (2002) 2995–2998
cannot be efficiently generated due to the mismatch
between the maximum absorption of B4 and the pho-
toirradiation wavelength. When the photoirradiation
wavelength was changed to 450 nm through a light
filter, B4 showed effective photocleavage at a concen-
tration of 5 mM (Fig. 1b).
formation of isomers in a Pschorr cyclization was
known previously in benzophenone derivatives, where a
phenyl radical-induced intramolecular aromatic 1,5-
hydrogen transfer gave two five-membered ring isomers
in a ratio of 55:45.17 No other similar situations have
been found, as aromatic compounds are usually poor
hydrogen donors in radical transformations.17 In our
case, naphthyl radical-induced intramolecular aromatic
hydrogen transfer in naphthalene derivatives (from A1
to produce B1) gave the five-membered A2 and the
six-membered B2 (Scheme 1). The isomeric ratio of A2
The photocleavage activity of B4 increased remarkably
with prolonged photoirradiation time (Fig. 1c), which
had not been seen in the case of 2 and the other organic
hydroperoxides.9 It was different from DNA photo-
cleavers involving metal complexes and classical Fenton
reactions, where radicals are produced in a rapid
burst.19 This continuous generation of reactive
hydroxyl radicals provided a novel alternative to the
well-known Fenton-based chemistry. Therefore, B4
might be more attractive for ‘time-resolved’ DNA
cleavage studies and for in vivo biomedical application
involving ‘photodynamic therapy’. In addition, the
photocleavage ability of compound B4 was increased
with the pH values of buffers, it was sensitive to the
outer environment and it gave good photocleaving
results at pH 8.5. While compound 2 showed no differ-
ence in photonicking efficiency in the pH-dependent
experiments.
1
and B2 was at 86:14 (via H NMR and HPLC) with A
as starting material and 25:75 with B as starting mate-
rial. The isomeric ratio of thio-compounds A2 and B2
was different from that of their oxo-counterpart (90:10
and 7:93, respectively) in the same reaction.18 It sug-
gests that the radical-induced intramolecular aromatic
1,5-hydrogen transfer in the thio-compounds is more
efficient compared with their oxo-counterpart and that
the presence of the sulfur atom promotes intramolecu-
lar migration of a hydrogen atom.
It can be seen in Table 1 that, because of the extended
conjugated planarity, the UV–vis absorption and
fluorescence maxima of B4, B2 and B3 are at longer
wavelength compared with those of compound 2. It can
also be seen in Table 1 that the fluorescence quantum
yield of compound B4 is approximately three times
higher than that corresponding to compound 2,
although they are isomers. In addition, B4 was also
very stable and did not give a decomposition product
(with a hydroxyl group) by comparison with the photo-
chemical behavior of 2 in chloroform solution under
scattered daylight.14 These differences imply possible
differences in photo-biology.
The apparent association constant Ka (for the interac-
tion between DNA and compound) of the hydroperox-
ides B4 (4.26×103 M−1) was smaller than that of 2
(3.82×105 M−1), and this might also have a negative
effect on the photocleavage of the former, but, it should
be stressed that for efficient photonick DNA at mM
level the photosensitizing wavelength of B4 was much
longer than that of 2 (at 366 nm, match with its
absorption).
The DNA photocleavage of B4 was evaluated using
supercoiled circular pBR322 (form I) DNA (50 mM/
base pair) under photoirradiation at a distance of 20 cm
at 0°C and then analyzed on a 1% agarose gel. The
efficiency was defined as the degree of relaxation of the
supercoiled DNA, relaxed circular DNA (single-strand
cleavage) as form II. When B4 and DNA were mixed
and photoirradiated with a transluminator (366 nm), no
obvious cleavage was observed at as high as 50 mM
concentration of B4 (Fig. 1a), as the hydroxyl radicals
Table 1. UV–vis and fluorescence spectral data for benzo-
(thio)xanthene dicarboximides
Compound
UV–vis umax/nm FL umax/nm (ƒ) Stoke shift
(log m)
(nm)
2
379 (4.05)
455 (4.24)
456 (3.78)
458 (4.40)
457 (0.045)
524 (0.12)
521 (0.21)
524 (0.11)
78
69
65
66
B4
B2
B3
Scheme 1. The synthesis of B4 and the Pschorr cyclization to give isomers A2 and B2.

X. Qian et al. / Tetrahedron Letters 43 (2002) 2995–2998
2997
Figure 1. The photocleavage to DNA by B4. (a) The photocleavage of DNA under irradiation with light at 366 nm. Lane 1: DNA
alone (no hw); Lane 2: DNA and B4 at concentrations of 50 mM; Lane 3: DNA and B4 at concentrations of 20 mM; Lane 4: DNA
alone (hw, 75 min). (b) Effect of concentrations on the photocleavage of DNA under the irradiation of light at 450 nm. Lane 1: DNA
alone (no hw); Lane 2–7: DNA and B4 at concentrations of 50, 20, 10, 5, 1 and 0.5 mM, respectively; Lane 8: DNA alone (hw,
75 min). (c) Effect of irradiation time on the photocleavage of DNA under the irradiation of light at 450 nm. Lane 1: DNA alone
(no hw); Lane 2: B4 and DNA (hw, 30 min); Lane 3: B4 and DNA (hw, 60 min); Lane 4: B4 and DNA (hw, 75 min); Lane 5: B4
and DNA (hw, 90 min); Lane 6: DNA alone (hw, 90 min). (d) Effect of pH of the aqueous buffer on the photocleavage of DNA at
450 nm. Lanes 1, 3, 5, 7: DNA and B4 at 50 mM, buffer pH 8.5, 8.0, 7.5, 7.0, respectively; Lanes 2, 4, 6, 8: DNA alone, buffer
pH 8.5, 8.0, 7.5, 7.0, respectively; hv, 75 min.
In conclusion, the above study on benzothioxanthene
dicarboximide hydroperoxide B4 probably provides the
first example of an organic hydroperoxide, which has
‘time-resolved’ photocleavage properties on DNA and
demonstrated photocleavage under irradiation with vis-
ible light (450 nm). And it also provided a novel
example of naphthyl radical-induced intramolecular
aromatic hydrogen transfer during Pschorr cyclization
in the preparation of B4.
Jpn. 1999, 72, 1571–1574; (e) Qian, X.; Huang, T.; Wei,
D.; Zhu, D.; Fan, M.; Yao, W. J. Chem. Soc., Perkin
Trans. 2 2000, 715–718.
10. Sarker, A. K.; Tabata, M.; Wasaaki, K. Anal. Sci. 1997,
13, 509–512.
11. Kumar, C. V.; Tan, W. B.; Bett, P. W. J. Inorg. Biochem.
1997, 68, 177–181.
12. Chatterjee, S. R.; Srivastava, T. S.; Kamat, J. P.;
Devasagayam, T. P. A. J. Porphyrins Phthalocyanins
1998, 2, 337–343.
13. (a) Theodorakis, E. A.; Wilcoxen, K. M. Chem. Commun.
1996, 1927–1928; (b) Blom, P.; Xiang, A. X.; Kao, D.;
Theodorakis, E. A. Bioorg. Med. Chem. 1999, 7, 727–736;
(c) Qian, X.; Yao, W.; Chen, G.; Huang, X.; Mao, P.
Tetrahedron Lett. 2001, 42, 6175.
14. Wei, Y.; Qian, X.; Hu, Q. Tetrahedron Lett. 2000, 41,
7711.
15. (a) Peters, A. T.; Bide, M. J. Dyes Pigments 1985, 6, 267;
(b) Peters, A. T.; Bide, M. J. Dyes Pigments 1985, 6, 417.
16. Grayshan, P. H.; Kashim, A. M.; Peters, A. T. J. Hetero-
cycl. Chem. 1974, 11, 33.
Acknowledgements
This work was financially supported by the Fuk Ying
Tung Foundation, The Ministry of Education of China,
National Natural Science Foundation of China and
Shanghai Foundation of Science and Technology.
References
17. Karady, S.; Abramson, N. L.; Dolling, U. H.; Douglas,
A. W.; McManemin, G. J.; Marcune, B. J. Am. Chem.
Soc. 1995, 117, 5425.
1. Satio, I.; Nakatani, K. Bull. Chem. Soc. Jpn. 1996, 69,
3007.
2. Saito, I.; Takayama, M.; Sugiyama, H.; Nakamura, T. In
DNA and RNA Cleavers and Chemotherapy of Cancer and
Viral Diseases; Menunier, B., Ed.; Kluwer: Netherlands,
1996; pp. 163–176.
3. Breslin, D. T.; Coury, J. E.; Anderson, J. R.; McFail-
Isom, L.; Kan, Y.; Williams, L. D.; Bottomley, L. A.;
Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 5043.
4. Sigman, D.; Mazumder, A.; Perrin, D. M. Chem. Rev.
1993, 93, 2295.
5. Ogata, Y.; Tomizawa, K.; Furuta, K. In The Chemistry
of Peroxides; Patai, S., Ed.; Wiley: New York, 1983; p.
711.
6. Matsugo, S.; Saito, I. Tetrahedron Lett. 1991, 25, 2949–
2950.
7. Armitage, B. Chem. Rev. 1998, 98, 1171–1200.
8. Mehroto, J.; Mishra, R. K. Nucleosides Nucleotides 1994,
13, 963.
9. (a) Tao, Z.; Qian, X.; Wei, D. Dyes Pigments 1996, 31,
245; (b) Tao, Z.; Qian, X. Dyes Pigments 1999, 43,
139–145; (c) Yao, W.; Qian, X. Dyes Pigments 2000, 106,
69–72; (d) Tao, Z.; Qian, X.; Fan, M. Bull. Chem. Soc.
18. Qian, X.; Cui, J.; Zhang, R. Chem. Commun. 2001,
2656–2657.
19. (a) Hess, K. M.; Dix, T. A. Anal. Biochem. 1992, 206,
309; (b) Boivin, J.; Crepon, E.; Zard, S. Z. Bull. Soc.
Chim. Fr. 1992, 129, 145; (c) Nocentini, G.; Castagnino,
E.; Salvatori, A.; Corsano, S.; Fioretti, M. C. Arzneim.-
Forsch./Drug Res. 1995, 10, 1127.
20. Compound A2: yellow solid, mp 284–286°C, lH (DMSO-
d6, 300 MHz): l 9.4 (s, 1H, 7-H), 8.75 (m, 2H, 3-H, 1-H),
8.59 (dd, J1=1.0 Hz, J2=7.4 Hz, 1H, 11-H), 8.28 (m, 1H,
2-H), 8.04 (dd, J1=8.2 Hz, J2=7.4 Hz, 1H, 8-H), 7.68
(m, 2H, 9-H, 10-H); EI MS (m/z, %): 306 ([M+2]+, 11.4),
304 (M+, 100). Compound B2: orange solid, mp >300°C,
lH (DMSO-d6, 300 MHz): l 8.55 (m, 3H, 2-H, 6-H, 7-H),
8.36 (d, J=8.0 Hz, 1H, 2-H), 7.83 (d, J=8.1 Hz, 1H,
9-H), 7.56–7.70 (m, 3H, 10-H, 11-H, 12-H); EI MS (m/z,
%): 306 ([M+2]+, 8.7), 304 (M+, 100). Compound B3: lH
(500 MHz, CDCl3): l 1.74 (s, 3H, 3%-CH3), 1.77 (s, 3H,
3%-CH3), 4.73 (d, J=7.5Hz, 2H, ꢀNCH2ꢀ), 5.35 (t, J1=
J2=7.5 Hz, 1H, 2%-H), 7.39 (m, 3H, 10-12H), 7.49 (d,
J=8.0 Hz, 1H, 9-H), 8.20 (m, 2H, 1-H, 7-H), 8.42 (d,

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X. Qian et al. / Tetrahedron Letters 43 (2002) 2995–2998
J=8.0 Hz, 1H, 6-H), 8.62 (d, J=8.0 Hz, 1H, 2-H); EI MS
(m/z, %): 373 ([M+2]+, 5.1), 371 (M+, 41.3), 302 ([M+
HꢀCH2CHꢁCMe2]+, 100); IR (KBr): 3080, 2960, 2930,
1690, 1645, 1585, 1380. Anal. calcd for C23H17NO2S: C,
74.39; H, 4.58; N, 3.77. Found: C, 74.08; H, 4.89; N, 3.79%.
Compound B4: lH (500 MHz, CDCl3): 2.01 (s, 3H,
3%-CH3), 4.70 (m, 3H, NꢀCH2ꢀCHꢀ), 5.12 (d, J=1.4 Hz,
2H, 3%-CH2ꢁ), 7.56 (m, 2H, 9-H, 10-H), 7.75 (m, 1H, 2-H),
7.92 (d, J=7.6 Hz, 1H, 8-H), 8.19 (d, J=7.6 Hz, 1H, 11-H),
8.32 (d, J=8.0 Hz, 1H, 1-H), 8.53 (d, J=7.1 Hz, 1H, 3-H),
9.04 (s, 1H, 7-H), 10.17 (br, 1H, ꢀOOH); EI MS (m/z, %):
386 ([M−OH]+, 5.7); IR (KBr): 3258, 1688, 1640, 1587,
1335, 906, 799, 780 cm−1; FT-Raman: 3065, 1689, 1587,
1400, 1377, 799 cm−1
.