Novel and highly efficient DNA photocleavers: Hydroperoxides of heterocyclic-fused naphthalimides

Article abstract of DOI:10.1016/S0040-4039(00)01304-6

A series of novel hydroperoxides of heterocyclic-fused naphthalimides were designed, synthesized and exhibited excellent DNA photonicking activities. Among these compounds, the benzothiophenonaphthalimide-type reagent proved to be the most efficient, it c

Full text of DOI:10.1016/S0040-4039(00)01304-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 7711–7715
Novel and highly efficient DNA photocleavers:
hydroperoxides of heterocyclic-fused naphthalimides
Wei Yao,a Xuhong Qianb,* and Qingyan Hua
aInstitute 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 21 February 2000; revised 27 July 2000; accepted 28 July 2000
Abstract
A series of novel hydroperoxides of heterocyclic-fused naphthalimides were designed, synthesized and
exhibited excellent DNA photonicking activities. Among these compounds, the benzothiophenonaphth-
alimide-type reagent proved to be the most efficient, it can induce single-strand nicks in duplex DNA at
micromolar concentration upon illumination. The relationship between the molecular structure and
cleaving activities was discussed. © 2000 Elsevier Science Ltd. All rights reserved.
DNA cleavage by synthetic nucleases is of great interest in biology and bio-organic chemistry.
During recent decades, many DNA cleavage reagents have been developed as potential
antitumor agents or prothetic groups for antisensoligonucleotides.1–4 One of the most well-
known DNA cleavage species is the hydroxyl radical, and much effort has been devoted to the
’
development of efficient methods for OH generation using organic precursors by low-energy
irradiation, such as long-wavelength UV-light (u>350 nm) or more preferably by visible light
irradiation.5,6 Among the exploited photochemical DNA-cleavers, hydroperoxides of naphth-
alimides, such as 1 and 2, have been proved to be very efficient and could effectively generate a
hydroxyl radical under the irradiation of long-wavelength UV-light, and exhibit promising DNA
photo-cleavage capabilities.6–8 The high efficiency of these reagents may also be attributed to
their high DNA-intercalating capability due to the large conjugated planarity of these com-
pounds. Encouraged by the excellent DNA photonicking capabilities of this type of reagent and
based upon our previous studies,8–10 we further explored a series of novel DNA cleaving
reagents bearing a larger conjugated planar, heterocyclic (oxazole and benzothiophene) fused
naphthalene ring and a hydroxyl radical generating group (hydroperoxide). The former func-
tions as the binding part, and the latter as the photonicking functionality. All of them
* Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01304-6

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(compounds 3–5) were observed to exhibit significantly higher cleaving abilities to the super-
coiled circular pBR322 DNA rather than the non-substituted counterpart, DNA cleaver 2, upon
photoirradiation.
These reagents were synthesized from 4-bromo-3-nitro-1,8-naphthalic anhydride11 as shown in
Scheme 1. Photooxidation of the corresponding N-isopentenyl-1,8-naphthalimides resulted in
the formation of compounds 3, 4 and 5 as shelf-stable crystals.12 All of these hydroperoxides
1
were identified via H NMR, EI-MS, IR and elemental analysis and displayed the characteristic
absorption peak of the OꢀO bond vibration around 800 cm−1 in FTIR or FT-Raman spectra.
Scheme 1. Synthesis of novel naphthalimide-derived DNA cleaving reagents. (a) PhSH, EtOH, reflux, 4 h, 51% yield;
(b) SnCl2/HCl, 92.6% yield; (c) Pschorr cyclization,13 83% yield; (d) 20% NaOH, 85°C, 8 h, 83.3% yield; (e) H2, Pd/C,
97% yield; (f) CH3COOH, PPA, 120°C, 3.5 h, 62% yield; (g) (CF3CO)2O, PPA, 90°C, 3 h, 75.9% yield; (h)
H2NCH2CHꢁC(CH3)2, EtOH, 3 h, >90% yields; (i) hw, O2, TPP/CH2Cl2, −10°C, 3.5 h, 32ꢀ40% yields
Interestingly, upon exposure of the chloroform solution of 3 to scattered daylight at room
temperature for about 3 h, the corresponding hydroxyl derivative 914 was generated. The
characteristic absorption peak of the OꢀO bond of 3 at 799 cm−1 gradually disappeared,
accompanied by the emergence of the broad absorption peak of the hydroxyl group at 3460
cm−1 by FT-Raman and IR monitoring. The sensitivity of this compound might be due to the
prolonged conjugated system of 3, which leads to stronger UV absorption than 2 at long-wave-
length UV-light even in daylight and more effectively absorbing energy to overcome the energy
barrier between the ground and the excited state. The formation of this hydroxyl compound was
proposed to be involved in intramolecular energy transfer from the photoexcited chromophore
to the hydroperoxy group and consequently homolysis of the OꢀO bond with the release of the
’
active species OH and the substituted allyloxy radical, which was quenched in a hydrogen-

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donating solvent to give compound 9. However, it is known that most organic hydroperoxides
are converted into the corresponding ketones derivatives via an g-H abstraction mechanism
under the irradiation of UV-light.6,15,16 The difference between the two pathways was probably
because the enlarged conjugated system decreases the energy gap between the LUMO and
HOMO, thus the released and transferred energy from the photoexicited chromophore can only
selectively cleave the OꢀO bond, due to its weaker bond energy, rather than the g-CꢀH bond.
The probable generation of a hydroxyl radical also suggested that compound 3 might exhibit
high DNA photocleaving activity.
The cleavage activities of 3, 4 and 5 were evaluated using supercoiled circular pBR322 (form
I) DNA (50 mM/base pair) under photoirradiation with a transluminator (366 nm) at a distance
of 20 cm at 0°C for 0.5 h and analyzed on a 1% agarose gel. DNA photocleavage efficiency was
defined as the degree of relaxation of the supercoiled DNA. Comparing to hydroperoxide 2 at
the same conditions, reagents 3, 4 and 5 demonstrated higher DNA cleavage ability as shown
in Figs. 1 and 2. All the three new hydroperoxides exhibited cleaving capabilities at the
concentrations below 50 mM, while no obvious cleavage was observed for 2 even at as high as
100 mM concentrations. As speculated above, compound 3 showed excellent cleavage activity
which can effectively photonick form I DNA into form II DNA at a concentration as low as 1
mM (Fig. 1).
Figure 1. Cleavage of supercoiled circular pBR322 DNA by hydroperoxides 3 and 2. Lane 1: DNA alone; lanes 2–5:
DNA and 3 at concentrations of 3: 0.5, 1, 5 and 10 mM, respectively; lanes 6–9: DNA and 2 at concentrations of 2:
1, 10, 50 and 100 mM, respectively; lane 10: DNA alone (no hw)
Figure 2. Cleavage of supercoiled circular pBR322 DNA by hydroperoxides 4 and 5. Lane 1: DNA alone (no hw);
lanes 2–5: DNA and 4 at concentrations of 4: 10, 20, 50 and 100 mM, respectively; lanes 6–9: DNA and 5 at
concentrations of 5: 10, 20, 50 and 100 mM, respectively; lane 10: DNA alone
The significantly enhanced cleavage activities of compounds 3, 4 and 5 also confirmed the
rationality of design of the molecular structures. UV spectra showed the wavelengths of the
maximum absorption of reagents 3, 4 and 5 were 379 nm (4.05), 335 nm (4.04), 328 nm (4.04),
respectively. It was obvious that the umax of compound 3 was the longest and the closest to the

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Table 1
Growth inhibitory activities against human tumor cell lines for 2, 3, 4 and 5
Tumor type
BEL-7404 Human liver cancera
HL-60 Human leukemiab
Conc. (mol/L)c
10−4
10−5
10−6
10−4
100
99.2
99.0
88.3
10−5
100
96.2
100.0
66.7
10−6
2
3
4
5
90.7
95.9
94.2
95.3
92.8
95.4
91.4
46.1
22.7
18.5
0.5
33.3
51.1
9.7
18.1
5.7
a Experiments were carried out with incubation for 72 h using an SRB assay.
b Experiments were carried out with incubation for 48 h using an MIT assay.
c The concentration of tested reagents 2, 3, 4, 5.
photoirradiation wavelength 365 nm, which may be responsible for the improvement of cleaving
activity since the increased absorption of energy would speed up the OꢀO bond homolysis to
form the active species, a hydroxyl radical. On the other hand, the enlargement of the molecular
plane of these compounds with the conjugated heterocycles enhanced their DNA-intercalating
capabilities. The binding constant of compounds 2, 3 and 4 to calf thymus DNA measured using
the fluorescence quenching method17 are 2.67×104, 8.72×105 and 8.31×104, respectively. The
higher intercalating capabilities of these compounds may be another contribution to their
plausible DNA cleaving capabilities.
The antitumor activities of 3, 4 and 5 were further examined by the growth inhibition against
human tumor cells, HL-60 and BEP-7404 tumor cell lines. The results were parallel to their
DNA cleavage activities, as shown in Table 1, hydroperoxide 3 also exhibited the highest
efficiency of inhibition.
In general, novel hydroperoxides of heterocyclic-fused naphthalimides 3, 4 and 5, which can
be synthesized from substituted 1,8-naphthalimides as shelf-stable crystals, were shown to be
very efficient DNA cleavage reagents, and were further studied as valuable leading compounds
of anti-tumor drugs.
Acknowledgements
This work was supported by the Fuk Ying Tung Foundation and the National Natural
Science Foundation of China.
References
1. Chatterjee, M.; Rokita, S. E. J. Am. Chem. Soc. 1991, 113, 5116.
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: The Netherlands, 1996; pp. 163–176.
3. Breslin, D. T.; Coury, J. E.; Anderson, J. R.; McFailIsom, L.; Kan, Y. Z.; Williams, L. D.; Bottomley, L. A.;
Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 5043.
4. Armitage, B. Chem. Rev. 1998, 98, 1171–1200.

7715
5. Bielski, B. H.; Gehicki, J. M. Free Radic. Biol. 1977, 1, 1.
6. Saito, I. Pure Appl. Chem. 1992, 64, 1305–1310.
7. Matsugo, S.; Saito, I. Tetrahedron Lett. 1991, 25, 2949–2950.
8. Tao, Z.; Qian, X.; Wei, D. Dyes Pigments 1996, 31, 245–251.
9. Qian, X.; Tang, J.; Zhang, J.; Zhang, Y. Dyes Pigments 1994, 25, 109.
10. Tao, Z.; Qian, X.; Tang, J. Dyes Pigments 1996, 30, 247–252.
11. Kadhim, A. M.; Peters, A. T. J. Soc. Dyers Colourists 1974, 90, 153.
12. Compound 3: lH (500 M, CDCl3) 2.01 (s, 3H), 4.70 (m, 3H), 5.08 (s, 1H), 5.13 (s, 1H), 7.56 (m, 2H), 7.75 (m,
1H), 7.92 (d, J=7.6 Hz, 1H), 8.19 (d, J=7.6 Hz, 1H), 8.32 (d, J=8.0 Hz, 1H), 8.53 (d, J=7.1 Hz, 1H), 9.04 (s,
1H), 10.17 (br, 1H); EI-MS m/z 386 (M−OH)+; IR (KBr): 3258, 1688, 1640, 1587, 1335, 906, 799, 780 cm−1
;
FT-Raman: 3065, 1689, 1587, 1400, 1377, 799 cm−1. Compound 4: lH (500 M, CDCl3): l 2.00 (s, 3H), 2.87 (s,
3H), 4.56, 4.59 (2d, J1=3.0, J2=5.9, 2H), 4.73–4.78 (dd, J=9.4, J%=14.5, 1H), 5.07, 5.12 (2d, J1=0.9, J2=0.9,
2H), 7.94 (dd, J=7.4, J%=8.2, 1H), 8.59 (dd, J=1.2, J%=8.2, 1H), 8.71 (dd, J=1.2, J%=7.4, 1H), 8.97 (s, 1H),
9.76 (br, 1H); EI-MS m/z 352 M+; IR (KBr): 3200, 1700, 1670, 1600, 1450, 1380, 910, 800 cm−1; anal. calcd for
1
C19H16N2O5: C, 64.77; H, 4.57; N, 7.95. Found: C, 64.28; H, 4.44; N, 8.08. Compound 5: H NMR (500 MHz,
CDCl3): l 2.00 (s, 3H), 4.57–4.60 (m, 2H), 4.74–4.79 (dd, J=9.1 Hz, J%=14.4 Hz, 1H), 5.06 (s, 1H), 5.12 (s, 1H),
8.05 (dd, J=7.9 Hz, J%=7.8 Hz, 1H), 8.72 (dd, J=0.8 Hz, J%=7.9 Hz, 1H), 8.82 (dd, J=0.7 Hz, J%=7.8 Hz, 1H),
9.12 (s, 1H), 9.79 (br, 1H); EI-MS m/z: 407 M++1; IR (KBr): 3360, 3090, 1700, 1665, 910, 800, 790 cm−1
C19H13F3N2O5 requires: C, 56.17; H, 3.22; N, 6.89. Found: C, 55.92; H, 3.03; N, 6.56.
13. Peters, A. T.; Bide, M. J. Dyes Pigments 1985, 6, 267–275.
.
14. Compound 9: lH (500 M, CDCl3) 1.96 (s, 3H), 4.42 (d, J=5.9 Hz, 2H), 4.53 (t, J=5.9 Hz, 1H), 4.98 (s, 1H), 5.18
(s, 1H), 7.56 (m, 2H), 7.78 (m, 1H), 7.94 (m, 1H), 8.23 (m, 1H), 8.35 (d, J=7.8 Hz, 1H), 8.56 (d, J=7.3 Hz, 1H),
9.11 (s, 1H), 9.78 (br, 1H); EI-MS m/z 387 M+; IR (KBr): 3458, 1699, 1652, 1587, 1334, 913, 780 cm−1
;
FT-Raman: 3061, 1700, 1588, 1401, 1377 cm−1
.
15. Howard, J. A.; Chenier, J. H. B. Can. J. Chem. 1980, 58, 2808–2812.
16. Saito, I.; Takayama, M.; Matsuura, T. J. Am. Chem. Soc. 1990, 112, 883–884.
17. Gupta, M.; Ali, R. J. Biochem. 1984, 95, 1253–1257.
.